Insect control nanobody and its use

Abstract

Insect control nanobodies are provided. Thus, the nanobodies specifically bind to an insect polypeptide selected from the group consisting of chitin-binding domain (CBD), V-ATPase subunit c, trehalase, cytochrome p450 monooxygenase, chitin deacetylase, chitin synthase, and NPC1 sterol transporter, where binding of the nanobody to the insect polypeptide confers insect control activity to the nanobody. Polynucleotides encoding the nanobodies, host cells expressing the nanobodies, and uses thereof are also provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Patent Application No. 62 / 933,533, filed on November 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] Statement regarding sequence listings A 387,592-bit ASCII file named 83672_ST25.txt, created on November 9, 2020, was submitted simultaneously with this application and is incorporated herein by reference.

[0003] Technical field In some embodiments, the present invention relates to insect control nanobodies and their use. [Background technology]

[0004] Pest control over the past 70 years has been achieved primarily through the adoption of synthetic insecticides. Since Paul Müller's discovery of DDT's insecticidal properties in 1939, hundreds of insecticidal compounds have been developed, accompanied by a relatively steady increase in insecticide use. Most chemical insecticides used in recent years have a high potential for ecosystem disruption, are toxic to humans directly or through the food chain, and their use is hindered by the development of genetic insect resistance. The most effective biological solutions currently in use include beneficial organisms or natural enemies that reduce insect damage, pheromones that function as decoys or are harmful to reproduction, the release of sterile males, the spraying of Bacillus thuringiensis toxin, or genetically modified plants carrying the gene for Bacillus thuringiensis toxin that is toxic to insects but harmless to humans. However, these biological solutions also have drawbacks, including low efficiency, the risk of disrupting the balance of natural ecosystems, and the development of genetic insect resistance (e.g., in the case of Bacillus toxin use).

[0005] Nanobodies, also known as VHH antibodies, are single-domain antibodies (HCAbs) that possess a heavy chain and completely lose their light chain. They were discovered in camel blood, and Raymond Hamers was credited as the discoverer at the Free University of Brussels in 1989. Nanobodies are the smallest intact antigen-binding fragments in existence (Cortez-Retamozo et al., 2004; Revets et al., 2005), with a size of approximately 15 kDa. Nanobodies offer significant advantages, including high production yields in a wide range of expression systems, high accessibility to their epitopes due to their minimal size, high physical and chemical stability, reversible refolding and high solubility in aqueous solutions, high homogeneity without signs of spontaneous dimerization, and the ability to specifically recognize unique epitopes with sub-nanomolecular affinity. The use of nanobodies as insecticides has already been suggested (see, for example, European Patent Application Publications No. 3415010 and 2609116, U.S. Patent No. 9516879, U.S. Patent No. 9803003, and International Publication No. 2014191146). [Overview of the project]

[0006] According to one aspect of several embodiments of the present invention, a nanobody specifically binds to an insect polypeptide, wherein the insect polypeptide is as follows: (i) A polypeptide comprising a chitin-binding domain (CBD), wherein the nanobody is bound to the CBD, (ii) V-ATPase subunit c, wherein the nanobody includes a complementarity-determining region (CDR) represented by SEQ ID NOs. 167-169, SEQ ID NOs. 171 and 168-169, SEQ ID NOs. 174 and 168-169, SEQ ID NOs. 167, 178 and 169, or SEQ ID NOs. 180-182, wherein the nanobody is continuously arranged on the nanobody in the direction from N to C. (iii) Trehalase, (iv) Cytochrome p450 monooxygenase, (v) Chitin deacetylase, (vi) Chitin synthase, and (vii) NPC1 Stealth Transporter A nanobody is provided, selected from the group consisting of the following, wherein the binding of the nanobody to an insect polypeptide confers insect control activity to the nanobody.

[0007] According to one aspect of several embodiments of the present invention, the present invention comprises a nanobody that specifically binds to an insect polypeptide and a toxin moiety having insect control activity, wherein the insect polypeptide is as follows: (i) A polypeptide comprising a chitin-binding domain (CBD), wherein the nanobody is bound to the CBD, (ii) V-ATPase subunit c, wherein the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, 180-182, 187-189, or 191-193, which are continuously arranged on the nanobody in the direction from N to C. (iii) Trehalase, (iv) Cytochrome p450 monooxygenase, (v) Chitin deacetylase, (vi) Chitin synthase, and (vii) NPC1 Stealth Transporter A composition selected from the group consisting of the following is provided.

[0008] According to some embodiments of the present invention, the binding of nanobodies to insect polypeptides confers insect control activity to the nanobodies. According to some embodiments of the present invention, the nanobodies downregulate the activity of insect polypeptides.

[0009] According to some embodiments of the present invention, nanobodies or compositions are formulated for delivery by spraying, irrigation, and / or fumigation. According to some embodiments of the present invention, CBD is a type 2 chitin-binding domain (ChtBD2).

[0010] According to some embodiments of the present invention, CBD comprises an amino acid sequence selected from SEQ ID NOs: 3 to 7.

[0011] According to some embodiments of the present invention, the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 15-17, SEQ ID NOs: 19-21, SEQ ID NOs: 23-25, or SEQ ID NOs: 15 and 28-29, which are specifically bound to CBD and arranged continuously in the direction from N to C on the nanobody.

[0012] According to some embodiments of the present invention, the nanobody is specifically bound to CBD and arranged continuously in the direction from N to C on the nanobody, such as SEQ ID NOs: 15-17, 19-21, 23-25, 15 and 28-29, 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- Includes complementary determination regions (CDRs) represented by 89, SEQ ID NOs. 91, 80 and 93, SEQ ID NOs. 95-97, SEQ ID NOs. 99-101, SEQ ID NOs. 103-105, SEQ ID NOs. 107-109, SEQ ID NOs. 111-113, SEQ ID NOs. 115-117, SEQ ID NOs. 119-121, SEQ ID NOs. 123-125, SEQ ID NOs. 127-129, SEQ ID NOs. 131-133, SEQ ID NOs. 135-137, SEQ ID NOs. 139-141, SEQ ID NOs. 143-145, SEQ ID NOs. 147-149, SEQ ID NOs. 151-153, SEQ ID NOs. 15 and 156-157, SEQ ID NOs. 159-161, or SEQ ID NOs. 163-165.

[0013] According to some embodiments of the present invention, the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, or 180-182, which specifically bind to the V-ATPase subunit c and are continuously arranged on the nanobody in the direction from N to C.

[0014] According to some embodiments of the present invention, the nanobodies are specifically bound to trehalase and arranged continuously in the direction from N to C on the nanobodies, such as SEQ ID NOs: 195-197, 199 and 196-197, 201-203, 201 and 205-206, 208-210, 212 and 209-210, 215-217, 219-221, 223- Includes the complementarity determination region (CDR) represented by 225, sequence numbers 227-229, sequence numbers 231, 228 and 232, sequence numbers 234-236, sequence numbers 238-240, sequence numbers 242-244, sequence numbers 246-248, sequence numbers 250-252, sequence numbers 254-256, sequence numbers 258-260, sequence numbers 588-590, sequence numbers 5889-589 and 592, or sequence numbers 595, 589 and 592.

[0015] According to some embodiments of the present invention, the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 195-197, 199 and 196-197, 201-203, 201 and 205-206, 208-210, 212 and 209-210, 215-217, 219-221, 223-225, 227-229, 231, 228 and 232, 234-236, 238-240, 242-244, 246-248, 250-252, 254-256, or 258-260, which specifically bind to trehalase and are continuously arranged in the direction from N to C on the nanobody.

[0016] According to some embodiments of the present invention, the nanobody specifically binds to trehalase and comprises complementarity-determining regions (CDRs) represented by SEQ ID NO: 201 and 205-206, SEQ ID NO: 208-210, SEQ ID NO: 212 and 209-210, SEQ ID NO: 223-225, SEQ ID NO: 238-240, SEQ ID NO: 242-244, SEQ ID NO: 246-248, SEQ ID NO: 588-590, or SEQ ID NO: 595, 58 and 592, which are arranged continuously in the direction from N to C on the nanobody.

[0017] According to some embodiments of the present invention, the nanobody specifically binds to cytochrome p450 monooxygenase and comprises complementarity-determining regions (CDRs) represented by SEQ ID NO: 262-264, SEQ ID NO: 262-263 and 266, SEQ ID NO: 262, 270 and 264, SEQ ID NO: 274-276, SEQ ID NO: 280 and 275-276, SEQ ID NO: 282 and 275-276, SEQ ID NO: 282, 275 and 285, SEQ ID NO: 287, 220 and 289, SEQ ID NO: 291, 220 and 289, SEQ ID NO: 239-294 and 289, SEQ ID NO: 296-298, SEQ ID NO: 300-302, SEQ ID NO: 304-306, SEQ ID NO: 308-310, SEQ ID NO: 312-314, or SEQ ID NO: 316-318, which are arranged continuously in the direction from N to C on the nanobody.

[0018] According to some embodiments of the present invention, the nanobody specifically binds to cytochrome p450 monooxygenase and comprises complementarity-determining regions (CDRs) represented by SEQ ID NO: 262-264, SEQ ID NO: 287, 220 and 289, SEQ ID NO: 296-298, SEQ ID NO: 300-302, SEQ ID NO: 304-306, or SEQ ID NO: 312-314, which are arranged continuously in the direction from N to C on the nanobody.

[0019] According to some embodiments of the present invention, the nanobody is specifically bound to chitin deacetylase and arranged continuously in the direction from N to C on the nanobody, such as SEQ ID NOs: 320-322, 320 and 324-325, 327, 321 and 328, 320-321 and 332, 208 and 336-337, 320, 324 and 339, 343-345, 347-349, 351-353, 351, 347 and 356, and SEQ ID NOs: 3 Includes complementary determination regions (CDRs) represented by 58-360, 358-359 and 362, 365, 359 and 366, 368-370, 372 and 369-370, 374, 369 and 375, 377-379, 381-383, 385 and 382-383, 387-389, 387, 391 and 389, 394-396, 398-400, 402-404, or 406-408.

[0020] According to some embodiments of the present invention, the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 320-322, SEQ ID NOs: 320 and 324-325, SEQ ID NOs: 372 and 369-370, SEQ ID NOs: 374, 369 and 375, SEQ ID NOs: 377-379, SEQ ID NOs: 387-389, or SEQ ID NOs: 398-400, which specifically bind to chitin deacetylase and are continuously arranged on the nanobody in the direction from N to C.

[0021] According to some embodiments of the present invention, the nanobodies are specifically bound to chitin synthase and arranged continuously in the direction from N to C on the nanobodies, such as SEQ ID NOs: 412-414, 418-420, 422-424, 426-428, 426-427 and 430, 432-434, 436, 140 and 437, 439-441, 443-445, 447-449, and 356 and 451-45. 2. Includes a complementarity determination region (CDR) represented by sequence numbers 454-456, 458-460, 462-464, 466-468, 470-472, 474-476, 478-480, 482, 479 and 483, 485-487, 489-491, 493-495, 426 and 497-498, 500-502, 504-506, or 508-510.

[0022] According to some embodiments of the present invention, the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 412-414, 418-420, 426-428, 432-434, 443-445, 447-449, 466-468, 482, 479 and 483, 426 and 497-498, or 504-506, which are specifically bound to chitin synthase and arranged continuously in the direction from N to C on the nanobody.

[0023] According to some embodiments of the present invention, the nanobody includes complementarity determination regions (CDRs) represented by SEQ ID NOs: 512-514, 517 and 513-514, 521-523, 521-522 and 526, 531-533, 539-541, 485 and 540-541, 545 and 540-451, 547-549, 552-554, 556-558, 561-563, 565-567, 569-571, 573-575, 485 and 577-578, 580-582, or 584-586, which are specifically bound to the NPC1 sterol transporter and arranged continuously in the direction from N to C on the nanobody.

[0024] According to some embodiments of the present invention, the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 517 and 513-514, SEQ ID NOs. 485 and 540-541, SEQ ID NOs. 545 and 540-541, SEQ ID NOs. 556-558, SEQ ID NOs. 565-567, SEQ ID NOs. 573-575, SEQ ID NOs. 485 and 577-578, or SEQ ID NOs. 580-582, which specifically bind to the NPC1 sterol transporter and are continuously arranged on the nanobody in the direction from N to C.

[0025] According to one aspect of several embodiments of the present invention, a polynucleotide encoding a nanobody or composition is provided.

[0026] According to one aspect of several embodiments of the present invention, a nucleic acid construct is provided comprising a polynucleotide and a cis-acting regulatory element for controlling the expression of the polynucleotide.

[0027] According to one aspect of several embodiments of the present invention, a host cell is provided comprising a nanobody or composition, or a polynucleotide or nucleic acid construct encoding it.

[0028] According to one aspect of several embodiments of the present invention, a method for producing insect control nanobodies is provided, comprising expressing a polynucleotide or nucleic acid construct in a host cell.

[0029] According to some embodiments of the present invention, the method includes the isolation of nanobodies.

[0030] According to one aspect of several embodiments of the present invention, a method for insect control is provided, which involves contacting an insect with a nanobody or composition, a polynucleotide or nucleic acid construct encoding it, or a host cell expressing it.

[0031] According to some embodiments of the present invention, contact includes directly imparting nanobodies or compositions to insects.

[0032] According to some embodiments of the present invention, contact includes imparting nanobodies or compositions to a living organism or surface that insects may come into contact with.

[0033] According to some embodiments of the present invention, the nanobody or composition is formulated as a liquid formulation.

[0034] According to some embodiments of the present invention, the nanobody or composition is formulated as a dry formulation.

[0035] According to one aspect of several embodiments of the present invention, a plant is provided comprising a nanobody or composition, or a polynucleotide or nucleic acid construct encoding it.

[0036] According to some embodiments of the present invention, the plant is a transgenic plant.

[0037] According to one aspect of several embodiments of the present invention, an industrial product comprising a nanobody or composition is provided.

[0038] According to some embodiments of the present invention, industrial products are manufactured from plants.

[0039] According to one aspect of several embodiments of the present invention, a method for manufacturing nanobodies, the following: (i) Polypeptides containing a chitin-binding domain (CBD), (ii) V-ATPase subunit c, (iii) Trehalase, (iv) Cytochrome p450 monooxygenase, (v) Chitin deacetylase, (vi) Chitin synthase, and (vii) NPC1 Stealth Transporter A method is provided which involves immunosuppressing camels with recombinant or purified insect polypeptides selected from the group consisting of the following, wherein the purity of the insect polypeptide in the insect polypeptide preparation is at least 80%.

[0040] According to some embodiments of the present invention, the method includes isolation of antibodies following immunization.

[0041] According to some embodiments of the present invention, camels are llamas.

[0042] According to some embodiments of the present invention, the insect is selected from the group consisting of moths, stink bugs, grasshoppers, beetles, aphids, and honeybees.

[0043] According to some embodiments of the present invention, the insect is a moth.

[0044] According to some embodiments of the present invention, the moth is selected from the group consisting of Helicoverpa armigera and Spodoptera frugiperda.

[0045] According to some embodiments of the present invention, the moth is Helicoverpa armigera.

[0046] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice or testing of embodiments of the invention, but exemplary methods and / or materials are described below. In case of any conflict, the patent specification, including definitions, shall prevail. In addition, materials, methods, and examples are illustrative and not necessarily intended to be limiting.

[0047] Several embodiments of the present invention are described herein with reference to the accompanying drawings for illustrative purposes only. I would like to emphasize that the details shown below, particularly with close reference to the drawings, are for illustrative purposes only and for the purpose of providing a detailed description of embodiments of the present invention. Similarly, by looking at the description together with the drawings, it will be clear to those skilled in the art how embodiments of the present invention can be put into practice. [Brief explanation of the drawing]

[0048] [Figure 1] Figure 1 is a graph showing the specific binding of the fabricated anti-CBD nanobodies to the target CBD antigen, as determined by ELISA, and referred to herein as CB20501, CB20801, CB20901, and CB201101. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 2] Figure 2 shows a graph comparing the stability of the prepared anti-CBD nanobodies CB20501, CB20801, CB20901, and CB201101 after 1 hour incubation with larval digestive tract fluid recovered from H. armigera larvae, as determined by ELISA, with that of the control PBS. Each experiment was performed three times, and the data are shown as mean ± SE. [Figure 3]Figure 3 is a graph comparing the stability of the fabricated anti-CBD nanobodies CB20501, CB20801, CB20901, and CB201101 after incubation for 1-3 hours in a field-like environment, as determined by ELISA, with that of the control under room temperature (RT) conditions. Each treatment included 5 repetitions, and the data are shown as mean ± SE. [Figure 4-1] Figures 4(A)-(C) show the effects of anti-CBD nanobodies CB20501, CB20801, CB20901, and CB20110 on the body length (Figure 4(A)), body weight (Figure 4(B)), and mortality rate (Figure 4(C)) of H. armigera after feeding with a single dose of 40 μg of the nanobodies shown. Measurements were taken on day 7, and mortality rates were measured on days 7 and 14. Each experiment contained 12 larvae and was repeated 3-5 times; error bars indicate the standard error. Shown here are the percentage of body length of larvae fed the specified nanobody relative to the body length of larval controls fed PBS (Figure 4(A)), the percentage of body weight of larvae fed the specified nanobody relative to the body weight of larval controls fed PBS (Figure 4(B)), and the percentage of larval deaths at 7 and 14 days (Figure 4(C)). Statistical significance is indicated by a (P<0.05) and b (P<0.01). [Figure 4-2] Same as above [Figure 5-1]Figures 5(A)-(C) show the presence of the fabricated anti-CBD nanobodies CB20901 in the digestive tract of insects after incubation with an artificial feed containing fluorescently Cy3-labeled CB20901. Figure 5(A) shows the fluorescently labeled nanobodies tracked from the larval feed after 48-hour and 6-day incubation with the larvae. The upper panel shows bright-field images, and the lower panel shows images reflecting the Cy3 signal of the feed given to the larvae. Furthermore, the yellow dashed line indicates two different regions: the right side of the dashed line shows the signal from intact feed, and the left side of the dashed line shows the signal from larval feces. The difference in fluorescence intensity between the feed and feces represents uptake by the larval digestive tract. Figure 5(B) shows the presence of Cy5-labeled CB20901 nanobodies in the digestive tract of live larvae after 48 hours of incubation with feed containing the indicated Cy3-labeled nanobodies, or after 36 hours of incubation with feed containing Cy3-labeled nanobodies followed by 12 hours of incubation with feed without nanobodies. [Figure 5-2] Figure 5(C) shows the presence of Cy5-labeled CB20901 nanobodies in the larval digestive tract, particularly in the periphaloblast, after a 36-hour incubation with a feed containing the indicated Cy3-labeled nanobodies and a subsequent 12-hour incubation with a feed without nanobodies, compared to 48 hours of feeding with a feed without nanobodies. The upper panel shows bright-field images, and the lower panel shows images reflecting the Cy3 signal of the feed given to the larvae. [Figure 6] Figure 6 is a graph showing the specific binding of the fabricated anti-ATPase V subunit c (hereinafter referred to as "VAT") nanobodies, designated as VAT0101A, VAT0101B, VAT0101C, VAT0101D, VAT0101E, VAT0101F, and VAT0101G herein, to the target VAT antigen, as determined by ELISA. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 7]Figure 7 shows a graph comparing the stability of the fabricated anti-VAT nanobodies VAT0101A and VAT0101G after 1 hour incubation with larval digestive tract fluid recovered from H. armigera larvae, as determined by ELISA, with that of the control PBS. Each experiment was performed three times, and the data are shown as mean ± SE. [Figure 8] Figure 8 is a graph comparing the stability of the fabricated anti-VAT nanobodies VAT0101A, VAT0101B, VAT0101C, VAT0101D, VAT0101E, VAT0101F, and VAT0101G after incubation for 1-3 hours in a field-like environment, as determined by ELISA, with that of the control under room temperature (RT) conditions. Each treatment included three repetitions, and the data are shown as mean ± SE. [Figure 9-1] Figures 9(A)–(C) show the effects of anti-VAT nanobodies VAT0101A, VAT0101B, VAT0101C, VAT0101D, VAT0101E, VAT0101F, VAT0101G, and VAT0201 on the body length (Figure 9(A)), body weight (Figure 9(B)), and mortality rate (Figure 9(C)) of H. armigera after feeding with a single dose of 40 μg of the nanobodies shown. Measurements were taken on day 7 or day 14 as shown. Each experiment contained 12 larvae and was repeated 3–5 times, with error bars indicating the standard error. Shown here are the percentage of body length of larvae fed the specified nanobody relative to the body length of larval controls fed PBS (Figure 9(A)), the percentage of body weight of larvae fed the specified nanobody relative to the body weight of larval controls fed PBS (Figure 9(B)), and the percentage of larval deaths at 7 and 14 days (Figure 9(C)). Statistical significance is expressed as a (P<0.05) and b (P<0.01). [Figure 9-2] Same as above [Figure 10]Figure 10 is a graph showing the specific binding of the fabricated anti-trehalase (hereinafter referred to as "TRH") nanobodies, referred to herein as TRH1A, TRH12, TRH13, and TRH14, to their target TRH antigens, as determined by ELISA. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 11-1] Figures 11(A) and 11(B) are graphs comparing the stability of fabricated anti-TRH nanobodies, referred to herein as TRH1A, TRH1D, TRH3B, TRH5A, and TRH5C, after incubation for 6 hours or overnight in a field-like environment (Figure 11(A)) and after incubation for 6 hours or overnight at 50°C (Figure 11(B)), as determined by ELISA, with that under control conditions at room temperature (RT). Each treatment consisted of three repetitions, and the data are shown as mean ± SE. [Figure 11-2] Same as above [Figure 12A] Figure 12A shows the effect of anti-TRH nanobodies TRH1A, TRH1D, TRH3B, TRH5A, TRH5B, TRH5C, and TRH8 on the mortality rate of H. armigera when fed an artificial diet containing the indicated nanobodies at a final concentration of 0.4 mg / ml on days 0 and 7. Measurements were performed on days 7 and 14 as shown. Each experiment contained 12 larvae and was repeated three times; error bars indicate the standard error. Statistical significance is expressed as a (P<0.05). [Figure 12B] Figure 12B shows the effect of anti-TRH3B nanobodies on the mortality of H. armigera when nanobodies at concentrations of 0.4 or 1 mg / ml were fed with disc-shaped cotton leaves on day 0. The larvae were transferred to fresh disc-shaped cotton leaves treated with similar concentrations of nanobodies on days 4 and 7. Mortality was measured on days 4, 7, and 10 as shown. Each experiment contained 12 larvae and was repeated twice; error bars indicate the standard error. [Figure 13]Figure 13 is a graph showing the specific binding of the fabricated anti-cytochrome p450 monooxygenase (hereinafter referred to as "P450") nanobodies, designated as P45001A, P45003A, P45004, P45005, and P45006 herein, to their target P450 antigens, as determined by ELISA. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 14] Figure 14 shows the effect of the fabricated anti-P450 nanobody, referred to herein as P45008, on the mortality rate of H. armigera when fed an artificial diet containing the nanobody at a final concentration of 0.4 mg / ml on days 0 and 7. Measurements were performed on days 7 and 14 as shown. Each experiment included 12 larvae and was repeated three times; error bars indicate the standard error. [Figure 15] Figure 15 is a graph showing the specific binding of the fabricated anti-chitin deacetylase (hereinafter referred to as "CDA") nanobodies, referred to herein as CDA01A, CDA05, CDA07A, and CDA10, to their target CDA antigens, as determined by ELISA. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 16] Figures 16(A) and 16(B) are graphs comparing the stability of fabricated CDA7A nanobodies after incubation in a field-like environment for 6 hours or overnight (Figure 16(A)) and after incubation at 50°C for 6 hours or overnight (Figure 16(B)), as determined by ELISA, with that of a control under room temperature (RT) conditions. Each treatment included three repetitions, and the data are shown as mean ± SE. [Figure 17A] Figure 17A shows the effect of anti-CDA nanobodies, referred to herein as CDA1B, CDA4B, CDA4C, and CDA7A, on the mortality rate of H. armigera when fed an artificial diet containing the nanobodies at a final concentration of 0.4 mg / ml on days 0 and 7. Measurements were performed on days 7 and 14 as shown. Each experiment included 12 larvae and was repeated three times; error bars indicate the standard error. Statistical significance is expressed as a (P<0.05). [Figure 17B] Figure 17B shows the effect of CDA7A nanobodies on the mortality of H. armigera when nanobodies at increasing concentrations (0.2, 0.4, 1, 2, or 4 mg / ml, as shown) were fed with disc-shaped cotton leaves on day 0. The larvae were transferred to fresh disc-shaped cotton leaves treated with similar concentrations of nanobodies shown on days 4 and 7. Mortality was measured on days 4, 7, and 10 as shown. Each experiment contained 12 larvae and was repeated twice; error bars indicate the standard error. [Figure 18] Figure 18 is a graph showing the specific binding of the fabricated anti-chitin synthase (hereinafter referred to as "CHS") nanobodies, referred to herein as CHS01A, CHS02A, CHS08, CHS13, CHS18, CHS22, and CHS24, to their target CHS antigens, as determined by ELISA. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 19-1] Figures 19(A) and 19(B) are graphs comparing the stability of the fabricated anti-CHS nanobodies, referred to herein as CHS3A, after incubation for 6 hours or overnight in a field-like environment (Figure 19(A)) and after incubation for 6 hours or overnight at 50°C (Figure 19(B)), as determined by ELISA, with that of the control under room temperature (RT) conditions. Each treatment included three repetitions, and the data are shown as mean ± SE. [Figure 19-2] Same as above [Figure 20A] Figure 20A shows the effect of anti-CHS nanobodies, referred to herein as CHS3A, CHS4, and CHS7, on the mortality rate of H. armigera when fed an artificial diet containing the nanobodies at a final concentration of 0.4 mg / ml on days 0 and 7. Measurements were performed on days 7 and 14 as shown. Each experiment included 12 larvae and was repeated three times; error bars indicate the standard error. Statistical significance is expressed as a (P<0.05). [Figure 20B]Figure 20B shows the effect of CHS3A nanobodies on the mortality of H. armigera when nanobodies at increasing concentrations (0.2, 0.4, 1, 2, or 4 mg / ml, as shown) were fed with disc-shaped cotton leaves on day 0. Larvae were transferred to fresh disc-shaped cotton leaves treated with similar concentrations of nanobodies shown on days 4 and 7. Mortality was measured on days 4, 7, and 10 as shown. Each experiment contained 12 larvae and was repeated three times; error bars indicate the standard error. Statistical significance is expressed as a (P<0.05). [Figure 21] Figure 21 is a graph showing the specific binding of the fabricated anti-NPC1 sterol transporter (hereinafter referred to as "NPC") nanobodies, designated herein as NPC01C01, NPC04C01, NPC04D01, NPC07A01, NPC0901, NPC01101, and NPC01201, to their target NPC antigens, as determined by ELISA. Each experiment was performed five times, and the data are shown as mean ± SE. [Figure 22] Figure 22 shows the effect of anti-NPC nanobodies, referred to herein as NPC4C and NPC4D, on the mortality rate of H. armigera when fed an artificial diet containing the nanobodies at a final concentration of 0.4 mg / ml on days 0 and 7. Measurements were performed on days 7 and 14 as shown. Each experiment included 12 larvae and was repeated three times; error bars indicate the standard error.

[0049] In some embodiments, the present invention relates to insect control nanobodies and their use.

[0050] Before describing in detail at least one embodiment of the present invention, it should be understood that the applications of the present invention are not necessarily limited to the configuration details and arrangement of elements and / or methods shown in the following description and / or illustrated in the drawings and / or embodiments. Other embodiments of the present invention are possible and can be carried out or implemented by various means.

[0051] Pest control over the past 70 years has been achieved primarily through the application of synthetic insecticides, much of which pose a high risk of ecosystem destruction, are toxic to humans directly or through the food chain, and their use is hampered by the development of genetic insect resistance. Even the most advanced biological solutions currently in use have drawbacks, including low efficiency, the risk of disrupting the balance of natural ecosystems, and the development of genetic insect resistance.

[0052] In carrying out the present invention, the inventors have hereby developed nanobodies that target several insect polypeptides having insecticidal activity. As a result, certain embodiments of the teachings of this application suggest their use as insecticides.

[0053] Therefore, according to the first embodiment of the present invention, a nanobody that specifically binds to an insect polypeptide, wherein the insect polypeptide is as follows: (i) A polypeptide comprising a chitin-binding domain (CBD), wherein the nanobody is bound to the CBD, (ii) V-ATPase subunit c, wherein the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, or 180-182, which are continuously arranged on the nanobody in the direction from N to C. (iii) Trehalase, (iv) Cytochrome p450 monooxygenase, (v) Chitin deacetylase, (vi) Chitin synthase, and (vii) NPC1 Stealth Transporter A nanobody is provided, selected from the group consisting of the above, wherein the binding of the nanobody to the insect polypeptide confers insect control activity to the nanobody.

[0054] According to further or alternative embodiments, the insect polypeptide comprises a nanobody that specifically binds to an insect polypeptide and a toxin moiety having insecticidal activity, wherein the insect polypeptide is as follows: (i) A polypeptide comprising a chitin-binding domain (CBD), wherein the nanobody is bound to the CBD, (ii) V-ATPase subunit c, wherein the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, 180-182, 187-189, or 191-193, which are continuously arranged on the nanobody in the direction from N to C. (iii) Trehalase, (iv) Cytochrome p450 monooxygenase, (v) Chitin deacetylase, (vi) Chitin synthase, and (vii) NPC1 Stealth Transporter A composition selected from the group consisting of the following is provided.

[0055] In this application, the term "nanobody" refers to a single-domain antigen-binding fragment.

[0056] According to certain embodiments, nanobodies are single variable domains derived from naturally occurring antibody heavy chains. Nanobodies are typically derived from heavy-chain-only antibodies (lacking light chains), commonly found in camels (Hamers-Casterman et al., 1993, Nature 363: 446-448, Desmyter et al., 1996, Nat. Struct. Biol. 803-811), and are therefore often referred to as VHH antibodies or VHH sequences. Camels include Old World camels (Camelus bactrianus and Camelus dromedarius) and New World camels (e.g., Lama paccos, Lama glama, Lama guanicoe, and Lama vicugna). Non-exclusive examples of camels include dromedary camels, Bactrian camels, wild Bactrian camels, llamas, alpacas, vicuñas, and guanacos. According to certain embodiments, the camel is a llama.

[0057] NANOBODY® and NANOBODIES® are registered trademarks of Ablynx NV (Belgium).

[0058] For a further explanation of VHHs or nanobodies, see the textbook “Single Domain Antibodies,” Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, Vol. 911, in particular chapter by Vincke and Muyldermans (2012), and the following non-limiting patent documents listed as general background technology: International Publications 94 / 04678, 95 / 04079, and 96 / 34103 of the Free University of Brussels; International Publications 94 / 25591, 99 / 37681, 00 / 40968, 00 / 43507, 00 / 65057, 01 / 40310, and 01 / 44301 of Unilever Corporation; Specification of European Patent No. 1134231; and International Publication 02 / 48193 of the Flemish Institute of Biotechnology (Vlaams Instituut voor Refer to International Publications 97 / 49805, 01 / 21817, 03 / 035694, 03 / 054016, and 03 / 055527 of Biotechnologie) (VIB), International Publications 04 / 041867, 04 / 041862, 04 / 041865, 04 / 041863, 04 / 062551, 05 / 044858, 06 / 40153, 06 / 079372, 06 / 122786, 06 / 122787, and 06 / 122825 of Ablynx NV, and other published patent applications of Ablynx NV. As will be apparent to those skilled in the art, nanobodies are characterized in particular by the presence of camel “Hallmark residues” in one or more (Kabat numbered) framework sequences, which are described, for example, in International Publication No. 08 / 020079, p. 75, Table A-3, incorporated herein by reference.

[0059] According to certain embodiments, a nanobody means an intact molecule (i.e., comprising four framework regions and three complementarity-determining regions), or a functional fragment thereof capable of binding to an antigen epitope to which the intact molecule binds.

[0060] As used in this application, the term "epitope" refers to any antigenic determinant of an antigen to which a paratope of a nanobody binds. Epitope determinants typically consist of chemically active surface groups such as amino acids or carbohydrate side chains, and usually possess specific three-dimensional structural properties as well as specific charge properties.

[0061] According to certain embodiments, the nanobody is a complete or intact nanobody.

[0062] According to a particular embodiment, the nanobody is a nanobody fragment.

[0063] According to a particular embodiment, the size of the nanobody is 5-30 kDa, 10-30 kDa, or 10-20 kDa.

[0064] According to a particular embodiment, the size of the nanobody is approximately 15 kDa.

[0065] The term "nanobody" also includes natural or synthetic analogues, similarities, variants, and variants of nanobody.

[0066] Typically, intact nanobodies contain three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3).

[0067] The terms “complementarity-determining region” or “CDR” as used herein are used alternatively to refer to antigen-binding regions found within the variable region of heavy chain polypeptides. The characteristics of amino acid residues forming a CDR within a particular nanobody can be determined by methods known in the art, such as those described in the paper by Riechmann and Muyldermans (1999) for camels V HHSequence diversity adapted to the domain, as defined by Kabat et al. (e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), location of structural loops as defined by Chothia et al. (e.g., Chothia et al., Nature 342:877-883, 1989), a hybrid method of 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 the worldwide website www(dot)bioinf-org(dot)uk / abs), and available composite crystal structures defined by contact definition (MacCallum et al., J. Mol. Biol. 262:732-745, Examples include the definition of conformation (see 1996) and "the definition of conformation" (see, for example, Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).

[0068] As used in this application, "CDR" may mean a CDR as defined by means known in the art (including combinations of means).

[0069] According to a particular embodiment, CDR is a V of camels in the paper by Riechmann and Muyldermans (1999). HH This is the same as the definition by Kabat et al. adapted to the domain.

[0070] Nanobodies can be single-specific (capable of recognizing one type of epitope or protein), bispecific (capable of binding to two types of epitopes or proteins), or polyspecific (capable of recognizing multiple types of epitopes or proteins).

[0071] According to a particular embodiment, the nanobody is a single-specific nanobody.

[0072] According to certain embodiments, the nanobody is multispecific (e.g., dispecific, trispecific, or tetraspecific).

[0073] According to certain embodiments, the nanobodies are bispecific nanobodies. Methods for fabricating bispecific nanobodies are publicly known in the art and are disclosed, for example, in Deffer 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), 2602, the contents of which are incorporated herein by reference.

[0074] The nanobodies disclosed herein bind specifically to the insect polypeptides described herein.

[0075] Preferably, the nanobody specifically binds to at least one epitope of the insect polypeptide described in this application.

[0076] Methods for testing binding are well known in the industry and include, but are not limited to, ELISA, radioimmunoassay (RIA), flow cytometry, BiaCore, Biolayer interferometry Blitz® assay, and HPLC.

[0077] According to a specific embodiment, the nanobody contains insect polypeptides with a Kd value of ≤ 10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 The terms are joined by M, and each possibility represents a different embodiment of the present invention.

[0078] According to certain embodiments, nanobodies can bind to insect polypeptides in the field or under controlled conditions, for example, in a greenhouse.

[0079] The term "insect" as used in this application is used in a broad, general sense and includes all species belonging to the superphylum Panarthropoda (classification: Systema Naturae, Brands, SJ (comp.) 1989-2005. Systema Naturae 2000. Amsterdam, Netherlands [www(dot)sn2000(dot)taxonomy(dot)nl / ]), including the phyla Arthropoda, Tardigrade, and Onychophora, and encompassing all stages of the life cycle, not limited to egg, larva, nymph, pupa, and adult. According to a particular embodiment, insects belong to the phylum Arthropoda (including, but not limited to, the orders Paleognatha, Silverfish, Paleoptera, and Neoptera, as well as ticks, mites, and spiders), more preferably belonging to the superclass Hexapoda, and most preferably belonging to the class Insecta. According to a particular embodiment, insects belong to the order Lepidoptera. Non-exclusive examples of insects include, but are not limited to, bed bugs, houseflies, moths, beetles, grasshoppers, caterpillars, aphids, mosquitoes, fleas, horseflies, wasps, cockroaches, and ants.

[0080] ·For example, the order of butterflies includes Acleris spp., Adoxophyes spp., Agrotis spp., Alabama argillacea, Amyelois spp., Anticarsia gemmatalis, Archips spp., Argyrotaenia spp., Autographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo suppressalis, Chilo spp., Choristoneura conflictana, Choristoneura fumiferana, Choristoneura occidentalis, Choristoneura rosaceana, Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydalima perspectalis, Cydia inopinata, Cydia spp., Diatraea spp., Diparopsis castanea, Earias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp.、Grapholita prunivora、Grapholita spp.、Hedya nubiferanal、Helicoverpa armigera、Helicoverpa zea、Helicoverpa spp.、Heliothis spp.、Hellula undalis、Hypantria cunea、Keiferia lycopersicella、Leucoptera scitella、Lithocolletis spp.、Lobesia botrana、Lymantria spp.、Lyonetia spp.、Malacosoma spp., Mamestra brassicae, Manduca sexta, 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 spp., Sesia spp., Sparganothis spp., Spodoptera dolichos, Spodoptera eridania, Spodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis, Spodoptera litura, Spodoptera spp., Synanthedon spp., Tecia solanivora, Thaumatotibia leucotreta, Thaumetopoea processionea, Thaumetopoea spp., Tortrix spp., Trichoplusia ni, and Yponomeuta spp.

[0081] · Coleoptera include, for example, Agrilus anxius, Agrilus planipennis, Agriotes spp., Anomala orientalis, Anoplophora chinensis, Anoplophora glabripennis, Anoplophora spp., Anthonomus bisignifer, Anthonomus eugenii, Anthonomus grandis, Anthonomus quadrigibbus, Anthonomus signatus, Anthonomus spp., Apriona spp., Arrhenodes minutus, Atomaria linearis, Chaetocnema tibialis, Conotrachelus nenuphar, Cosmopolites spp., Curculio spp., Dendroctonus micans, Dendrolimus sibiricus, Dermestes spp., Diabrotica virgifera, Diabrotica virgifera zeae, Diabrotica virgifera, Diabrotica balteata, Diabrotica barberi, Diabrotica undecimpunctata howardi, Diabrotica undecimpunctata tenella, Diabrotica undecimpunctata, Diabrotica undecimpunctata, Diabrotica spp., Epilachna varivestis, Epilachna spp., Epitrix cucumeris, Eremnus cerealis, Eremnus spp., Gonipterus scutellatus, Ips amitinus, Ips cembrae, Ips duplicatus, Ips sexdentatus, Ips typographus, Ips spp., Leptinotarsa ​​decemlineata, Leptinotarsa ​​juncta, Leptinotarsa ​​texana, Lissorhoptrus spp., Listronotus bonariensis, 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, Scarabidae species, Bark beetle species, Sitophilus spp., Sitotroga Examples include *Sternochetus mangiferae*, *Tenebrio* spp., *Tribolium castaneum*, *Tribolium* spp., and *Trogoderma* spp.

[0082] Examples of orthopteran species include Gryllotalpa spp., Locusta spp., and Schistocerca spp.

[0083] Examples of cockroaches include Blatta spp., Blattella spp., Periplaneta spp., and Leucophaea maderae.

[0084] Examples of isopterans include Coptotermes spp. and Reticulitermes spp.

[0085] Examples of the order Psocoptera include Liposcelis spp.

[0086] Examples of lice species in the order Liceformes include Haematopinus spp., Linognathus spp., Pediculus spp., and Trichodectes spp.

[0087] Examples of the suborder Damalinia within the order Lice include Damalinia spp.

[0088] Examples of thrips include Frankliniella occidentalis, Frankliniella platensis, Frankliniella spp., Hercinosa spp., Taeniothrips spp., Thrips palmi, Thrips tabaci, Scirtothrips aurantii, Scirtothrips citri, Scirtothrips dorsalis, and Scirtothrips spp.

[0089] Examples of species in the Hemiptera suborder Hemiptera include Cimex spp., Distantiella theobroma, Dysdercus spp., Euschistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp., Triatoma spp., species of the family Lygus hesperus and Lygus lineoloris, species of the family Lycanthidae (Blissus leucopterus, etc.), and the family Pentatomidae.

[0090] ·                         Aleurocanthus spiniferus, Aleurocanthus woglumi, Aleurocanthus spp., Aleurothrixus floccosus, Aleyrodes brassicae, Aonidella citrine, Aonidiella spp., Acyrthosiphon spp., Aphis fabae, Aphis glycines, Aphis gossypii, Aphis spp., Aspidiotus spp., Bemisia tabaci, Ceroplasts spp.. Chrysomphalus aonidium japonica、Macrosiphus spp.、Margarodes prieskaensis、Margarodes vitis、Margarodes vredendalensis、Myzus persicae、Myzus spp.Parasaissetia nigra.Pemphigus spp.Phylloxera spp.Planococcus spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp., Rhopalosiphum spp., Ripersiella hibisci, Saissetia spp., Schizaphis spp., Sitobion spp. Toxoptera citricida, Trialeurodes vaporariorum, Trioza erytreae, and Unaspis citri.

[0091] Examples of species in the Hemiptera suborder Dorciromorpha include Circulifer haematoceps, Circulifer tenellus, Draeculacephala minerva, Empoasca spp., Erythroneura spp., Graphocephala atropunctata, Hishimonus phycitis, Myndus crudus, Laodelphax spp., Nephotettix spp., Nilaparvata spp., Scaphoideus luteolus, Scaphoideus spp., and Xyphon fulgida.

[0092] Examples of Hymenoptera include Acromyrmex, Atta spp., Cephus spp., species of the Tenthredinidae family (such as Diprion spp. and Gilpinia polytoma), Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., and species of the Formididae family (such as Solenopsis spp. and Vespa spp.).

[0093] · Diptera include, for example, Aedes albopictus, Aedes cinereus, Aedes polynesiensis, Aedes spp., Amauromyza maculosa, Anastrepha fraterculus, Anastrepha ludens, Anastrepha obliqua, Anastrepha suspensa, Anastrepha spp., Anopheles gambiae, Anopheles spp., Aschistonyx eppoi, Atherigona soccata, Bactrocera spp., Bibio hortulanus, Calliphora erythrocephala, 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 spp., Liriomyza bryoniae, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii、Liriomyza spp.、Lucilia spp.、Melanagromyza spp.、Musca spp.、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. can be mentioned.

[0094] Examples of the Cryptoptera include Ceratophyllus spp. and Xenopsylla cheopis.

[0095] • An example of a member of the order Lepisma, subclass Lepisma, is Lepisma saccharina.

[0096] According to certain embodiments, insects are considered pests. The term "pest" as used in this application includes, but is not limited to, agriculturally harmful organisms (aphids, grasshoppers, caterpillars, beetles, moths, stink bugs, thrips, whiteflies, etc.), household pests (cockroaches, ants, wasps, flies, house crickets, bed bugs, cigarette beetle larvae, darkling beetles, earwigs, silverfish, termites, etc.), and blood-sucking pests (mosquitoes, fleas, lice, etc.). According to certain embodiments, insects are agriculturally harmful organisms.

[0097] According to a particular embodiment, the insect is selected from the group consisting of moths, stink bugs, grasshoppers, beetles, aphids, and honeybees.

[0098] According to a particular embodiment, the insect is a moth.

[0099] According to a particular embodiment, the insect is a nocturnal moth.

[0100] Non-specific examples of moths include Helicoverpa armigera, Cydia pomonella, and Spodoptera frugiperda.

[0101] According to certain embodiments, the moth is Helicoverpa armigera and / or Spodoptera frugiperda.

[0102] According to a particular embodiment, the moth is Helicoverpa armigera.

[0103] According to a particular embodiment, the insect is selected from the group consisting of Helicoverpa armigera, Spodoptera frugiperda, Nezara viridula, Nilaraparvata lugens, Aphis gossypii, Cydia pomonella, leaf-eating beetles (e.g., Leptinotarsa ​​decemlineata or Phaedon cochleariae) and honeybees.

[0104] According to a particular embodiment, the nanobody binds to an insect polypeptide selected from the group consisting of the following. (i) A polypeptide comprising a chitin-binding domain (CBD), wherein the nanobody is bound to the CBD, (ii) V-ATPase subunit c, wherein the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, 180-182, 187-189, or 191-193, which are continuously arranged on the nanobody in the direction from N to C. (iii) Trehalase, (iv) Cytochrome p450 monooxygenase, (v) Chitin deacetylase, (vi) Chitin synthase, and (vii) NPC1 Stealth transport vehicle.

[0105] As used in this application, the term "specifically binds" means that the nanobody binds to the target insect polypeptide with a higher affinity than other polypeptides present in the environment under physiological conditions, such as within the body of an insect.

[0106] According to certain embodiments, nanobodies bind to insect polypeptides without cross-reactivity with non-insect (e.g., plant, human) polypeptides.

[0107] According to a particular embodiment, the nanobody specifically binds to one of the insect polypeptides (i) to (vii) and does not cross-react with other insect polypeptides.

[0108] According to a particular embodiment, the nanobody is bound to at least two insect polypeptides (i) to (vii) by multi-property such as bispecificity.

[0109] According to a particular embodiment, the nanobody is a combination of at least two nanobodies to which each of two different insect polypeptides selected from the group consisting of insect polypeptides (i) to (vii) is bound.

[0110] According to a particular embodiment, a nanobody or combination of nanobodies combines (i)+(ii), (i)+(iii), (i)+(iv), (i)+(v), (i)+(vi), (i)+(vii), (ii)+(iii), (ii)+(iv), (ii)+(v), (ii)+(vi), (ii)+(vii), (iii)+(iv), (iii)+(v), (iii)+(vi), (iii)+(vii), (iv)+(v), (iv)+(vi), (iv)+(vii), (v)+(vi), (v)+(vii), (vi)+(vii).

[0111] According to a particular embodiment, the nanobody binds to a polypeptide containing a chitin-binding domain (CBD), and the nanobody binds to the CBD.

[0112] As used herein, the term “chitin-binding domain (CBD)” means an insect amino acid domain capable of binding to insect chitin (a linear polysaccharide consisting mostly of N-acetylated (1→4) beta-linked D-glucosamine residues) or its antigenic fragments, and capable of inducing a specific immune response to said fragments. Non-limiting examples of CBD include type 2 chitin-binding domains (ChtBD2) and the Rebers and Riddiform consensus sequence. According to other specific embodiments, CBD includes the Rebers and Riddiform consensus sequence.

[0113] As used herein, the term “Rebers and Riddiform Consensus Sequence” means the consensus sequence listed in the Pfam database, registration number pfam00379 (see Rebers and Riddiford, 1988, Anderson, 2010, Karouzou et al., 2007, and Willis, 2010, the contents of which are fully incorporated into this application with this reference).

[0114] According to certain embodiments, CBD is a type 2 chitin-binding domain (ChtBD2).

[0115] The term "type 2 chitin-binding domain (ChtBD2)" as used in this application is also referred to as the peritrophin A domain and refers to an amino acid sequence having six cysteine ​​groups that form three disulfide bonds. Such CBDs are known in the art and are disclosed in Tetreau, Guillaume, et al. Insect biochemistry and molecular biology 62 (2015): 127-141. According to a particular embodiment, ChtBD2 has the consensus sequence CX11-30CX5-6CX9-24CX12-17CX6-12C (SEQ ID NO: 409).

[0116] According to a particular embodiment, ChtBD2 includes an amino acid sequence that has at least 60%, 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 NOs: 3 to 7.

[0117] According to a particular embodiment, ChtBD2 includes an amino acid sequence having at least 60%, 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 sequence provided by GeneBank accession number XP_021181774 (SEQ ID NO: 410).

[0118] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW, and MUSCLE.

[0119] According to a particular embodiment, ChtBD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 7.

[0120] Non-exclusive examples of polypeptides containing ChtBD2 include peritrophic matrix proteins (PMPs) such as TcPMP5-B and HaIIM86, as well as peritrophin-like cuticle proteins (CPAPs) such as BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1, and BmCPAP3-D2.

[0121] According to certain embodiments, the polypeptide containing ChtBD2 is a PMP protein.

[0122] According to a particular embodiment, the polypeptide containing ChtBD2 is TcPMP5-B (PMP5-B of Tribolium castaneum).

[0123] According to a particular embodiment, the polypeptide containing ChtBD2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-2.

[0124] According to a particular embodiment, the polypeptide containing ChtBD2 consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 1-2.

[0125] Non-limiting examples of nanobodies that specifically bind to ChtBD2, and their corresponding CDRs, are shown in Tables 2A and 2B below.

[0126] According to a particular embodiment, the nanobody is specifically bound to ChtBD2 and arranged continuously on the nanobody in the direction from N to C, such as SEQ ID NOs: 15-17, 19-21, 23-25, 15 and 28-29, 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, 80 and 93, 95-97, 99-101, 103-105, 107- Includes a CDR having an amino acid sequence that is 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% identical to the amino acid sequence shown in 109, SEQ ID NOs: 111-113, SEQ ID NOs: 115-117, SEQ ID NOs: 119-121, SEQ ID NOs: 123-125, SEQ ID NOs: 127-129, SEQ ID NOs: 131-133, SEQ ID NOs: 135-137, SEQ ID NOs: 139-141, SEQ ID NOs: 143-145, SEQ ID NOs: 147-149, SEQ ID NOs: 151-153, SEQ ID NOs: 15 and 156-157, SEQ ID NOs: 159-161, or SEQ ID NOs: 163-165.

[0127] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to ChtBD2 and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 15-17, SEQ ID NOs. 19-21, SEQ ID NOs. 23-25, or SEQ ID NOs. 15 and 28-29.

[0128] According to a particular embodiment, the nanobody is specifically bound to ChtBD2 and arranged continuously on the nanobody in the direction from N to C, as shown in SEQ ID NOs: 15-17, 19-21, 23-25, 15 and 28-29, 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. This includes CDRs having the amino acid sequences shown in SEQ ID NOs. 91, 80 and 93, SEQ ID NOs. 95-97, SEQ ID NOs. 99-101, SEQ ID NOs. 103-105, SEQ ID NOs. 107-109, SEQ ID NOs. 111-113, SEQ ID NOs. 115-117, SEQ ID NOs. 119-121, SEQ ID NOs. 123-125, SEQ ID NOs. 127-129, SEQ ID NOs. 131-133, SEQ ID NOs. 135-137, SEQ ID NOs. 139-141, SEQ ID NOs. 143-145, SEQ ID NOs. 147-149, SEQ ID NOs. 151-153, SEQ ID NOs. 15 and 156-157, SEQ ID NOs. 159-161, or SEQ ID NOs. 163-165.

[0129] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 15-17, 19-21, 23-25, or 15 and 28-29, which are specifically bound to ChtBD2 and arranged continuously on the nanobody in the direction from N to C.

[0130] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 15-17, which are specifically bound to ChtBD2 and arranged continuously on the nanobody in a direction from N to C.

[0131] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 19-21, which are specifically bound to ChtBD2 and arranged continuously on the nanobody in a direction from N to C.

[0132] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 23-25, which are specifically bound to ChtBD2 and arranged continuously on the nanobody in a direction from N to C.

[0133] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 15 and 28-29, which are specifically bound to ChtBD2 and arranged continuously on the nanobody in a direction from N to C.

[0134] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, and 162, each possibility representing another embodiment of the present invention.

[0135] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 18, 22, and 26.

[0136] According to a particular embodiment, the nanobody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, and 162.

[0137] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 18, 22, and 26.

[0138] According to a particular embodiment, the nanobody includes SEQ ID NO: 14.

[0139] According to a particular embodiment, the nanobody includes SEQ ID NO: 18.

[0140] According to a particular embodiment, the nanobody includes SEQ ID NO: 22.

[0141] According to a particular embodiment, the nanobody includes sequence number 26.

[0142] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 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, and 162.

[0143] According to a particular embodiment, the nanobody comprises SEQ ID NOs: 14, 18, 22, or 26.

[0144] According to a particular embodiment, the nanobody consists of Sequence ID No. 14.

[0145] According to a particular embodiment, the nanobody consists of Sequence ID No. 18.

[0146] According to a particular embodiment, the nanobody consists of Sequence ID No. 22.

[0147] According to a particular embodiment, the nanobody consists of Sequence ID No. 26.

[0148] According to a particular embodiment, the nanobody binds to the V-ATPase subunit c.

[0149] Non-limiting examples of nanobodies that specifically bind to the V-ATPase subunit c, and their corresponding CDRs, are shown in Tables 3A-B below.

[0150] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, 180-182, 187-189, or 191-193, which are arranged continuously on the nanobody in the direction from N to C.

[0151] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 167-169, 171 and 168-169, 174 and 168-169, 167, 178 and 169, or 180-182, which are arranged continuously on the nanobody in the direction from N to C.

[0152] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 167-169, or SEQ ID NOs: 167, 178, and 169, which are arranged continuously on the nanobody in the direction from N to C.

[0153] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 167-169, which are continuously arranged on the nanobody in the direction from N to C.

[0154] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 171 and 168-169, which are continuously arranged on the nanobody in the direction from N to C.

[0155] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs. 174 and 168-169, which are continuously arranged on the nanobody in the direction from N to C.

[0156] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 167, 178, and 169, which are continuously arranged on the nanobody in the direction from N to C.

[0157] According to a particular embodiment, the nanobody binds a V-ATPase subunit c, and the nanobody includes complementarity-determining regions (CDRs) represented by SEQ ID NOs: 180-182, which are continuously arranged on the nanobody in the direction from N to C.

[0158] In this application, the term "V-ATPase subunit c" means Pfam PF03223, an insect enzyme that catalyzes the gradient of the electron chemical potential of protons, or an antigenic fragment thereof, the amino acid sequence of a fragment capable of eliciting an immune response to said fragment.

[0159] According to a particular embodiment, V-ATPase subunit c is the V-ATPase subunit c of Helicoverpa armigera, provided under GeneBank accession number XP_021198264, etc.

[0160] According to a particular embodiment, the V-ATPase subunit c includes SEQ ID NO: 8.

[0161] According to a particular embodiment, it consists of V-ATPase subunit c, sequence number 8.

[0162] According to a particular 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 NOs: 166, 170, 172, 173, 175, 176, 177, 183, 185, 186, and 190, each possibility representing another embodiment of the present invention.

[0163] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 170, 172, 173, 175, 176, 177, and 179.

[0164] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 176 and 177.

[0165] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 170, 172, 173, 175, 176, 177, 179, 183, 185, 186, and 190.

[0166] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 170, 172, 173, 175, 176, 177, and 179. According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 176 and 177.

[0167] According to a particular embodiment, the nanobody includes SEQ ID NO: 166.

[0168] According to a particular embodiment, the nanobody includes SEQ ID NO: 170.

[0169] According to a particular embodiment, the nanobody includes SEQ ID NO: 172.

[0170] According to a particular embodiment, the nanobody includes SEQ ID NO: 173.

[0171] According to a particular embodiment, the nanobody includes SEQ ID NO: 175.

[0172] According to a particular embodiment, the nanobody includes SEQ ID NO: 176.

[0173] According to a particular embodiment, the nanobody includes SEQ ID NO: 177.

[0174] According to a particular embodiment, the nanobody includes SEQ ID NO: 179.

[0175] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 170, 172, 173, 175, 176, 177, 179, 183, 185, 186, and 190.

[0176] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 166, 170, 172, 173, 175, 176, 177, and 179.

[0177] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 176 and 177.

[0178] According to a particular embodiment, the nanobody consists of sequence number 166.

[0179] According to a particular embodiment, the nanobody consists of Sequence ID No. 170.

[0180] According to a particular embodiment, the nanobody consists of Sequence ID No. 172.

[0181] According to a particular embodiment, the nanobody consists of Sequence ID No. 173.

[0182] According to a particular embodiment, the nanobody consists of Sequence ID No. 175.

[0183] According to a particular embodiment, the nanobody consists of sequence number 176.

[0184] According to a particular embodiment, the nanobody consists of Sequence ID No. 177.

[0185] According to a particular embodiment, the nanobody consists of sequence number 179.

[0186] According to a particular embodiment, the nanobody binds to trehalase.

[0187] The term "trehalase" as used in this application means the insect enzyme EC3.2.1.2, which catalyzes the conversion of trehalose to glucose, or the amino acid sequence of an antigenic fragment thereof that can produce an immune response to said fragment.

[0188] According to a particular embodiment, the trehalase is the trehalase of Helicoverpa armigera, provided under GeneBank accession number AJK29979. According to a particular embodiment, the trehalase comprises SEQ ID NO: 10. According to a particular embodiment, the trehalase is comprised of SEQ ID NO: 10. Table 4 below shows non-limiting examples of nanobodies that specifically bind to trehalase, and their corresponding CDRs.

[0189] According to a particular embodiment, the nanobody is specifically bound to trehalase and arranged continuously on the nanobody in the direction from N to C, as in SEQ ID NOs. 195-197, 199 and 196-197, 201-203, 201 and 205-206, 208-210, 212 and 209-210, 215-217, 219-221, 223-225, 227-229, 231, 228 and 232, 234-236, 238-240, sequence Includes a CDR having an amino acid sequence that 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 shown in sequences 242-244, 246-248, 250-252, 254-256, 258-260, 588-590, 588-589 and 592, or 595, 589 and 592.

[0190] According to a particular embodiment, the nanobodies are specifically bound to trehalase and arranged continuously on the nanobodies in the direction from N to C, such as sequence numbers 195-197, 199 and 196-197, 201-203, 201 and 205-206, 208-210, 212 and 209-210, 215-217, 219-221, 223-225, 227-229, 231, 228 and 232, sequence number Includes a CDR having an amino acid sequence that is 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% identical to the amino acid sequences shown in numbers 234-236, SEQ ID NOs. 238-240, SEQ ID NOs. 242-244, SEQ ID NOs. 246-248, SEQ ID NOs. 250-252, SEQ ID NOs. 254-256, or SEQ ID NOs. 2258-260.

[0191] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to trehalase and is continuously arranged on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 212 and 209-210, SEQ ID NOs. 223-225, SEQ ID NOs. 238-240, SEQ ID NOs. 242-244, SEQ ID NOs. 246-248, SEQ ID NOs. 588-590, or SEQ ID NOs. 595, 589 and 592.

[0192] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to trehalase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 212 and 209-210, SEQ ID NOs. 223-225, SEQ ID NOs. 588-590, or SEQ ID NOs. 595, 589 and 592.

[0193] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to trehalase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 223-225, or SEQ ID NOs. 588-590.

[0194] According to a particular embodiment, the nanobody is specifically bound to trehalase and arranged continuously on the nanobody in the direction from N to C, as in SEQ ID NOs. 195-197, 199 and 196-197, 201-203, 201 and 205-206, 208-210, 212 and 209-210, 215-217, 219-221, and 22 Includes CDRs shown in 3-225, SEQ ID NOs. 227-229, SEQ ID NOs. 231, 228 and 232, SEQ ID NOs. 234-236, SEQ ID NOs. 238-240, SEQ ID NOs. 242-244, SEQ ID NOs. 246-248, SEQ ID NOs. 250-252, SEQ ID NOs. 254-256, SEQ ID NOs. 258-260, SEQ ID NOs. 588-590, SEQ ID NOs. 5889-589 and 592, or SEQ ID NOs. 595, 589 and 592.

[0195] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 195-197, SEQ ID NOs. 199 and 196-197, SEQ ID NOs. 201-203, SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 212 and 209-210, SEQ ID NOs. 215-217, SEQ ID NOs. 219-221, SEQ ID NOs. 223-225, SEQ ID NOs. 227-229, SEQ ID NOs. 231, 228 and 232, SEQ ID NOs. 234-236, SEQ ID NOs. 238-240, SEQ ID NOs. 242-244, SEQ ID NOs. 246-248, SEQ ID NOs. 250-252, SEQ ID NOs. 254-256, or SEQ ID NOs. 258-260, which specifically bind to trehalase and are arranged continuously on the nanobody in the direction from N to C.

[0196] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 212 and 209-210, SEQ ID NOs. 223-225, SEQ ID NOs. 238-240, SEQ ID NOs. 242-244, SEQ ID NOs. 246-248, SEQ ID NOs. 588-590, or SEQ ID NOs. 595, 589 and 592, which specifically bind to trehalase and are arranged continuously on the nanobody in the direction from N to C.

[0197] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 212 and 209-210, SEQ ID NOs. 223-225, SEQ ID NOs. 588-590, or SEQ ID NOs. 595, 589 and 592, which specifically bind to trehalase and are arranged continuously on the nanobody in the direction from N to C.

[0198] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 201 and 205-206, SEQ ID NOs. 208-210, SEQ ID NOs. 223-225, or SEQ ID NOs. 588-590, which specifically bind to trehalase and are arranged continuously on the nanobody in a direction from N to C.

[0199] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 198, 200, 204, 207, 211, 213, 214, 218, 222, 226, 230, 233, 237, 241, 245, 249, 253, 257, 587, 591, 593, 594, and 596, and an amino acid sequence of 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 another embodiment of the present invention.

[0200] According to a particular 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 NOs: 194, 198, 200, 204, 207, 211, 213, 214, 218, 222, 226, 230, 233, 237, 241, 245, 249, 253, and 257, each possibility representing another embodiment of the present invention.

[0201] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 211, 213, 222, 237, 241, 245, 587, and 594.

[0202] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 211, 213, 222, 587, and 594.

[0203] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 222, and 587.

[0204] According to a particular embodiment, the nanobody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 198, 200, 204, 207, 211, 213, 214, 218, 222, 226, 230, 233, 237, 241, 245, 249, 253, 257, 587, 591, 593, 594, and 596. According to a particular embodiment, the nanobody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 198, 200, 204, 207, 211, 213, 214, 218, 222, 226, 230, 233, 237, 241, 245, 249, 253, and 257.

[0205] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 211, 213, 222, 237, 241, 245, 587, and 594.

[0206] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 211, 213, 222, 587, and 594.

[0207] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 222, and 587.

[0208] According to a particular embodiment, the nanobody includes SEQ ID NO: 204.

[0209] According to a particular embodiment, the nanobody includes SEQ ID NO: 207.

[0210] According to a particular embodiment, the nanobody includes SEQ ID NO: 211.

[0211] According to a particular embodiment, the nanobody includes SEQ ID NO: 213.

[0212] According to a particular embodiment, the nanobody includes Sequence ID No. 222.

[0213] According to a particular embodiment, the nanobody includes SEQ ID NO: 587.

[0214] According to a particular embodiment, the nanobody includes SEQ ID NO: 594.

[0215] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 198, 200, 204, 207, 211, 213, 214, 218, 222, 226, 230, 233, 237, 241, 245, 249, 253, 257, 587, 591, 593, 594, and 596.

[0216] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 194, 198, 200, 204, 207, 211, 213, 214, 218, 222, 226, 230, 233, 237, 241, 245, 249, 253, and 257.

[0217] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 211, 213, 222, 237, 241, 245, 587, and 594.

[0218] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 211, 213, 222, 587, and 594.

[0219] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 204, 207, 222, and 587.

[0220] According to a particular embodiment, the nanobody consists of Sequence ID No. 204.

[0221] According to a particular embodiment, the nanobody consists of Sequence ID No. 207.

[0222] According to a particular embodiment, the nanobody consists of Sequence ID No. 211.

[0223] According to a particular embodiment, the nanobody consists of Sequence ID No. 213.

[0224] According to a particular embodiment, the nanobody consists of Sequence ID No. 222.

[0225] According to a particular embodiment, the nanobody consists of Sequence ID No. 587.

[0226] According to a particular embodiment, the nanobody consists of sequence number 594.

[0227] According to a particular embodiment, the nanobody binds to cytochrome p450 monooxygenase.

[0228] The term "cytochrome p450 monooxygenase" as used in this application refers to the insect enzyme EC1.14, or an antigenic fragment thereof, which contains heme as a cofactor and, as a result of introducing one hydroxyl group into the substrate, reduces two atoms of a diatomic oxygen molecule to one hydroxyl group and one H2O molecule by oxidation of the accompanying NAD(P)H, and has an amino acid sequence capable of eliciting an immune response to the fragment.

[0229] According to a particular embodiment, the cytochrome p450 monooxygenase is Helicoverpa armigera cytochrome p450 monooxygenase, provided under GeneBank accession number AKS48889, etc.

[0230] According to a particular embodiment, the cytochrome p450 monooxygenase comprises SEQ ID NO: 11.

[0231] According to a particular embodiment, the cytochrome p450 monooxygenase is represented by SEQ ID NO: 11.

[0232] Non-limiting examples of nanobodies that specifically bind to cytochrome p450 monooxygenase, and their corresponding CDRs, are shown in Table 5 below.

[0233] According to a particular embodiment, the nanobody is specifically bound to cytochrome p450 monooxygenase and arranged continuously on the nanobody in the direction from N to C, as shown in SEQ ID NOs. 262-264, 262-263 and 266, 262, 270 and 264, 274-276, 280 and 275-276, 282 and 275-276, 282, 275 and 285, 287, 220 and 289, 291, 220 and 289, Includes a CDR having an amino acid sequence that is 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% identical to the amino acid sequences shown in SEQ ID NOs. 239-294 and 289, SEQ ID NOs. 296-298, SEQ ID NOs. 300-302, SEQ ID NOs. 304-306, SEQ ID NOs. 308-310, SEQ ID NOs. 312-314, or SEQ ID NOs. 316-318.

[0234] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to cytochrome p450 monooxygenase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 262-264, SEQ ID NOs. 287, 220, and 289, SEQ ID NOs. 296-298, SEQ ID NOs. 300-302, SEQ ID NOs. 304-306, or SEQ ID NOs. 312-314.

[0235] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to cytochrome p450 monooxygenase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 300-302 or SEQ ID NOs. 312-314.

[0236] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to cytochrome p450 monooxygenase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 312-314.

[0237] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 262-264, 262-263 and 266, 262, 270 and 264, 274-276, 280 and 275-276, 282 and 275-276, 282, 275 and 285, 287, 220 and 289, 291, 220 and 289, 239-294 and 289, 296-298, 300-302, 304-306, 308-310, 312-314, or 316-318, which specifically bind to cytochrome p450 monooxygenase and are arranged continuously on the nanobody in the direction from N to C.

[0238] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 262-264, SEQ ID NOs: 287, 220 and 289, SEQ ID NOs: 296-298, SEQ ID NOs: 300-302, SEQ ID NOs: 304-306, or SEQ ID NOs: 312-314, which specifically bind to cytochrome p450 monooxygenase and are arranged continuously on the nanobody in the direction from N to C.

[0239] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 300-302 or SEQ ID NOs: 312-314, which are specifically bound to cytochrome p450 monooxygenase and arranged continuously on the nanobody in the direction from N to C.

[0240] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 312-314, which are specifically bound to cytochrome p450 monooxygenase and arranged continuously on the nanobody in a direction from N to C.

[0241] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 261, 265, 267, 268, 269, 271, 272, 273, 277, 278, 279, 281, 283, 284, 286, 29, 292, 295, 299, 303, 307, 311, and 315, each possibility representing another embodiment of the present invention.

[0242] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 261, 286, 295, 299, 303, and 311.

[0243] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 299 and 311.

[0244] According to a particular embodiment, the nanobody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 261, 265, 267, 268, 269, 271, 272, 273, 277, 278, 279, 281, 283, 284, 286, 29, 292, 295, 299, 303, 307, 311, and 315.

[0245] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 261, 286, 295, 299, 303, and 311.

[0246] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 299 and 311.

[0247] According to a particular embodiment, the nanobody includes SEQ ID NO: 311.

[0248] According to a particular embodiment, the nanobody includes Sequence ID No. 299.

[0249] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 261, 265, 267, 268, 269, 271, 272, 273, 277, 278, 279, 281, 283, 284, 286, 29, 292, 295, 299, 303, 307, 311, and 315.

[0250] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 261, 286, 295, 299, 303, and 311.

[0251] According to certain embodiments, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 299 and 311.

[0252] According to certain embodiments, the nanobody consists of SEQ ID NO: 311.

[0253] According to certain embodiments, the nanobody consists of SEQ ID NO: 299.

[0254] According to certain embodiments, the nanobody binds to chitin deacetylase.

[0255] As used herein, the term "chitin deacetylase" means an insect enzyme EC 3.5.1.41 that converts chitin and H2O into chitosan and acetic acid, or an antigenic fragment thereof, which means an amino acid sequence capable of generating an immune response against the fragment.

[0256] According to certain embodiments, the chitin deacetylase is the chitin deacetylase of Helicoverpa armigera provided by GeneBank accession number AJA30435 etc.

[0257] According to certain embodiments, the chitin deacetylase contains SEQ ID NO: 9.

[0258] According to certain embodiments, the chitin deacetylase consists of SEQ ID NO: 9.

[0259] Non-limiting examples of nanobodies that specifically bind to chitin deacetylase and their corresponding CDRs are shown in Table 6 below.

[0260] According to a particular embodiment, the nanobody is specifically bound to chitin deacetylase and arranged continuously on the nanobody in the direction from N to C, such as SEQ ID NOs. 320-322, SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 327, 321 and 328, SEQ ID NOs. 320-321 and 332, SEQ ID NOs. 208 and 336-337, SEQ ID NOs. 320, 324 and 339, SEQ ID NOs. 343-345, SEQ ID NOs. 347-349, SEQ ID NOs. 351-353, SEQ ID NOs. 351, 347 and 356, SEQ ID NOs. 358-360, SEQ ID NOs. 358-359 and 362, SEQ ID NOs. 365, 359 and 366, SEQ ID NOs. 368-370, SEQ ID NOs. 372 and Includes a CDR having an amino acid sequence that is 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% identical to the amino acid sequences shown in 369-370, SEQ ID NOs. 374, 369 and 375, SEQ ID NOs. 377-379, SEQ ID NOs. 381-383, SEQ ID NOs. 385 and 382-383, SEQ ID NOs. 387-389, SEQ ID NOs. 387, 391 and 389, SEQ ID NOs. 394-396, SEQ ID NOs. 398-400, SEQ ID NOs. 402-404, or SEQ ID NOs. 406-408.

[0261] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to a chitin deacetylase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 320-322, SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 372 and 369-370, SEQ ID NOs. 374, 369 and 375, SEQ ID NOs. 377-379, SEQ ID NOs. 387-389, or SEQ ID NOs. 398-400.

[0262] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to a chitin deacetylase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 372 and 369-370, SEQ ID NOs. 374, 369 and 375, or SEQ ID NOs. 387-389.

[0263] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to a chitin deacetylase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 374, 369 and 375, or SEQ ID NOs. 387-389.

[0264] According to a particular embodiment, the nanobody is specifically bound to chitin deacetylase and arranged continuously on the nanobody in the direction from N to C, such as sequence numbers 320-322, 320 and 324-325, 327, 321 and 328, 320-321 and 332, 208 and 336-337, 320, 324 and 339, 343-345, 347-349, 351-353, 351, 347 and 356, sequence number Includes CDRs shown in numbers 358-360, sequence numbers 358-359 and 362, sequence numbers 365, 359 and 366, sequence numbers 368-370, sequence numbers 372 and 369-370, sequence numbers 374, 369 and 375, sequence numbers 377-379, sequence numbers 381-383, sequence numbers 385 and 382-383, sequence numbers 387-389, sequence numbers 387, 391 and 389, sequence numbers 394-396, sequence numbers 398-400, sequence numbers 402-404, or sequence numbers 406-408.

[0265] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 320-322, SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 372 and 369-370, SEQ ID NOs. 374, 369 and 375, SEQ ID NOs. 377-379, SEQ ID NOs. 387-389, or SEQ ID NOs. 398-400, which are specifically bound to chitin deacetylase and arranged continuously on the nanobody in the direction from N to C.

[0266] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 372 and 369-370, SEQ ID NOs. 374, 369 and 375, or SEQ ID NOs. 387-389, which are specifically bound to chitin deacetylase and arranged continuously on the nanobody in the direction from N to C.

[0267] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 320 and 324-325, SEQ ID NOs. 374, 369 and 375, or SEQ ID NOs. 387-389, which are specifically bound to chitin deacetylase and arranged continuously on the nanobody in a direction from N to C.

[0268] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 323, 326, 329, 331, 333, 334, 338, 340, 341, 342, 346, 350, 354, 355, 357, 361, 363, 364, 367, 371, 373, 376, 380, 384, 386, 390, 392, 393, 397, 401, and 405, each possibility representing another embodiment of the present invention.

[0269] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 323, 371, 373, 376, 386, and 397.

[0270] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 371, 373, and 386.

[0271] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 373, and 386.

[0272] According to certain embodiments, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 323, 326, 329, 331, 333, 334, 338, 340, 341, 342, 346, 350, 354, 355, 357, 361, 363, 364, 367, 371, 373, 376, 380, 384, 386, 390, 392, 393, 397, 401 and 405.

[0273] According to certain embodiments, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 323, 371, 373, 376, 386 and 397.

[0274] According to certain embodiments, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 371, 373 and 386.

[0275] According to certain embodiments, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 373 and 386.

[0276] According to certain embodiments, the nanobody comprises SEQ ID NO: 323.

[0277] <0oo0971>According to certain embodiments, the nanobody comprises SEQ ID NO: 371.

[0278] According to certain embodiments, the nanobody comprises SEQ ID NO: 373.

[0279] According to certain embodiments, the nanobody comprises SEQ ID NO: 386.

[0280] According to certain embodiments, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 323, 326, 329, 331, 333, 334, 338, 340, 341, 342, 346, 350, 354, 355, 357, 361, 363, 364, 367, 371, 373, 376, 380, 384, 386, 390, 392, 393, 397, 401 and 405.

[0281] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 319, 323, 371, 373, 376, 386, and 397.

[0282] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 371, 373, and 386.

[0283] According to a particular embodiment, the nanobody consists of Sequence ID No. 323.

[0284] According to a particular embodiment, the nanobody consists of Sequence ID No. 371.

[0285] According to a particular embodiment, the nanobody consists of Sequence ID No. 373.

[0286] According to a particular embodiment, the nanobody consists of Sequence ID No. 386.

[0287] According to a particular embodiment, the nanobody binds to chitin synthase.

[0288] The term "chitin synthase" as used in this application refers to UDP-N-acetyl-D-glucosamine and [1,4-(N-acetyl-beta-D-glucosaminyl)] n UDP + [1,4-(N--acetyl-beta-D-glucosaminyl)] n+1 This refers to an enzyme that converts to an enzyme with enzyme number (EC) EC2.4.1.16 in insects, or an antigenic fragment thereof, which has an amino acid sequence capable of eliciting an immune response to the fragment.

[0289] According to certain embodiments, chitin synthase is a chitin deacetylase of Helicoverpa armigera, provided under GeneBank accession number AKZ08594, etc.

[0290] According to a particular embodiment, the chitin synthase includes SEQ ID NO: 12.

[0291] According to a particular embodiment, the chitin synthase consists of an amino acid sequence selected from the group comprising SEQ ID NO: 12.

[0292] Non-limiting examples of nanobodies that specifically bind to chitin synthase, and their corresponding CDRs, are shown in Table 7 below.

[0293] According to a particular embodiment, the nanobody is specifically bound to chitin synthase and arranged continuously on the nanobody in the direction from N to C, such as SEQ ID NOs: 412-414, 418-420, 422-424, 426-428, 426-427 and 430, 432-434, 436, 140 and 437, 439-441, 443-445, 447-449, 356 and 451-452, 454-456, 458-460, 462-464, 466-468, and 470-4 72, includes a CDR having 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 the amino acid sequences shown in SEQ ID NOs. 474-476, SEQ ID NOs. 478-480, SEQ ID NOs. 482, 479 and 483, SEQ ID NOs. 485-487, SEQ ID NOs. 489-491, SEQ ID NOs. 493-495, SEQ ID NOs. 426 and 497-498, SEQ ID NOs. 500-502, SEQ ID NOs. 504-506, or SEQ ID NOs. 508-510.

[0294] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to chitin synthase and is continuously arranged on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 412-414, SEQ ID NOs. 418-420, SEQ ID NOs. 426-428, SEQ ID NOs. 432-434, SEQ ID NOs. 443-445, SEQ ID NOs. 447-449, SEQ ID NOs. 466-468, SEQ ID NOs. 482, 479 and 483, SEQ ID NOs. 426 and 497-498, or SEQ ID NOs. 504-506.

[0295] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to chitin synthase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 426-428, SEQ ID NOs. 432-434, or SEQ ID NOs. 443-445.

[0296] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to chitin synthase and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 426-428 or SEQ ID NOs. 443-445.

[0297] According to a particular embodiment, the nanobody is specifically bound to chitin synthase and arranged continuously on the nanobody in the direction from N to C, such as SEQ ID NOs: 412-414, 418-420, 422-424, 426-428, 426-427 and 430, 432-434, 436, 140 and 437, 439-441, 443-445, 447-449, 356 and 451- Includes CDRs shown in 452, SEQ ID NOs. 454-456, SEQ ID NOs. 458-460, SEQ ID NOs. 462-464, SEQ ID NOs. 466-468, SEQ ID NOs. 470-472, SEQ ID NOs. 474-476, SEQ ID NOs. 478-480, SEQ ID NOs. 482, 479 and 483, SEQ ID NOs. 485-487, SEQ ID NOs. 489-491, SEQ ID NOs. 493-495, SEQ ID NOs. 426 and 497-498, SEQ ID NOs. 500-502, SEQ ID NOs. 504-506, or SEQ ID NOs. 508-510.

[0298] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 412-414, 418-420, 426-428, 432-434, 443-445, 447-449, 466-468, 482, 479 and 483, 426 and 497-498, or 504-506, which are specifically bound to chitin synthase and arranged continuously on the nanobody in the direction from N to C.

[0299] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 426-428, 432-434, or 443-445, which are specifically bound to chitin synthase and arranged continuously on the nanobody in a direction from N to C.

[0300] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs: 426-428 or 443-445, which are specifically bound to chitin synthase and arranged continuously on the nanobody in the direction from N to C.

[0301] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 411, 415, 416, 417, 421, 425, 429, 431, 435, 438, 442, 446, 450, 453, 457, 461, 465, 469, 473, 477, 481, 484, 488, 492, 496, 499, 503, and 507, each possibility representing another embodiment of the present invention.

[0302] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 411, 417, 425, 431, 442, 446, 465, 481, 496, and 503.

[0303] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 425, 431, and 442.

[0304] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 425 and 442.

[0305] According to a particular embodiment, the nanobody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 411, 415, 416, 417, 421, 425, 429, 431, 435, 438, 442, 446, 450, 453, 457, 461, 465, 469, 473, 477, 481, 484, 488, 492, 496, 499, 503, and 507.

[0306] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 411, 417, 425, 431, 442, 446, 465, 481, 496, and 503.

[0307] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 425, 431, and 442.

[0308] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 425 and 442.

[0309] According to a particular embodiment, the nanobody includes SEQ ID NO: 425.

[0310] According to a particular embodiment, the nanobody includes SEQ ID NO: 431.

[0311] According to a particular embodiment, the nanobody includes SEQ ID NO: 442.

[0312] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 411, 415, 416, 417, 421, 425, 429, 431, 435, 438, 442, 446, 450, 453, 457, 461, 465, 469, 473, 477, 481, 484, 488, 492, 496, 499, 503, and 507.

[0313] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 411, 417, 425, 431, 442, 446, 465, 481, 496, and 503.

[0314] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 425, 431, and 442.

[0315] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group comprising SEQ ID NOs: 425 and 442.

[0316] According to a particular embodiment, the nanobody consists of Sequence ID No. 425.

[0317] According to a particular embodiment, the nanobody consists of Sequence ID No. 431.

[0318] According to a particular embodiment, the nanobody consists of Sequence ID No. 442.

[0319] According to a particular embodiment, the nanobody binds to the NPC1 sterol transporter.

[0320] In this application, the term "NPC1 sterol transporter" means an insect protein encoded by the NPC1 gene (Gene ID: 4864), or an antigenic fragment thereof, which has an amino acid sequence capable of eliciting an immune response to the fragment.

[0321] According to a particular embodiment, the NPC1 sterol transporter is the NPC1 sterol transporter of Helicoverpa armigera, provided under GeneBank accession number XP_02118611, etc.

[0322] According to a particular embodiment, the NPC1 sterol transporter includes SEQ ID NO: 13.

[0323] According to a particular embodiment, it consists of NPC1 sterol transporter sequence number 13.

[0324] Non-limiting examples of nanobodies that specifically bind to the NPC1 sterol transporter, and their corresponding CDRs, are shown in Table 8 below.

[0325] According to a particular embodiment, the nanobodies are specifically bound to the NPC1 sterol transporter and arranged continuously on the nanobodies in the direction from N to C, such as sequence numbers 512-514, 517 and 513-514, 521-523, 521-522 and 526, 531-533, 539-541, 485 and 540-541, 545 and 540-451, 547-549, 552-554, 556-558, sequence number Includes a CDR having an amino acid sequence that 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 shown in numbers 561-563, SEQ ID NOs. 565-567, SEQ ID NOs. 569-571, SEQ ID NOs. 573-575, SEQ ID NOs. 485 and 577-578, SEQ ID NOs. 580-582, or SEQ ID NOs. 584-586.

[0326] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to the NPC1 sterol transporter and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequences shown in SEQ ID NOs. 517 and 513-514, SEQ ID NOs. 485 and 540-541, SEQ ID NOs. 556-558, SEQ ID NOs. 565-567, SEQ ID NOs. 573-575, SEQ ID NOs. 485 and 577-578, or SEQ ID NOs. 580-582.

[0327] According to a particular embodiment, the nanobody includes a CDR having an amino acid sequence that specifically binds to the NPC1 sterol transporter and is arranged continuously on the nanobody in the direction from N to C, 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 the amino acid sequence shown in SEQ ID NOs. 485 and 540-541, or SEQ ID NOs. 545 and 540-541.

[0328] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 512-514, SEQ ID NOs. 517 and 513-514, SEQ ID NOs. 521-523, SEQ ID NOs. 521-522 and 526, SEQ ID NOs. 531-533, SEQ ID NOs. 539-541, SEQ ID NOs. 485 and 540-541, SEQ ID NOs. 545 and 540-451, SEQ ID NOs. 547-549, SEQ ID NOs. 552-554, SEQ ID NOs. 556-558, SEQ ID NOs. 561-563, SEQ ID NOs. 565-567, SEQ ID NOs. 569-571, SEQ ID NOs. 573-575, SEQ ID NOs. 485 and 577-578, SEQ ID NOs. 580-582, or SEQ ID NOs. 584-586, which are specifically bound to the NPC1 sterol transporter and arranged continuously on the nanobody in the direction from N to C.

[0329] According to a particular embodiment, the nanobody includes CDRs shown in SEQ ID NOs. 517 and 513-514, SEQ ID NOs. 485 and 540-541, SEQ ID NOs. 545 and 540-541, SEQ ID NOs. 556-558, SEQ ID NOs. 565-567, SEQ ID NOs. 573-575, SEQ ID NOs. 485 and 577-578, or SEQ ID NOs. 580-582, which are specifically bound to the NPC1 sterol transporter and arranged continuously on the nanobody in the direction from N to C.

[0330] According to a particular embodiment, the nanobody includes a CDR shown in SEQ ID NOs. 485 and 540-541, or SEQ ID NOs. 545 and 540-541, which is specifically bound to the NPC1 sterol transporter and arranged continuously on the nanobody in the direction from N to C.

[0331] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 511, 515, 516, 518, 518, 519, 519, 520, 524, 525, 527, 528, 529, 530, 534, 535, 536, 537, 538, 542, 543, 544, 546, 550, 551, 555, 559, 560, 564, 568, 572, 576, 579, and 583, each possibility representing another embodiment of the present invention.

[0332] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 516, 543, 544, 555, 564, 572, 576, and 579.

[0333] According to a particular embodiment, the nanobody includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs. 543 and 544.

[0334] According to a particular embodiment, the nanobody contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 511, 515, 516, 518, 519, 520, 524, 525, 527, 528, 529, 530, 534, 535, 536, 537, 538, 542, 543, 544, 546, 550, 551, 555, 559, 560, 564, 568, 572, 576, 579, and 583.

[0335] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 516, 543, 544, 555, 564, 572, 576, and 579.

[0336] According to a particular embodiment, the nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 543 and 544.

[0337] According to a particular embodiment, the nanobody includes SEQ ID NO: 543.

[0338] According to a particular embodiment, the nanobody includes SEQ ID NO: 544.

[0339] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 511, 515, 516, 518, 519, 520, 524, 525, 527, 528, 529, 530, 534, 535, 536, 537, 538, 542, 543, 544, 546, 550, 551, 555, 559, 560, 564, 568, 572, 576, 579, and 583.

[0340] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 516, 543, 544, 555, 564, 572, 576, and 579.

[0341] According to a particular embodiment, the nanobody consists of an amino acid sequence selected from the group comprising SEQ ID NOs. 543 and 544.

[0342] According to a particular embodiment, the nanobody consists of Sequence ID No. 543.

[0343] According to a particular embodiment, the nanobody consists of sequence number 544.

[0344] It should be noted that the nanobodies of some embodiments of the present invention are not limited in their broadest interpretation to specific biological raw materials or specific preparation methods. For example, nanobodies can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy-chain antibody; (2) expressing a nucleotide sequence encoding the naturally occurring VHH domain; (3) "humanizing" the naturally occurring VHH domain, or expressing a nucleic acid encoding such a humanized VHH domain; (4) "camelizing" a naturally occurring VH domain from any animal species, particularly mammalian species, e.g., human, or expressing a nucleic acid encoding such a camelized VH domain; (5) "camelizing" a "domain antibody" or "Dab" known in the art, or expressing a nucleic acid encoding such a camelized VH domain; (6) producing a protein, polypeptide or other amino acid sequence known in itself using synthetic or semi-synthetic techniques; (7) producing a nucleic acid encoding a nanobodies using techniques known in itself for nucleic acid synthesis, followed by expression of the obtained nucleic acid; and / or (8) any combination of one or more of the above. Descriptions of nanobodies, including humanized and / or camelized nanobodies, can be found in addition to this application, for example, in International Publications 08 / 101985 and 08 / 142164. A recently reported finding on a fully in vitro platform-based yeast surface display of nanobodies is disclosed in McMahon, Conor, et al. "Nature structural & molecular biology" 25.3 (2018): 289.

[0345] According to certain embodiments, nanobodies are “camelized.” For example, “camelized” can be achieved by providing a nucleotide sequence encoding a naturally occurring VH domain and modifying one or more codons in the nucleotide sequence by means known by itself, such that each new nucleotide sequence encodes a “camelized” nanobody. This nucleic acid is then expressed by means known by itself to provide the desired nanobodies.

[0346] Starting from a naturally occurring VH sequence, preferably a V H H sequence, other suitable methods and techniques for obtaining a nanobody and / or a nucleic acid sequence encoding the same will be apparent to those skilled in the art. For example, one or more portions of a naturally occurring VH sequence (such as one or more FR sequences and / or CDR sequences, etc.), one or more portions of one or more naturally occurring V H H sequences (such as one or more FR sequences or CDR sequences, etc.), and / or one or more synthetic or semi-synthetic sequences may be combined in a suitable procedure to provide a nanobody or a nucleic acid sequence encoding the same.

[0347] Specific methods for making nanobodies are described herein.

[0348] According to one aspect of the present invention, a method for producing a nanobody, comprising: (i) a polypeptide comprising a chitin-binding domain (CBD), (ii) V-ATPase subunit c, (iii) trehalase, (iv) cytochrome p450 monooxygenase, (v) chitin deacetylase, (vi) chitin synthase, and (vii) NPC1 sterol transporter immunizing camels with a recombinant or purified insect polypeptide selected from the group consisting of, and the purity of the insect polypeptide in the insect polypeptide preparation is at least 80%, a method is provided.

[0349] As used herein, the term "purified insect polypeptide" means a polypeptide purified from an insect, the purity of which is at least 80% compared to other polypeptides present in the protein preparation.

[0350] According to certain embodiments, the purity of the insect polypeptide 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 different embodiment of the present invention.

[0351] According to certain embodiments, the method further includes the isolation of nanobodies.

[0352] Methods for isolating nanobodies are known in the industry and are disclosed, for example, in Pardon et al. Nat Protoc. 2014 Mar, 9(3): 674-693, the details of which are fully incorporated herein by reference, and in the examples described below.

[0353] Once nanobodies are obtained, their binding and / or biological activity (e.g., insect control activity) can be analyzed in vivo or in vitro. Such methods are well known in the industry and are disclosed above and below.

[0354] Therefore, according to certain embodiments, the method further includes the selection of nanobodies that exhibit insect control activity in a biological assay. Such assays are known in the art and are described further below.

[0355] According to certain embodiments, the nanobody is a naked nanobody.

[0356] In this application, the term "as-is nanobody" means a nanobody that does not contain heterogeneous effector parts such as toxin parts or detection parts.

[0357] According to certain embodiments, the nanobody includes heterogeneous effector portions, such as a toxin portion or a detection portion. The effector portions may be proteinaceous or non-proteinaceous (e.g., small molecule compounds), the latter of which are typically fabricated using functional groups on the nanobody or on its binding partners.

[0358] Therefore, for example, various types of detectable portions or reporter portions can be attached to the nanobodies of the present invention. These include radioactive isotopes (

[0125] Examples include, but are not limited to, molecules detectable by positron emission tomography (PET) or magnetic resonance imaging (MRI) (contrast agents), phosphorescent compounds, chemiluminescent compounds, fluorescent compounds or polypeptides (e.g., phycoerythrin (PE), fluorescein, fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas Red, Cy5, PE-Cy5, etc.), enzymes (e.g., horseradish peroxidase (HPR), beta-galactosidase, and alkaline phosphatase (AP)), affinity tags [e.g., antigens identifiable by corresponding antibodies (e.g., digoxigenin (DIG) identifiable by anti-DIG antibodies), or molecules with high affinity for tags (e.g., streptavidin and biotin)], and molecules detectable by positron emission tomography (PET) or magnetic resonance imaging (MRI).

[0359] According to certain embodiments, the nanobody contains toxins.

[0360] In this application, the terms "toxin" or "toxic portion" refer to a compound having insecticidal activity (as defined later), and include, but are not limited to, polypeptides, polynucleotides, small molecules, etc.

[0361] Non-specific examples of the toxic portion include δ-endotoxins [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] etc., colicin (colicin E1, colicin la, colicin A, colicin N, etc.), actinoporins (equinatoxin II, stycolicin II, fragacetoxin C, etc.), ClyA family toxins (cytolysin A, nonhemolytic tripartite enterotoxin, hemolysin BL, etc.), hemolysin (α-hemolysin, γ-hemolysin, leucocidine, necrotizing panenteroenterotoxin B, δ-toxin, Vibrio cholerae cytolysin, Vibrio Vulnificus hemolysin, etc.), erollysine family toxins (erollysine, α-toxin, hydralysin, ε-toxin, enterotoxin, hemolytic lectin, xenin, etc.), cholesterol-dependent cytolysins (perphlingolicin, suiricin, intermedilisin, listeriolisin O, rectinolysin, anthrolysin, streptorisin, etc.), membrane invasion complex components / perforins (Plu-MACPF, Bth-MACPF, etc.), repeat sequences in toxins (repeats-in-toxins) (HlyA, bifunctional hemolysin-adenylyl cyclase toxin, MARTX, etc.) (Dal Peraro and van der Goot, 2016), spider toxin, scorpion toxin, patatin, Bacillus thuringiensis insecticidal protein, Xenorhabdus insecticidal protein, Photorhabdus insecticidal protein, Bacillus laterosporous insecticidal protein, Bacillus Examples include bombysepticus insecticidal proteins, Bacillus sphaericus insecticidal proteins, and insect control double-stranded RNA.

[0362] The effector portion, such as the toxic portion or the detectable portion, can be attached to or bound to the nanobody of the present invention in various ways, depending on the environment, application, and purpose.

[0363] The effector portion can be coupled directly or indirectly (e.g., contained within a single element) to the nanobody. The coupling can be covalent or non-covalent.

[0364] If the effector portion is a polypeptide, the immune complex can be manufactured by recombinant techniques. For example, a nucleic acid sequence encoding a toxin or fluorescent protein can be ligated into a frame of nucleic acid sequences encoding nanobodies and expressed in host cells to produce recombinant antibody complexes. Alternatively, the effector portion can be chemically synthesized, for example, by solid-phase peptide synthesis techniques that involve the stepwise addition of one or more amino acid residues in a specific order.

[0365] The effector portion can also be attached to the nanobody using standard chemical synthesis techniques widely practiced in the industry [see, for example, worldwideweb(dot)chemistry(dot)org / portal / Chemistry], such as direct or indirect appropriate chemical bonding via peptide bonds (when the effector portion is a polypeptide), or covalent bonding to an intervening linker factor (e.g., linker peptide) or other chemical moiety (e.g., organic polymer). Chimeric peptides can be linked by bonding to the carboxyl (C) or amino (N) terminus of the peptide, or by bonding to linear, branched, or cyclic side chains, or to internal chemical groups such as internal carbon or nitrogen atoms. Fluorescent labeling is described in detail in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110.

[0366] Examples of methods for attaching the peptide portion to the nanobody include, but are not limited to, SPDP bonds, glutaraldehyde bonds, and carbodiimide bonds.

[0367] The nanobody can be attached to the particle containing the effector portion or to the particle alone. Methods for covalently bonding nanobody to encapsulating particles are known in the industry and are disclosed, for example, in U.S. Patent Nos. 5,171,578, 5,204,096, and 5,258,499.

[0368] Any polypeptide described herein (e.g., nanobodies and proteinaceous compositions) may be encoded by polynucleotides. These polynucleotides can be used as is or in recombinant production of the polypeptides disclosed herein.

[0369] Therefore, according to one aspect of the present invention, a polynucleotide is provided which codes for a composition comprising a nanobody or a nanobody and a toxin.

[0370] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid sequence that is 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).

[0371] In certain embodiments, any polynucleotide and nucleic acid sequence disclosed herein may have conserved nucleic acid substitutions. A conservedly modified polynucleotide refers to a nucleic acid that codes for the same or substantially identical amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, a substantially identical or related (e.g., naturally consecutive) sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for most proteins. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at all positions where alanine is identified by the codon, this codon can be modified to replace the corresponding codon described herein without altering the encoded polypeptide. Such nucleic acid mutations are called "silent variations" and are a type of conservedly modified polynucleotide. In certain embodiments, all polynucleotides and nucleic acid sequences herein that code for polypeptides also represent silent variations of that nucleic acid. Those skilled in the art will recognize that, in certain contexts, each codon within a nucleic acid (with the exception of AUG, usually the sole codon of methionine, and TGG, usually the sole codon of tryptophan) can be modified to result in a functionally identical molecule. Therefore, silent mutations in nucleic acids encoding polypeptides are implicitly present in the sequences described with respect to the expression product.

[0372] According to certain embodiments, the nucleic acid sequences disclosed herein are optimized for codons, for example, expression in mammals or plants.

[0373] Methods for codon optimization are publicly known in the industry and are disclosed, for example, in the codon usage database of the DNA Bank of Japan, NIAS (National Institute of Agrobiological Sciences) (www(dot)kazusa(dot)or(dot)jp / codon / ), International Publication No. 93 / 07278, and Grote et al. Nucleic Acid Res. Nucleic Acids Res. (2005) Jul 1, 33 (web version): W526-W531).

[0374] Therefore, some embodiments of the present invention include the nucleic acid sequences described above, fragments thereof, sequences that can hybridize with them, sequences similar thereto, sequences orthologous thereto, sequences encoding similar polypeptides using different codons, and modified sequences characterized by spontaneously occurring or human-induced random or targeted mutations (such as deletions, insertions, or substitutions of one or more nucleotides).

[0375] To express an exogenous polypeptide in cells, the polynucleotide sequence encoding the polypeptide is preferably ligated into a nucleic acid construct suitable for cell expression. Such a nucleic acid construct includes a promoter sequence that constitutively or inducibly directs the transcription of the polynucleotide sequence in cells.

[0376] Therefore, according to one aspect of the present invention, a nucleic acid construct is provided which comprises a polynucleotide and a cis-acting regulatory element that guides the expression of the polynucleotide.

[0377] According to a particular embodiment, the regulator is a heterogeneous regulator.

[0378] Nucleic acid constructs of some embodiments of the present invention (also referred to herein as “expression vectors”) include further sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals. For example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, a second-strand DNA synthesis origin, and a 3'LTR or a portion thereof.

[0379] Nucleic acid constructs of some embodiments of the present invention typically include a signal sequence for causing the cell containing the construct to secrete a polypeptide. According to certain embodiments, the signal sequence is the intrinsic signal sequence of the polypeptide (e.g., nanobody) of some embodiments of the present invention.

[0380] Eukaryotic promoters typically contain two types of recognition sequences: a TATA box and an upstream promoter element. The TATA box, located 25–30 base pairs upstream of the transcription start site, is thought to be involved in the initiation of RNA synthesis by RNA polymerase. The other upstream promoter element determines the frequency of transcription initiation.

[0381] The promoters used in the nucleic acid constructs of some embodiments of the present invention are preferably those that exhibit activity in a specific transformed cell population. Examples of cell type-specific and / or tissue-specific promoters include liver-specific promoters such as albumin [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid-specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275], in particular T cell receptor promoters [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulin promoters [Banerji et al. (1983) Cell 33729-740], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916], or mammary gland-specific promoters, such as whey promoters (U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166).

[0382] When using plant expression vectors, the expression of the coding sequence can be driven by several promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters for CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter for TMV [Takamatsu et al. (1987) EMBO J. 6:307-311] can be used. Alternatively, plant promoters, such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J. 3:1671-1680 and Brogli et al., (1984) Science 224:838-843], the GR fusion GVG, and pOp / LhGR (dexamethasone-inducible), XVE / OlexA (beta-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] may be used.

[0383] Enhancer elements can increase transcription by the bound homologous or heterologous promoter by up to 1,000-fold. Enhancers are active whether located downstream or upstream of the transcription start site. Many virus-derived enhancer elements have a broad host range and exhibit activity in various tissues. For example, the SV40 initial gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the present invention include those derived from polyomaviruses, human or mouse cytomegalovirus (CMV), and long terminal repeat sequences derived from various retroviruses such as mouse leukemia virus, mouse or Rous sarcoma virus, and HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1983, incorporated herein by reference. Enhancers specific to certain neuronal cell types that can be incorporated into AAV expression vectors to obtain specificity without a Cre driver system have also been reported in the industry, for example, as described in Hrvatin et al. (doi:www(dot)doi(dot)org / 10.1101 / 570895), which are incorporated herein by reference.

[0384] In constructing an expression vector, it is preferable that the promoter be located approximately the same distance from the heterologous transcription initiation site as it is from the transcription initiation site in its natural environment. However, as is well known in the art, some variation in this distance may be acceptable without loss of promoter function.

[0385] To increase mRNA translation efficiency, polyadenylation sequences can also be added to expression vectors. Accurate and efficient polyadenylation requires two characteristic sequence elements: a GU-rich or U-rich sequence located downstream of the polyadenylation site, and a highly conserved AAUAAA sequence consisting of six nucleotides located 11 to 30 nucleotides upstream. Suitable termination and polyadenylation signals for some embodiments of the present invention include those derived from SV40.

[0386] Expression vectors in some embodiments of the present invention may, in addition to the elements already described, typically contain other specialized elements intended to increase the expression level of cloned nucleic acids or to facilitate the identification of cells having recombinant DNA. For example, some animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in tolerant cell types. Plasmids having replicas of such viruses replicate as episomes, provided that the appropriate factors are provided by genes on the plasmid or on the host cell genome.

[0387] Vectors may or may not contain eukaryotic replicons. If eukaryotic replicons are present, the vector can be amplified in eukaryotic cells using appropriate selectable markers. If the vector does not contain eukaryotic replicons, episome amplification is not possible. Instead, recombinant DNA is integrated into the genome of an engineered cell, where a promoter expresses the desired nucleic acid.

[0388] Expression vectors in some embodiments of the present invention may further include additional polynucleotide sequences, such as an internal ribosome entry site (IRES) and a sequence for genomic integration of a promoter-chimeric polypeptide, which enable the translation of several proteins from a single mRNA.

[0389] It is understood that the individual elements contained in an expression vector can be arranged in various configurations. For example, enhancer elements, promoters, and even polynucleotide sequences encoding polypeptides may be arranged in a "head-to-tail" configuration, and may exist as an inverse complementary strand or as a complementary strand in an antiparallel strand. Such diversity of configurations is more likely to occur in the non-coding elements of an expression vector, but alternative configurations of coding sequences in an expression vector are also conceivable.

[0390] 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 from Invitrogen, pCI from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV from Strategene, pTRES from Clontech, and their derivatives.

[0391] Expression vectors containing regulatory elements derived from eukaryotic viruses such as retroviruses can also be used. SV40 vectors include pSVT7 and pMT2. Examples of bovine papillomavirus-derived vectors include pBV-1MTHA, and examples of Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG and pAV009 / A. + pMTO10 / A +Examples include pMAMneo-5, baculovirus pDSVE, and any other vectors that enable protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or any other promoter that has been reported to be effective for expression in eukaryotic cells.

[0392] As mentioned above, viruses are highly specialized infectious agents that have often evolved to evade the host's defense mechanisms. Typically, viruses infect and transmit to specific cell types. The targeting specificity of viral vectors introduces recombinant genes into infected cells by utilizing their innate specificity to specifically target a given cell type. Therefore, the type of vector used in some embodiments of the present invention depends on the cell type being transformed. The ability to select a suitable vector based on the cell type being transformed is well within the capabilities of those skilled in the art, and therefore no overview of selection considerations is provided herein.

[0393] Cells can be stably or transiently transformed by nucleic acid constructs of several embodiments of the present invention. In stable transformation, the nucleic acid molecules of several embodiments of the present invention are incorporated into the cell genome, and therefore exhibit stable heritability. In transient transformation, the nucleic acid molecules are expressed by the transformed cells but are not incorporated into the genome, and therefore exhibit transient nature.

[0394] Various methods can be used to introduce expression vectors of several embodiments of the present invention into cells. Such methods include Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs 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 at. [Biotechniques 4 (6): 504-512]. This is outlined in

[1986] and includes, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, for positive-negative selection methods, see U.S. Patent Nos. 5,464,764 and 5,487,992. 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 & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0395] Introducing nucleic acids through viral infection offers several advantages compared to other methods such as lipofection and electroporation. This is because the infectious nature of the virus allows for higher transfection efficiency.

[0396] Currently preferred in vivo nucleic acid transfer techniques include transfection of viral or non-viral constructs, such as adenoviruses, lentiviruses, herpes simplex virus type 1, or adeno-associated viruses (AAVs), and lipid-based systems. Lipids useful 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 construct for use in gene therapy is a virus, most preferably adenovirus, AAV, lentivirus, or retrovirus. Viral constructs, such as retroviral constructs, include at least one transcription promoter / enhancer or locus-determining element, or other elements that control gene expression by other means such as alternative splicing of messengers, nuclear RNA transport, or post-translational modification. Such vector constructs also include a packaging signal, a terminal repeat sequence (LTR) or a portion thereof, and positive and negative primer binding sites appropriate for the virus used, unless already present in the viral construct. In addition, such constructs typically include a signal sequence for causing the host cell containing the construct to secrete a peptide. Preferably, the signal sequence for this purpose is a mammalian signal sequence or a signal sequence of a polypeptide variant in some embodiments of the present invention. Optionally, the construct may also include a signal resulting in polyadenylation, as well as one or more restriction sites and translation termination sequences. For example, such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, a second-strand DNA synthesis origin, and a 3'LTR or a portion thereof. Other non-viral vectors, such as cationic lipids, polylysine, and dendrimers, may also be used.

[0397] Expression constructs of some embodiments of the present invention may, in addition to containing elements necessary for the transcription and translation of the inserted coding sequence, also contain sequences engineered to enhance the stability, generation, purification, yield, or toxicity of the expressed monomer or heterodimer. For example, the expression of a fusion protein or cleavable fusion protein comprising a polypeptide of some embodiments of the present invention and a heterologous protein can be engineered. Such fusion proteins can be designed so that they can be easily isolated by affinity chromatography, for example, by immobilization on a column specific to the heterologous protein. If a cleavage site is engineered between the polypeptide and the heterologous protein, the polypeptide can be released from the chromatographic column by treatment with a suitable enzyme or agent that disrupts the cleavage site [see, for example, Booth et al. (1988) Immunol. Lett. 19:65-70 and Gardella et al., (1990) J. Biol. Chem. 265:15854-15859].

[0398] The present invention also intends to include cells comprising polypeptides, polynucleotides, and nucleic acid constructs as described herein.

[0399] Therefore, according to one aspect of the present 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 the same.

[0400] As described herein, various prokaryotic or eukaryotic cells can be used as host expression systems for expressing polypeptides of some embodiments of the present invention. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing coding sequences, yeast transformed with recombinant yeast expression vectors containing coding sequences, and plant cell lines infected with recombinant virus expression vectors containing coding sequences (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors such as Ti plasmids. Mammalian expression systems can also be used to express polypeptides of some embodiments of the present invention.

[0401] According to certain embodiments, the cells are mammalian cells.

[0402] According to a particular embodiment, the cells are camel cells.

[0403] Suitable mammalian cells include primary cells and immortalized cell lines.

[0404] According to other specific embodiments, mammalian cells are immortalized cell lines.

[0405] Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), HEK293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, NS0, Sp2 / 0, BHK, Namalwa, etc.

[0406] According to a particular embodiment, the cells are Escherichia coli (E. coli), for example, SHuffle T7 Express & BL21.

[0407] According to a particular embodiment, the cells are plant cells.

[0408] According to one aspect of the present invention, a method for producing insect control nanobodies is provided, which includes expressing a polynucleotide or nucleic acid construct disclosed herein in a host cell.

[0409] According to a particular embodiment, the method further includes the isolation of nanobodies.

[0410] The isolation or recovery of any recombinant polypeptide (e.g., nanobodies) can be carried out by any method known in the art. According to certain embodiments, the isolation or recovery of recombinant polypeptides is performed after culturing for a suitable period of time. The phrases “recovery of recombinant polypeptides” or “isolation of recombinant polypeptides” refer to the collection of the entire culture medium containing the polypeptides and do not necessarily require further separation or purification steps. Notwithstanding the foregoing, polypeptides of some embodiments of the present invention can be purified using a variety of standard protein purification techniques, including, 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, chromatofocusing, and differential solubilization (DS).

[0411] According to certain embodiments, following synthesis and purification, the binding and / or insect control activity of nanobodies can be detected either in vivo or in vitro. Such methods are known in the art and are further disclosed above and below.

[0412] The compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotides and nucleic acid constructs encoding them, and host cells expressing them) may be formulated by appropriate means such as lyophilization, freeze-drying, or drying, or by aqueous carriers, culture media, or appropriate diluents (physiological saline or other buffers). According to certain embodiments, the formulated compositions may take the form of fine or granular materials, powders, gels, creams, pastes, pellets, tablets, or suspensions in oil (vegetable oil or mineral oil) or water, or o / w emulsions, capsule suspensions, emulsions, or wettable powders, wettable granules, water-dispersible granules, aerosols, foams, slurries, or flowable concentrates.

[0413] According to certain embodiments, the composition is formulated as a liquid concentrate, a dry powder, a tablet, a capsule suspension, a slurry, or a "wet cake," which are appropriately diluted, dispersed, suspended, emulsified, or otherwise reconstituted before final specification by the end user.

[0414] According to certain embodiments, the composition is formulated as a liquid formulation.

[0415] According to certain embodiments, the composition is formulated as a dry formulation.

[0416] According to certain embodiments, the composition is formulated for delivery by spraying, irrigation, and / or fumigation.

[0417] According to certain embodiments, the compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotides and nucleic acid constructs encoding them, and host cells expressing them) are stable both during storage and in use, meaning that the composition maintains its complete state under storage and / or use conditions of the composition, such as temperature increases, freeze-thaw cycles, changes in pH or ionic intensity, UV irradiation, and the presence of hazardous chemicals.

[0418] According to certain embodiments, the complete state and activity of the composition are maintained under storage and / or usage conditions of the composition, such as temperature increases, freeze-thaw cycles, changes in pH or ionic strength, UV irradiation, and the presence of hazardous chemicals.

[0419] According to certain embodiments, the complete state and activity of the composition are maintained under field conditions or controlled conditions, such as in a greenhouse.

[0420] It should be noted that the compositions disclosed herein can be formulated, for example, with various carriers designed to improve deliverability, stability, penetration, etc.

[0421] As used in this application, "carrier" means a solid, semi-solid, or liquid carrier capable of appropriately introducing, i.e., immobilizing, adsorbing, absorbing, binding, encapsulating, embedding, attaching to, or containing compounds (e.g., nanobodies and / or toxins, compositions thereof).

[0422] Non-limiting examples of such carriers include nanocapsules, microcapsules, nanospheres, microspheres, nanoparticles, microparticles, liposomes, vesicles, beads, gels, weakly ionic resin particles, liposomes, cocreate delivery vehicles, small granules, granules, nanotubes, fullerenes (bucky-balls), water droplets as part of W / O emulsions, oil droplets as part of O / W emulsions, organic materials such as cork, wood or other plant-derived materials (e.g., seed hulls, wood chips, pulp, spheres, beads, sheets or any other suitable shape), inorganic materials such as paper or cardboard, talc, clay, microcrystalline cellulose, silica, alumina, silicates and zeolites, or even microbial cells (such as yeast cells) or suitable fractions or fragments thereof.

[0423] According to certain embodiments, the carrier has immediate-release, sustained-release, or delayed-release characteristics, for example, being released over minutes, hours, days, or weeks. Furthermore, the carrier is formed of a material (e.g., polymer) that disintegrates or slowly decomposes over time (e.g., due to high or low temperatures, sunlight, high or low humidity, or other environmental factors or conditions) and can release compounds (e.g., nanobodies and / or toxins, or compositions containing them) from the carrier. According to certain embodiments, the carrier is coupled, bonded, linked, attached to, or associated with the compound. According to certain embodiments, the carrier is covalently coupled to the compound.

[0424] The compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotides and nucleic acid constructs encoding them, and host cells expressing them) can be formulated into compositions such as pesticides or insecticidal compositions, which are mixtures of the compositions with appropriate physiologically acceptable carriers or excipients.

[0425] In this specification, the term “active ingredient” means nanobodies that are the cause of biological effects, compositions comprising nanobodies and toxins, polynucleotides and nucleic acid constructs encoding them, and host cells that express them.

[0426] The term "agricultural chemical formulation" as used in this application refers to a composition for agricultural use comprising one or more active ingredients described in this application and other compounds such as agriculturally acceptable carriers and excipients.

[0427] Hereafter, the expression "physiologically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not inactivate the biological activity and properties of the administered compound. These expressions include adjuvants.

[0428] According to certain embodiments, the composition is formulated with an agriculturally acceptable carrier. Suitable agricultural carriers may be solid or liquid, and these are widely known in the art. The term "agriculturally acceptable carrier" includes all adjuvants, such as inert components, dispersants, surfactants, tackifiers, binders, etc., which are commonly used in insecticide formulations and are well known to those skilled in the art.

[0429] In this specification, the term “excipient” means an inert substance added to a composition to further facilitate the administration of the active ingredient. Examples of excipients, but not limited to, include calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, surfactants, gelatin, vegetable oils, polyethylene glycol, wetting agents, spreading agents, buffering agents, acidifying agents, and the like.

[0430] According to certain embodiments, the compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotides and nucleic acid constructs encoding them, and host cells expressing them) may be the sole active ingredient in the composition.

[0431] According to other specific embodiments, the composition comprises one or more additional activators in addition to the compositions disclosed herein (e.g., nanobodies, compositions comprising nanobodies and toxins, polynucleotides and nucleic acid constructs encoding them, and host cells expressing them). Non-limiting examples of such components include herbicides, insecticides, plant growth regulators, phytotoxicity reducers, and the like.

[0432] According to certain embodiments, the composition disclosed herein and additional activators are co-formulated.

[0433] According to certain embodiments, the composition disclosed herein and additional activators are placed in separate containers.

[0434] According to other specific embodiments, the composition may include an insect attractant. The attractant may be a pheromone, such as a male or female pheromone. For example, pheromones described in the book “Insect Pheromones and their use in Pest Management” (Howse et al, Chapman and Hall, 1998) may be used in the present invention.

[0435] The attractant can be included in the formulation or applied separately to ensure that insects are attracted to the area where the formulation is applied.

[0436] Nanobodies of several embodiments of the present invention, as well as compositions containing nanobodies and toxins, are conferred with insect control activity.

[0437] As used in this application, the term "insect control" means the prevention, inhibition, or reduction of insect feeding, growth, movement, spread, occurrence, survival, and / or reproduction, and / or the limitation of insect-related damage, which can be achieved by, for example, killing insects, reducing insect survival rates or lifespan, reducing insect productivity and / or reproductive capacity, reducing or preventing insect feeding, reducing or preventing insect growth, reducing or preventing insect occurrence, reducing or preventing insect mobility, and / or preventing insect infestation. According to certain embodiments, insect control activity is achieved by killing insects.

[0438] Methods for determining insect control activity are well known to those skilled in the art and are disclosed in the following examples. These methods include, but are not limited to, determining mortality, body weight, body length, pupation, and timing of adulthood by rearing larvae in vitro in the presence of nanobodies or compositions and comparing the results with conditions in the absence of nanobodies or compositions.

[0439] The nanobodies disclosed herein may have insect control activity themselves, or they may exert insect control activity by delivering toxins to insects.

[0440] Therefore, according to certain embodiments, the binding of nanobodies to insect polypeptides confers insect control activity to the nanobodies.

[0441] According to certain embodiments, the nanobody downregulates the activity of the insect polypeptide to which it binds.

[0442] In this application, the phrase "downregulating activity" means that the biological function of an insect polypeptide, as determined by a method suitable for determining the activity of an insect polypeptide, is reduced by at least 5% in the presence of nanobodies compared to the absence of nanobodies. Therefore, for example, the activity of a polypeptide containing CBD can be determined by ELISA, Western blot analysis, immunoprecipitation, or flow cytometry. The activity of V-ATPase subunit c can be determined by ATPase activity analysis and / or ATP-dependent proton transporter assay. The activity of trehalase can be determined by ferric-ferrocyanide reaction for determining reducing sugars, or by quantification of released glucose using glucose oxidase / peroxidase following incubation with trehalase. The activity of cytochrome p450 monooxygenase can be determined by measurement of oxidation of reagents or surrogate compounds, heme quantification assay, or P450-Glo luminescence assay. The activity of chitin deacetylase can be determined by radiometric assays of radiolabeling of N-acetyl groups using partially O-hydroxyethylated chitin (glycol chitin) as a substrate, or by Bergmeyer enzyme assays that determine acetates released by the action of chitin deacetylase on various chitinous substrates. The activity of chitin synthase is [ 14 Radioactive assay using [C]UDP-N-acetyl-D-glucosamine (GlcNAc) as a substrate, and subsequent insolubility after acid precipitation. 14This can be determined by quantitative analysis of C-labeled chitin, etc. The activity of the NPC1 sterol transporter can be determined by cholesterol absorption assays, sterol quantification assays, etc.

[0443] According to other specific embodiments, the reduction is a reduction of 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, as determined, for example, by any of the methods described above.

[0444] According to further or alternative embodiments, the nanobody is a targeting agent that functions to provide specific delivery of a toxin having insecticidal activity against insects, for example.

[0445] Non-limiting examples of toxins and methods for coupling them to nanobodies will be discussed further later.

[0446] Since some embodiments of the present invention, and compositions containing nanobodies and toxins, are conferred with insect control activity, the present invention also encompasses methods for insect control.

[0447] Therefore, according to one aspect of the present invention, a method for insect control is provided, comprising contacting insects with a composition comprising nanobodies or nanobodies and toxins, polynucleotides or nucleic acid constructs encoding them, or host cells expressing them.

[0448] According to certain embodiments, contact involves directly imparting a nanobody, or a composition containing a nanobody and a toxin, to an insect.

[0449] According to a particular embodiment, contact involves imparting a nanobody, or a composition comprising a nanobody and a toxin, to an organism or surface that may come into contact with the insect.

[0450] According to another embodiment, a method is provided for preventing insect infestation in industrial products, comprising adding a nanobody or a composition containing a nanobody and a toxin to the product.

[0451] According to another embodiment, a method is provided for preventing insect infestation of industrial products, comprising packaging the product in a packaging material comprising a composition comprising nanobodies or nanobodies and toxins.

[0452] The above contact or addition may be carried out by any suitable method known in the art, such as spraying (including high volume (HV), low volume (LV), and low volume (ULV) spraying), misting, brushing, coating, dropping, painting, dipping, immersion, submersion, coating, foaming, fogging, injection, addition to culture medium, irrigation, application as droplets, mist or aerosol, and recombinant expression of nucleic acid constructs in the cells of organisms (as already further disclosed).

[0453] According to certain embodiments, contact and application are carried out by spraying, irrigation, and / or fumigation.

[0454] According to certain embodiments, contact and impartation are carried out by introducing polynucleotides or nucleic acid constructs into the cells of an organism.

[0455] According to certain embodiments, the organism is a plant.

[0456] As used in this application, the term "surface" means any object that can come into contact with insects. Non-limiting examples of surfaces include nets (e.g., mosquito nets), light sources, colored objects, shapes or contours that stand out against a contrasting background in a greenhouse, camping equipment, soil, etc.

[0457] According to certain embodiments, industrial products are manufactured from plants.

[0458] As used in this application, the term “plant” encompasses the whole plant, scions, ancestors or descendants of plants and plant parts, and specifically includes seeds, buds, stems, roots (including rhizomes), rootstocks, scions, fruits, vegetables, flowers, as well as plant cells, tissues, and organs. A plant can be any form including a suspension culture, embryo, growth region, callus tissue, leaf, gametophyte, sporophyte, pollen, and microspores.

[0459] According to certain embodiments, plants are crops. As used herein, the term “crop” means a species or variety of plant cultivated for food, animal feed, fuel, or other economic purposes. Non-limiting examples of crops include grains such as corn, wheat, rye, barley, and oats; sorghum, rice, sugar beets and fodder beets; fruits such as pears (e.g., apples and pears), citrus fruits (e.g., oranges, lemons, limes, grapefruit, or mandarins), drupes (e.g., peaches, nectarines, or plums), nuts (e.g., almonds, or walnuts), and muscovite (e.g., cherries, strawberries, blackberries, or raspberries); plantaginaceae or vines; legumes such as beans, lentils, peas, and soybeans; oil plants such as sunflowers, safflower, rapeseed, canola, perilla, or olives; cucumbers, Examples include cucurbitaceous crops such as melons or pumpkins, fiber crops such as cotton, flax, or hemp, fuel crops such as sugarcane, pampas grass, or switchgrass, vegetables such as potatoes, tomatoes, bell peppers, lettuce, spinach, onions, carrots, eggplants, asparagus, or cabbage, ornamental plants such as flowers (e.g., petunias, geraniums, roses, tulips, lilies, or chrysanthemums), shrubs, broad-leaved trees (e.g., poplars or willows), and evergreen trees (e.g., conifers), grasses such as lawns, turfgrass, or grasses, or other useful plants such as coffee, tea leaves, tobacco, hops, pepper, rubber, or latex plants.

[0460] According to a particular embodiment, the plants may be selected from the group consisting of: corn, soybean, al-Fa, cotton, sunflower, Brassica napus (e.g., canola, rapeseed), Brassica rapa, Brassica juncea (e.g., mustard greens), and Brassica Seeds for rapeseed oil such as carinata, palm species (e.g., oil palm, coconut), rice, wheat, sugar beet, sugarcane, oats, barley, millet and sorghum, rye wheat, flax, nuts, grapes and ivy, and fruits and vegetables belonging to various plant taxa, such as rose species (e.g., not only pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds, plums and peaches, and berries such as strawberries, raspberries, red currants and black currants and gooseberries), Libesioides species, walnut species, birch species, sumac species, beech species, mulberry species, osmanthus species (e.g., olive tree), Actinidia species, camphor species (e.g., avocado, cinnamon, camphor), banana species (e.g., banana trees and banana orchards).Plantations)), Rubia species (e.g., coffee), Camellia species (e.g., tea), Firmithium species, Citrus species (e.g., lemon, orange, mandarin and grapefruit), Solanum species (e.g., tomato, potato, pepper, chili pepper, eggplant, tobacco), Lilium species, Aster species (e.g., lettuce, Korean thistle and chicory (this includes root chicory, endive or chrysanthemum)), Apiaceae species (e.g., carrot, parsley, celery and celeriac), Cucurbitaceae species (e.g., cucumber including gherkin, pumpkin, watermelon, gourd and melon), Allium species (e.g., leek and onion), Brassica species (e.g., These include white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, bok choy, kohlrabi, radish, horseradish, celery, and Chinese cabbage), legumes (e.g., peas, peas, lentils, and other legumes (e.g., green beans and broad beans)), goosefoot (e.g., Swiss chard, fodder beets, spinach, and beet roots), flax (e.g., hemp), cannabis (e.g., cannabis), mallow (e.g., okra, cacao), poppy (e.g., poppy), asparagus (e.g., asparagus), useful and ornamental plants for gardens and forests, such as lawns, turfgrasses, grasses and Stevia rebaudiana, and genetically modified plants of these species.

[0461] According to a particular embodiment, the plant may be a harvestable part of a plant selected from the group consisting of fruits, flowers, nuts, vegetables, avocados, bananas, plantains, lemons, grapefruits, melons, oranges, pineapples, kiwifruits, guavas, mandarins, mangoes, and pumpkins.

[0462] According to a particular embodiment, the plant is a forest plant, as described, for example, in www(dot)fao(dot)org / 3 / i0640e / i0640e13.pdf.

[0463] According to a particular embodiment, the plant is an ornamental plant.

[0464] According to a particular embodiment, the plant may be an ornamental cut flower, preferably selected from alstroemeria, carnation, chrysanthemum, freesia, gerbera, gladiolus, baby's breath (Gypsophila spp.), sunflower, hydrangea, lily, lisianthus, rose, and summer flowers.

[0465] According to certain embodiments, the plant is cut grass or wood.

[0466] According to a particular embodiment, the plant is cotton.

[0467] According to certain embodiments, the plant is a transgenic plant.

[0468] According to a particular embodiment, the plant is a transgenic plant that recombinantly expresses nanobodies, or a composition comprising nanobodies and toxins.

[0469] Methods for creating transgenic plants are widely known in the industry and have been described above in this specification.

[0470] Next, the present invention also encompasses products comprising nanobodies or compositions comprising nanobodies and toxins, polynucleotides or nucleic acid constructs encoding them, or host cells expressing them.

[0471] According to certain embodiments, such products are more resistant to insect infestation and damage than products that do not contain nanobodies or compositions comprising nanobodies and toxins, polynucleotides or nucleic acid constructs encoding them, or host cells expressing them.

[0472] Accordingly, according to one aspect of the present invention, a plant is provided comprising a nanobody or a composition comprising a nanobody and a toxin, and a polynucleotide or nucleic acid construct encoding the same.

[0473] According to another embodiment, an industrial product is provided which comprises a nanobody or a composition comprising a nanobody and a toxin.

[0474] According to another embodiment, a surface is provided that is coated with a nanobody or a composition comprising a nanobody and a toxin.

[0475] According to another embodiment, a packaged product is provided which includes an industrial product contained within a packaging material and which includes a nanobody or a composition comprising a nanobody and a toxin.

[0476] Tables 1-8 below list polypeptides and nanobodies that can be used in specific embodiments of the present invention.

[0477] [Table 1-1]

[0478] [Table 1-2]

[0479] [Table 2-1]

[0480] [Table 2-2]

[0481] [Table 2-3]

[0482] [Table 2-4]

[0483] [Table 2-5]

[0484] Table 3-1

[0485] Table 3-2

[0486] Table 4-1

[0487] Table 4-2

[0488] Table 4-3

[0489] Table 5-1

[0490] Table 5-2

[0491] Table 5-3

[0492] Table 6-1

[0493] Table 6-2

[0494] Table 6-3

[0495] [Table 6-4]

[0496] [Table 7-1]

[0497] [Table 7-2]

[0498] [Table 7-3]

[0499] [Table 8-1]

[0500] [Table 8-2]

[0501] [Table 8-3]

[0502] [Table 8-4]

[0503] As used in this specification, "approximately" refers to ±10%.

[0504] The terms "comprises," "comprising," "includes," "including," and "having," along with their conjugations, all mean "including but not limited to."

[0505] The term "consisting of" means "to include or be limited to."

[0506] The term "substantially derived from" means that the composition, method, or structure may include additional components, steps, and / or parts, provided that these additional components, steps, and / or parts do not substantially alter the basic and novel properties of the composition, method, or structure described in the claim.

[0507] In this specification, the singular pronouns "a," "an," and "the" also refer to plural nouns unless the context clearly indicates otherwise. For example, "a compound" or "at least one compound" may include multiple compounds, and may also include mixtures thereof.

[0508] Throughout this application, various embodiments of the invention may be presented in range form. It should be understood that the use of range form is merely for convenience and brevity, and not as a limitation that restricts the flexibility of the scope of the invention. Therefore, a range description should be considered to specifically disclose all possible sub-ranges and the individual numerical values ​​within those ranges. For example, a range description such as 1-6 specifically discloses not only sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, but also the individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the size of the range.

[0509] Where a numerical range is indicated herein, it is always intended to include any quoted number (fraction or integer) within that range. The expressions “range between” the first indicator and the second indicator, and “range from” the first indicator to the second indicator, are used interchangeably herein and are intended to include the first and second indicators and all fractions and integers between them.

[0510] As used herein, the term “method” means a form, means, technique, and procedure for achieving a given task, and includes, but is not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or those readily available to practitioners from known forms, means, techniques, and procedures.

[0511] When referring to a specific sequence listing, it should be understood that such reference also includes sequences that substantially correspond to complementary sequences containing minor sequence changes resulting from, for example, sequencing errors, cloning errors, or other modifications resulting in base substitutions, base deletions, or base additions. However, the frequency of such changes is limited to less than 1 per 50 nucleotides, alternatively less than 1 per 100 nucleotides, alternatively less than 1 per 200 nucleotides, alternatively less than 1 per 500 nucleotides, alternatively less than 1 per 1000 nucleotides, alternatively less than 1 per 5,000 nucleotides, and alternatively less than 1 per 10,000 nucleotides.

[0512] Features of the present invention described as separate embodiments for clarity should be understood to be able to be combined and provided as a single embodiment. Conversely, various features of the present invention described as a single embodiment for brevity can also be provided individually, in any suitable partial combination, or in a suitable combination with other embodiments described herein. Features described in relation to various embodiments are not considered essential requirements of those embodiments unless the embodiment is inoperable without those features.

[0513] The following examples provide experimental evidence for the various embodiments and aspects of the present invention listed above and described in the claims below. [Examples]

[0514] Hereinafter, the present invention will be described in conjunction with the above description, and will not be limited to the following embodiments.

[0515] The nomenclature and experimental procedures used herein typically include molecular techniques, biochemical techniques, microbiological techniques, and recombinant DNA techniques. Such techniques are well described in the literature. For example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989), "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994), Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989), Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988), Watson et al., "Recombinant DNA", Scientific American Books, New York, Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998), methods described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057, "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994), "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition, "Current Protocols in Immunology" Volumes I-III Coligan, JE, ed.See also Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), and Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). Available immunoassays are widely described in patents and scientific literature, for example, in U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, “Oligonucleotide Synthesis” Gait, MJ. (1984), “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, eds. (1985), “Transcription and Translation” Hames, BD, and Higgins SJ, Eds. (1984), “Animal Cell Culture” Freshney, RI, ed. (1986), “Immobilized Cells and Enzymes” IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990), Marshak et al.See "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996). All of the above references are fully incorporated herein by reference. Other general references are provided herein. The procedures described therein are considered well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0516] material and method Preparation of Target Proteins for Immunity - The DNA sequence of the target protein of Helicoverpa armigera (see Table 1 above) was optimized and synthesized. The synthesized sequence was cloned with a His tag into pET-28a or pET-30a vectors and expressed in E. coli BL21 Star (DE3) strain. Single colonies were inoculated into kanamycin-containing LB medium, and the cultures were incubated at 37°C and 200 rpm, followed by induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). SDS-PAGE analysis was used to monitor protein expression. Subsequently, BL21 Star (DE3) cells stored in glycerol were inoculated into kanamycin-containing TB medium and cultured at 37°C. Once the OD600 reached 1.2, the cell cultures were induced with IPTG at 15°C for 16 hours. Cells were collected by centrifugation, and the pellet was resuspended in lysis buffer (20 mM NaPi, pH 7.4, 500 mM NaCl, 20 mM imidazole, cOmplete® (a protease inhibitor cocktail), and Roche (an EDTA-free protease inhibitor cocktail, cat#4693132001)). This was followed by sonication and centrifugation. The precipitate was dissolved with urea, and the denatured supernatant was retained for further purification. Subsequently, proteins were purified from the supernatant using a Ni-NTA column (GE-Healthcare, Cat No. 17-5318-02). The column was washed with 10 times its total volume of washing buffer (20 mM NaPi, pH 7.4, 500 mM NaCl, 50 mM imidazole), and the proteins were eluted with elution buffer (20 mM NaPi, pH 7.4, 500 mM NaCl, 500 mM imidazole). Following elution, the fractions were placed in MEGA tubes (Tivan Biotech MEGA3-50) with a membrane cutoff value of 3.5 kDa, dialyzed overnight at 4°C in 1 L of PBS (pH 7.4), and then dialyzed a second time under the same conditions. Some target proteins (e.g., V-ATPase subunit c target protein) were further refolded and sterilized through a 0.22 μm filter before storage in equal volumes. Concentrations were determined using nano-drops.Protein purity and molecular weight were determined using standard SDS-PAGE.

[0517] Subcutaneous injections were administered to llamas on immunization days 0, 7, 14, 21, 28, and 35, with each injection consisting of approximately 125 or 150 μg of target protein combined 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.

[0518] Construction of a VHH Library - To screen for the presence of antigen-specific nanobodies, a VHH library was constructed from llama lymphocytes. For this purpose, total RNA derived from peripheral blood lymphocytes was used as a template for first-strand cDNA synthesis with oligo(dT) primers. Using this cDNA, the VHH coding sequence was amplified by PCR, digested with PstI (Thermofischer #ER0611) and NotI (Sigma #11037668001), and cloned into the PstI and NotI sites of the phagemide vector pMECS. The VHH library was approximately 10 8 The study included several independent transformants, and approximately 80–92% of the transformants possessed vectors containing correctly sized inserts.

[0519] 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)). Enrichment of antigen-specific phages in each panning cycle was determined by comparing the number of phagemids eluted from the antigen-coated wells with the number of phagemids eluted from the negative control (uncoated and blocked) wells. These experiments demonstrated that antigen-specific phages within the phage population were enriched after three cycles. The antigen used for panning and ELISA screening was the same as that used for immunization, and the uncoated and blocked wells were used as negative controls. The amino acid sequences of the fabricated nanobodies are shown in Tables 2A to 6 above.

[0520] Nanobody Expression and Purification - Nanobody DNA sequences were optimized and synthesized using GenScript. The synthesized sequences were cloned with His tags into a pET-28b+ vector for protein expression in E. coli. Subsequently, E. coli SHuffle T7 cells were transformed with recombinant plasmids. Single colonies were inoculated into kanamycin-containing LB medium, incubated at 37°C and 220 rpm, and then induced with 1 M IPTG. SDS-PAGE analysis was used to monitor expression. Subsequently, SHuffle T7 cells stored in glycerol were inoculated into kanamycin-containing TB medium and cultured at 37°C. When the OD600 reached approximately 1.2, the cell cultures were induced with 1 M IPTG at 30°C for 20-22 hours. Cells were harvested by centrifugation, and the pellet was resuspended in lysis buffer (20 mM NaPi, pH 7.5, 500 mM NaCl, 20 mM imidazole), followed by sonication and centrifugation. The precipitate was dissolved using urea, and the denatured supernatant was retained for further purification. Subsequently, the protein was purified from the supernatant using a Ni-NTA column. The column was washed with 10 times its total volume of washing 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 fraction was then dialyzed overnight at 4°C in dialysis buffer (PBS, pH 7.4). The concentration was determined using nanodrops. Protein purity and molecular weight were determined by standard SDS-PAGE.

[0521] The fabricated nanobodies were bound to the target protein and coated with 10 μg / ml of the target protein by incubation (overnight at 4°C). The coated plates were washed with PBS and blocked at 37°C for 1 hour in blocking buffer containing PBS + 0.5% tween + 1% BSA, followed by incubation with 2 μg / ml of test nanobodies at room temperature for 1 hour. Following washing with PBS, the plates were incubated with MonoRab® rabbit anti-camel VHH antibody-HRP (Genscript, Cat no. A01860) in blocking buffer. Finally, the plates were washed with PBS, incubated with TMB solution for 30 minutes, followed by the addition of H2SO4 and measurement of absorbance at 450 nm.

[0522] Stability assay - The stability of the fabricated nanobodies was tested under "field-like" conditions by incubation under natural sunlight, natural UV irradiation, and high temperatures in outdoor / outdoor environments during summer. In addition, 4 instars (4 thEnzymatic proteolytic activity and stability at high pH were also evaluated by incubation with luminal fluid recovered from the start larval digestive tracts (start larvae guts) of H. armigera [see, for example, Purcell, John P., John T. Greenplate, and R. Douglas Sammons. Insect Biochemistry and Molecular Biology 22.1 (1992): 41-47, Pang, AS, & Gringorten, JL (1998). FEMS microbiology letters, 167(2), 281-285, and Ma, Gang, et al. Insect biochemistry and molecular biology 35.7 (2005): 729-739]. Specifically, Maxisorb 96-well plates were coated with 2 μg / ml test nanobodies by overnight incubation at 4°C. The coated plates were washed with PBS and blocked at 37°C for 1 hour in blocking buffer containing PBS + 0.5% tween + 1% BSA. The coated plates were incubated with PBS under "field-like conditions" for 1–3 hours, or at room temperature for 1 hour in the presence of luminal gastrointestinal fluid. Following incubation, the plates were washed with PBS and incubated with MonoRab® rabbit anti-camel VHH antibody-HRP in blocking buffer. Finally, the plates were washed with PBS, incubated with TMB solution for 30 minutes, followed by the addition of H2SO4, and the absorbance at 450 nm was measured.

[0523] Insect Culture - Cotton bollworms were collected from cotton fields in Israel and reared in the laboratory under conditions of 24°C, 70% relative humidity, and 10 hours of light / 14 hours of darkness. The collected larvae were reared on an artificial feed, Ward's diet powder Stonefly Heliothis Diet (product number 38-0600, Ward's Natural Science, Rochester, New York), which contains a vitamin mixture (nicotinic acid: 40 μM, calcium pantothenate: 5 μM, riboflavin: 5 μM, thiamine hydrochloride: 4 μM, pyridoxine hydrochloride: 5 μM, folic acid: 2.5 μM, D-biotin: 4 μM, 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%.

[0524] Feed Bioassay - 0.5 grams of artificial feed for the genus *Cypripedium* was added to each well of a 24-well plate, and a 100 μl suspension containing either a test nanobody at a concentration of 0.4 mg / ml or PBS as a negative control was added on top. Twelve sets were prepared, with one larva aged 24–48 hours added to each well. The plates were covered with polyofin sealing foil (HJ-BIOANALITIC, Cat No. 900371) and incubated in a laboratory at 24°C, 70% relative humidity, and in the dark. Each study for each nanobody was repeated 3–5 times. Following a 7-day incubation, the larvae were transferred to larger, 5 cm diameter petri dishes containing the same artificial feed without the addition of nanobodies. The body length and weight of each larva were measured on days 7 and 14 of incubation, and the timing of pupation and adult development was observed.

[0525] Cotton cultivation – The cotton cultivar Akalpi (an interspecific hybrid (ISH) between G. hirsutum and G. barbadense, 1432 Intercott Hazera) was cultivated under controlled conditions of 25°C / 18°C day / night. Temperature monitoring was performed and maintained using a SAS automation system. In addition, fertilization and irrigation were carried out using Sheffer 7-7-7 plus calcium, maintaining the leachate fraction at 30%. Pest control was carried out mechanically using only sticky, colored traps. The cultivation cycle lasted between 6 and 8 weeks. After this period, the plants were moved to the laboratory for use as a plant model in bioassay experiments.

[0526] Leaf Bioassay - 1 ml of sterile agar was added to each well of a 12-well plate, and 2 cm diameter disc-shaped cottony plant leaves were placed on top of the agar in each well. A 50 μl suspension containing tested nanobodies at concentrations of 0.4, 1, 2, or 4 mg / ml, or PBS as a negative control, was overlaid on each disc-shaped leaf. Twelve sets were prepared, each containing one 24-48 hour larva. The plates were covered with polyofin sealing foil (HJ-BIOANALITIC, Cat No. 900371) and incubated in the laboratory at 24°C and 70% relative humidity. Following a 4-day incubation, the larvae were transferred to a new plate containing fresh, disc-shaped cotton leaves and treated with the same concentration of nanobodies or PBS. On day 7, the larvae were again transferred to a larger well (6-well plate) containing agar and fresh, 2.8 cm diameter disc-shaped cotton leaves treated with 100 μl of the same concentration of the same nanobodies. Larval mortality was assessed on days 4, 7, and 10. Each study for each nanobody was repeated 2-3 times.

[0527] Purified anti-CBD nanobodies, referred to herein as "CB20901," at an immunofluorescence concentration of 1 mg / ml were conjugated to fluorescently labeled Cy3 (ab188287-Cy3 Fast Conjugation Kit, Cambridge, UK) according to the manufacturer's instructions. The feed bioassay was performed as described above. Specifically, 12 larvae were fed 100 μl of Cy3-fluorescently labeled nanobodies, and 12 larvae fed 100 μl of PBS were used as controls. Following a 36-hour incubation, the treated larvae were transferred to a clean, freshly prepared artificial feed for the genus *Cyclops* for a further 12 hours to eliminate false-positive signals, followed by a first imaging session. In the next step, the larvae were returned to the nanobodies / PBS feed until they were 6 days old, fed a nanobodies-free feed for the next 12 hours, and finally, a second imaging session was performed on live larvae and extracted gastrointestinal tracts. The following settings were maintained for effective image comparison. Nikon Eclipse 80i microscope, NIS sensor software, DS-Ri2 camera, 1X lens with 2.0x zoom, 10ms exposure, and 7.6x gain (for imaging feed), 1X lens with 1.4x zoom, 200ms exposure, and 2.0x gain (for 48-hour larvae), and 1X lens with 1.4x zoom, 400ms exposure, and 6.2x gain (for 6-day-old larvae and extracted digestive tracts).

[0528] Actual Example 1 Anti-chitin-binding domain nanobody In this specification, we have constructed several nanobodies targeting the chitin-binding domain (CBD) antigen of Helicoverpa armigera, referred to as Ha-PMP5B1 and Ha-PMP5B2 (see Tables 2A and 2B above). The Ha-PMP5B1 protein contains 506 amino acids, including five different CBDs derived from Helicoverpa armigera, while the Ha-PMP5B2 protein is a partial protein of Ha-PMP5B1, containing 192 amino acids, including two CBDs. ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bind to the target CBD antigen (Figure 1). Since certain embodiments disclose that when nanobodies are administered orally to Helicoverpa armigera larvae, the nanobodies are absorbed in the lumen of the larval digestive tract (which has a high pH and contains a large amount of proteolytic enzymes) before reaching the target site within the periphaloblastic membrane of the larval digestive tract, we studied the stability of anti-CBD nanobodies in the lumen. An ELISA assay using plates coated with the fabricated anti-CBD nanobodies and incubated for 1 hour in the presence of collected lumen fluid showed that 40–70% of the nanobodies remained intact after 1 hour of incubation (Figure 2), demonstrating that the fabricated anti-CBD nanobodies are viable under lumen conditions. In addition, the stability of the fabricated anti-CBD nanobodies under conditions mimicking outdoor conditions was also tested by incubation for 1–3 hours under natural sunlight and a high ambient temperature of 30°C. The results showed that after 3 hours of incubation under outdoor-like conditions of 30°C, 60–70% humidity, and a UV index of 4–5, there was only a slight decrease in nanobody content compared to the control (Figure 3).

[0529] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae 48 hours or younger were reared on artificial feed supplemented with a single dose of the anti-CBD nanobodies. Observations were performed throughout the insect's entire life cycle, including measurements of larval weight and length at days 7 and 14, as well as observation of the number of individuals that subsequently metamorphosed from pupae to adults. The results showed that larvae fed several types of anti-CBD nanobodies for 7 days exhibited a decrease in body length (Figure 4(A)) and / or weight (Figure 4(B)) and / or an increase in mortality (Figure 4(C)), depending on the nanobodies tested.

[0530] To track the trajectory of nanobodies in the larval digestive tract, Helicoverpa armigera larvae were reared on an artificial diet supplemented with the fluorescently labeled Cy3 anti-CBD nanobodies CB12901. As shown in Figure 5(A), the labeled nanobodies remained stable in the diet for at least one week. In addition, a decrease in the fluorescence signal was observed between the feed and the larval feces following feeding, suggesting uptake of the labeled nanobodies by the larval digestive tract (Figure 5(A)). Furthermore, the labeled nanobodies were detected in the larvae 12 hours after they were moved to a feed without nanobodies, suggesting that the labeled nanobodies are retained in the larval digestive tract (Figure 5B). Furthermore, the ability of the labeled nanobodies to reach the periphaloblastic membrane was confirmed on day 6 after feeding, at which point a positive signal was observed throughout the entire digestive tract, particularly from the periphaloblastic membrane following separation from the lumen of the digestive tract (Figure 5(C)).

[0531] Example 2 Anti-V-ATPase subunit c nanobody Several nanobodies were constructed targeting the V-ATPase subunit C (hereinafter referred to as "VAT") antigen of Helicoverpa armigera (see Tables 3A-3B above). ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bound to the target VAT antigen (Figure 6). The stability of the anti-VAT nanobodies under luminal and field-like conditions was then investigated (Figures 7 and 8). The results showed that 40-70% of the nanobodies remained intact after 1 hour of incubation in the presence of luminal fluid, indicating the survival property of the constructed anti-VAT nanobodies under luminal conditions. Furthermore, the results showed only a slight decrease in nanobody content compared to the control after 3 hours of incubation under field-like conditions of 30°C, 60-70% humidity, and a UV index of 4-5.

[0532] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae 48 hours or younger were reared on artificial feed supplemented with a single dose of the fabricated anti-VAT nanobodies. Observations were performed throughout the insect's life cycle, including measurements of larval body weight and length at days 7 and 14, and observation of the number of individuals that subsequently metamorphosed from pupae to adults. The results showed that, depending on the nanobodies tested, larvae fed the fabricated anti-VAT nanobodies for 7 days showed a decrease in body length (Figure 9(A)) and / or a decrease in body weight and / or an increase in mortality (Figure 9(C)).

[0533] In addition, we determined the percentage of larvae that survived and completed the complete life cycle, from larva to pupa and then to adult moth. As shown in Table 9 below, the percentage of larvae that became adult moths and completed the complete life cycle decreased in larvae treated with anti-VAT nanobodies for 7 days. Importantly, the results showed that even though the larvae were exposed to anti-VAT nanobodies for only the first 7 days of the 25-30 day experimental period, the nanobodies exerted a long-term, stable effect on Helicoverpa armigera, influencing even the later developmental stages of pupation and adulthood.

[0534] [Table 9]

[0535] Example 3 Anti-trehalase nanobody Several nanobodies targeting the trehalase antigen (hereinafter referred to as "TRH") of Helicoverpa armigera were constructed (see Table 4 above). ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bound to the target trehalase antigen (Figure 10). Next, the stability of the anti-TRH nanobodies under field-like conditions and at a high temperature of 50°C was investigated (Figure 11(A)-B). The results showed that overnight incubation under field-like conditions of 30°C, 60-70% humidity, and a UV index of 4-5 induced no effect or only a slight decrease in nanobody content compared to the control. Similar results were observed after overnight incubation at 50°C.

[0536] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae (48 hours or less in age) were reared on artificial feed supplemented with a single dose of the fabricated anti-TRH nanobodies, and mortality rates were measured on days 7 and 14. The results showed a significant increase in larval mortality following treatment with the fabricated anti-TRH nanobodies (Figure 12A). Subsequently, young Helicoverpa armigera larvae (48 hours or less in age) were reared after being administered the fabricated anti-TRH nanobodies by adding them to disc-shaped cotton leaves on days 0, 4, and 7, and mortality rates were measured on days 4, 7, and 10. The results showed a significantly dose-dependent increase in larval mortality following treatment with the anti-TRH nanobodies (Figure 12B).

[0537] In addition, we conducted observations of the entire life cycle of the insects, including measuring the weight and length of the larvae, and observing the number of individuals that later metamorphosed from pupae to adults. Furthermore, we determined the percentage of larvae that survived and completed the complete life cycle from larva to pupa and then to adult moth.

[0538] Example 4 Anti-cytochrome P450 monooxygenase nanobody Several nanobodies targeting the cytochrome p450 monooxygenase (hereinafter referred to as "P450") antigen of Helicoverpa armigera were constructed (see Table 5 above). ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bound to the target P450 antigen (Figure 13). Subsequently, the stability of the anti-P450 nanobodies under luminal and field-like conditions was investigated.

[0539] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae (48 hours old or younger) were reared on artificial feed supplemented with the fabricated anti-P450 nanobodies, and mortality rates were measured on days 7 and 14. The results showed a significant increase in larval mortality following treatment with the fabricated anti-P450 nanobodies (Figure 14).

[0540] In addition, we conducted observations of the entire life cycle of the insects, including measuring the weight and length of the larvae, and observing the number of individuals that later metamorphosed from pupae to adults. Furthermore, we determined the percentage of larvae that survived and completed the complete life cycle from larva to pupa and then to adult moth.

[0541] Example 5 Anti-chitin deacetylase nanobody Several nanobodies targeting the chitin deacetylase (hereinafter referred to as "CDA") antigen of Helicoverpa armigera were constructed (see Table 6 above). ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bound to the target CDA antigen (Figure 15). Next, the stability of the anti-CDA nanobodies under field-like conditions and at a high temperature of 50°C was investigated (Figure 16 (A)-B). The results showed that overnight incubation under field-like conditions of 30°C, 60-70% humidity, and a UV index of 4-5 had no effect on nanobody content compared to the control. Similar results were observed after overnight incubation at 50°C.

[0542] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae (48 hours or less in age) were reared on artificial feed supplemented with the fabricated anti-CDA nanobodies, and mortality rates were measured on days 7 and 14. The results showed a significant increase in larval mortality following treatment with the anti-CDA nanobodies (Figure 17A). Subsequently, young Helicoverpa armigera larvae (48 hours or less in age) were reared after being administered the fabricated anti-CDA nanobodies by adding them to disc-shaped cotton leaves on days 0, 4, and 7, and mortality rates were measured on days 4, 7, and 10. The results showed a significantly dose-dependent increase in larval mortality following treatment with the anti-CDA nanobodies (Figure 17B).

[0543] In addition, we conducted observations of the entire life cycle of the insects, including measuring the weight and length of the larvae, and observing the number of individuals that later metamorphosed from pupae to adults. Furthermore, we determined the percentage of larvae that survived and completed the complete life cycle from larva to pupa and then to adult moth.

[0544] Example 6 Anti-chitin synthase nanobody Several nanobodies targeting the chitin synthase (hereinafter referred to as "CHS") antigen of Helicoverpa armigera were constructed (see Table 7 above). ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bound to the target CHS (Figure 18). Next, the stability of the anti-CHS nanobodies under field-like conditions and at a high temperature of 50°C was investigated (Figure 19 (A)-B). The results showed that overnight incubation under field-like conditions of 30°C, 60-70% humidity, and a UV index of 4-5 induced only a slight decrease in nanobody content compared to the control. Similar results were observed after overnight incubation at 50°C.

[0545] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae (48 hours or less in age) were reared on artificial feed supplemented with the fabricated anti-CHS nanobodies, and mortality rates were measured on days 7 and 14. The results showed a significant increase in larval mortality following treatment with the fabricated anti-CHS nanobodies (Figure 20A). Subsequently, young Helicoverpa armigera larvae (48 hours or less in age) were reared after being administered the fabricated anti-CHS nanobodies by adding them to disc-shaped cotton leaves on days 0, 4, and 7, and mortality rates were measured on days 4, 7, and 10. The results showed a significantly dose-dependent increase in larval mortality following treatment with the anti-CHS nanobodies (Figure 20B).

[0546] In addition, we conducted observations of the entire life cycle of the insects, including measuring the weight and length of the larvae, and observing the number of individuals that later metamorphosed from pupae to adults. Furthermore, we determined the percentage of larvae that survived and completed the complete life cycle from larva to pupa and then to adult moth.

[0547] Example 7 Anti-NPC1 sterol transporter / nanobody Several nanobodies targeting the NPC1 sterol transporter antigen of Helicoverpa armigera (referred to herein as "NPC1") were constructed (see Table 8 above). ELISA assays using plates coated with the target antigen demonstrated that the constructed nanobodies bound to the target NPC1 antigen (Figure 21). Subsequently, the stability of the anti-NPC1 sterol transporter nanobodies under luminal and field-like conditions was investigated.

[0548] To determine the insecticidal activity of the fabricated nanobodies, young Helicoverpa armigera larvae (48 hours old or younger) were reared on artificial feed supplemented with the fabricated anti-NPC1 nanobodies, and mortality rates were measured on days 7 and 14. The results showed a significant increase in larval mortality following treatment with the fabricated anti-NPC1 nanobodies (Figure 22).

[0549] In addition, we conducted observations of the entire insect life cycle, including measuring the weight and length of the larvae on day 7 and day 14, and observing the number of individuals that later metamorphosed from pupae to adults. Furthermore, we determined the percentage of larvae that survived and completed the complete life cycle from larva to pupa and then to adult moth.

[0550] Although the present invention has been described in relation to its specific embodiments, numerous alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, all such alternatives, modifications, and variations are intended to be included within the spirit and broader scope of the appended claims.

[0551] All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole to the same extent that each individual publication, patent, and patent application is incorporated herein by specific and individual reference. In addition, no citation or specification of any reference in this application should be construed as an acceptance that such reference can be used as prior art of the present invention. Nor should the titles of each section be construed as limitations to the extent in which they are used. Furthermore, any basic application of this application is incorporated herein by reference in whole. [Sequence Listing Free Text]

[0552] Sequence ID 1: Chitin-binding domain CBD-containing polypeptide - HaPMP5B1 Sequence ID 2: Chitin-binding domain CBD-containing polypeptide - HaPMP5B1 Sequence ID 3: Chitin-binding domain (CBD) Sequence ID 4: Chitin-binding domain (CBD) Sequence ID 5: Chitin-binding domain (CBD) Sequence ID 6: Chitin-binding domain (CBD) Sequence ID 7: Chitin-binding domain (CBD) Sequence ID 8: V-ATPase subunit c Sequence ID 9: Chitin deacetylase Sequence ID 10: Trehalase Sequence ID 11: Cytochrome p450 monooxygenase Sequence ID 12: Chitin Synthase Sequence ID 13: NPC1 Sterol Transporter Sequence ID 14: Amino acid sequence of a nanobody Sequence ID 15: Amino acid sequence of the complementarity-determining region Sequence ID 16: Amino acid sequence of the complementarity-determining region Sequence ID 17: Amino acid sequence of the complementarity-determining region Sequence ID 18: Amino acid sequence of a nanobody Sequence ID 19: Amino acid sequence of the complementarity-determining region Sequence ID 20: Amino acid sequence of the complementarity-determining region Sequence ID 21: Amino acid sequence of the complementarity-determining region Sequence ID 22: Amino acid sequence of a nanobody Sequence ID 23: Amino acid sequence of the complementarity-determining region Sequence ID 24: Amino acid sequence of the complementarity-determining region Sequence ID 25: Amino acid sequence of the complementarity-determining region Sequence ID 26: Amino acid sequence of a nanobody Sequence ID 28: Amino acid sequence of the complementarity-determining region Sequence ID 29: Amino acid sequence of the complementarity-determining region Sequence ID 30: Amino acid sequence of a nanobody Sequence ID 31: Amino acid sequence of the complementarity-determining region Sequence ID 32: Amino acid sequence of the complementarity-determining region Sequence ID 33: Amino acid sequence of the complementarity-determining region Sequence ID 34: Amino acid sequence of a nanobody Sequence ID 35: Amino acid sequence of the complementarity-determining region Sequence ID 36: Amino acid sequence of the complementarity-determining region Sequence ID 37: Amino acid sequence of the complementarity-determining region Sequence ID 38: Amino acid sequence of a nanobody Sequence ID 39: Amino acid sequence of the complementarity-determining region Sequence ID 40: Amino acid sequence of the complementarity-determining region Sequence ID 41: Amino acid sequence of the complementarity-determining region Sequence ID 42: Amino acid sequence of a nanobody Sequence ID 43: Amino acid sequence of the complementarity-determining region Sequence ID 44: Amino acid sequence of the complementarity-determining region Sequence ID 45: Amino acid sequence of the complementarity-determining region Sequence ID 46: Amino acid sequence of a nanobody Sequence ID 47: Amino acid sequence of the complementarity-determining region Sequence ID 48: Amino acid sequence of the complementarity-determining region Sequence ID 49: Amino acid sequence of the complementarity-determining region Sequence ID 50: Amino acid sequence of a nanobody Sequence ID 51: Amino acid sequence of the complementarity-determining region Sequence ID 52: Amino acid sequence of the complementarity-determining region Sequence ID 53: Amino acid sequence of the complementarity-determining region Sequence ID 54: Amino acid sequence of a nanobody Sequence ID 55: Amino acid sequence of the complementarity-determining region Sequence ID 56: Amino acid sequence of the complementarity-determining region Sequence ID 57: Amino acid sequence of the complementarity-determining region Sequence ID 58: Amino acid sequence of a nanobody Sequence ID 59: Amino acid sequence of the complementarity-determining region Sequence ID 60: Amino acid sequence of the complementarity-determining region Sequence ID 61: Amino acid sequence of the complementarity-determining region Sequence ID 62: Amino acid sequence of a nanobody Sequence ID 63: Amino acid sequence of the complementarity-determining region Sequence ID 64: Amino acid sequence of the complementarity-determining region Sequence ID 65: Amino acid sequence of the complementarity-determining region Sequence ID 66: Amino acid sequence of a nanobody Sequence ID 67: Amino acid sequence of the complementarity-determining region Sequence ID 68: Amino acid sequence of the complementarity-determining region Sequence ID 69: Amino acid sequence of the complementarity-determining region Sequence ID 70: Amino acid sequence of a nanobody Sequence ID 71: Amino acid sequence of the complementarity-determining region Sequence ID 72: Amino acid sequence of the complementarity-determining region Sequence ID 73: Amino acid sequence of the complementarity-determining region Sequence ID 74: Amino acid sequence of a nanobody Sequence ID 75: Amino acid sequence of the complementarity-determining region Sequence ID 76: Amino acid sequence of the complementarity-determining region Sequence ID 77: Amino acid sequence of the complementarity-determining region Sequence ID 78: Amino acid sequence of a nanobody Sequence ID 79: Amino acid sequence of the complementarity-determining region Sequence ID 80: Amino acid sequence of the complementarity-determining region Sequence ID 81: Amino acid sequence of the complementarity-determining region Sequence ID 82: Amino acid sequence of a nanobody Sequence ID 83: Amino acid sequence of the complementarity-determining region Sequence ID 84: Amino acid sequence of the complementarity-determining region Sequence ID 85: Amino acid sequence of the complementarity-determining region Sequence ID 86: Amino acid sequence of a nanobody Sequence ID 87: Amino acid sequence of the complementarity-determining region Sequence ID 88: Amino acid sequence of the complementarity-determining region Sequence ID 89: Amino acid sequence of the complementarity-determining region Sequence ID 90: Amino acid sequence of a nanobody Sequence ID 91: Amino acid sequence of the complementarity-determining region Sequence ID 93: Amino acid sequence of the complementarity-determining region Sequence ID 94: Amino acid sequence of a nanobody Sequence ID 95: Amino acid sequence of the complementarity-determining region Sequence ID 96: Amino acid sequence of the complementarity-determining region Sequence ID 97: Amino acid sequence of the complementarity-determining region Sequence ID 98: Amino acid sequence of a nanobody Sequence ID 99: Amino acid sequence of the complementarity-determining region Sequence ID 100: Amino acid sequence of the complementarity-determining region

[0553] Sequence ID 101: Amino acid sequence of the complementarity-determining region Sequence ID 102: Amino acid sequence of a nanobody Sequence ID 103: Amino acid sequence of the complementarity-determining region Sequence ID 104: Amino acid sequence of the complementarity-determining region Sequence ID 105: Amino acid sequence of the complementarity-determining region Sequence ID 106: Amino acid sequence of a nanobody Sequence ID 107: Amino acid sequence of the complementarity-determining region Sequence ID 108: Amino acid sequence of the complementarity-determining region Sequence ID 109: Amino acid sequence of the complementarity-determining region Sequence ID 110: Amino acid sequence of a nanobody Sequence ID 111: Amino acid sequence of the complementarity-determining region Sequence ID 112: Amino acid sequence of the complementarity-determining region Sequence ID 113: Amino acid sequence of the complementarity-determining region Sequence ID 114: Amino acid sequence of a nanobody Sequence ID 115: Amino acid sequence of the complementarity-determining region Sequence ID 116: Amino acid sequence of the complementarity-determining region Sequence ID 117: Amino acid sequence of the complementarity-determining region Sequence ID 118: Amino acid sequence of a nanobody Sequence ID 119: Amino acid sequence of the complementarity-determining region Sequence ID 120: Amino acid sequence of the complementarity-determining region Sequence ID 121: Amino acid sequence of the complementarity-determining region Sequence ID 122: Amino acid sequence of a nanobody Sequence ID 123: Amino acid sequence of the complementarity-determining region Sequence ID 124: Amino acid sequence of the complementarity-determining region Sequence ID 125: Amino acid sequence of the complementarity-determining region Sequence ID 126: Amino acid sequence of a nanobody Sequence ID 127: Amino acid sequence of the complementarity-determining region Sequence ID 128: Amino acid sequence of the complementarity-determining region Sequence ID 129: Amino acid sequence of the complementarity-determining region Sequence ID 130: Amino acid sequence of a nanobody Sequence ID 131: Amino acid sequence of the complementarity-determining region Sequence ID 132: Amino acid sequence of the complementarity-determining region Sequence ID 133: Amino acid sequence of the complementarity-determining region Sequence ID 134: Amino acid sequence of a nanobody Sequence ID 135: Amino acid sequence of the complementarity-determining region Sequence ID 136: Amino acid sequence of the complementarity-determining region Sequence ID 137: Amino acid sequence of the complementarity-determining region Sequence ID 138: Amino acid sequence of a nanobody Sequence ID 139: Amino acid sequence of the complementarity-determining region Sequence ID 140: Amino acid sequence of the complementarity-determining region Sequence ID 141: Amino acid sequence of the complementarity-determining region Sequence ID 142: Amino acid sequence of a nanobody Sequence ID 143: Amino acid sequence of the complementarity-determining region Sequence ID 144: Amino acid sequence of the complementarity-determining region Sequence ID 145: Amino acid sequence of the complementarity-determining region Sequence ID 146: Amino acid sequence of a nanobody Sequence ID 147: Amino acid sequence of the complementarity-determining region Sequence ID 148: Amino acid sequence of the complementarity-determining region Sequence ID 149: Amino acid sequence of the complementarity-determining region Sequence ID 150: Amino acid sequence of a nanobody Sequence ID 151: Amino acid sequence of the complementarity-determining region Sequence ID 152: Amino acid sequence of the complementarity-determining region Sequence ID 153: Amino acid sequence of the complementarity-determining region Sequence ID 154: Amino acid sequence of a nanobody Sequence ID 156: Amino acid sequence of the complementarity-determining region Sequence ID 157: Amino acid sequence of the complementarity-determining region Sequence ID 158: Amino acid sequence of a nanobody Sequence ID 159: Amino acid sequence of the complementarity-determining region Sequence ID 160: Amino acid sequence of the complementarity-determining region Sequence ID 161: Amino acid sequence of the complementarity-determining region Sequence ID 162: Amino acid sequence of a nanobody Sequence ID 163: Amino acid sequence of the complementarity-determining region Sequence ID 164: Amino acid sequence of the complementarity-determining region Sequence ID 165: Amino acid sequence of the complementarity-determining region Sequence ID 166: Amino acid sequence of a nanobody Sequence ID 167: Amino acid sequence of the complementarity-determining region Sequence ID 168: Amino acid sequence of the complementarity-determining region Sequence ID 169: Amino acid sequence of the complementarity-determining region Sequence ID 170: Amino acid sequence of a nanobody Sequence ID 171: Amino acid sequence of the complementarity-determining region Sequence ID 172: Amino acid sequence of a nanobody Sequence ID 173: Amino acid sequence of a nanobody Sequence ID 174: Amino acid sequence of the complementarity-determining region Sequence ID 175: Amino acid sequence of a nanobody Sequence ID 176: Amino acid sequence of a nanobody Sequence ID 177: Amino acid sequence of a nanobody Sequence ID 178: Amino acid sequence of the complementarity-determining region Sequence ID 179: Amino acid sequence of a nanobody Sequence ID 180: Amino acid sequence of the complementarity-determining region Sequence ID 181: Amino acid sequence of the complementarity-determining region Sequence ID 182: Amino acid sequence of the complementarity-determining region Sequence ID 183: Amino acid sequence of a nanobody Sequence ID 185: Amino acid sequence of a nanobody Sequence ID 186: Amino acid sequence of a nanobody Sequence ID 187: Amino acid sequence of the complementarity-determining region Sequence ID 188: Amino acid sequence of the complementarity-determining region Sequence ID 189: Amino acid sequence of the complementarity-determining region Sequence ID 190: Amino acid sequence of a nanobody Sequence ID 191: Amino acid sequence of the complementarity-determining region Sequence ID 192: Amino acid sequence of the complementarity-determining region Sequence ID 193: Amino acid sequence of the complementarity-determining region Sequence ID 194: Amino acid sequence of a nanobody Sequence ID 195: Amino acid sequence of the complementarity-determining region Sequence ID 196: Amino acid sequence of the complementarity-determining region Sequence ID 197: Amino acid sequence of the complementarity-determining region Sequence ID 198: Amino acid sequence of a nanobody Sequence ID 199: Amino acid sequence of the complementarity-determining region Sequence ID 200: Amino acid sequence of a nanobody

[0554] Sequence ID 201: Amino acid sequence of the complementarity-determining region Sequence ID 202: Amino acid sequence of the complementarity-determining region Sequence ID 203: Amino acid sequence of the complementarity-determining region Sequence ID 204: Amino acid sequence of a nanobody Sequence ID 205: Amino acid sequence of the complementarity-determining region Sequence ID 206: Amino acid sequence of the complementarity-determining region Sequence ID 207: Amino acid sequence of a nanobody Sequence ID 208: Amino acid sequence of the complementarity-determining region Sequence ID 209: Amino acid sequence of the complementarity-determining region Sequence ID 210: Amino acid sequence of the complementarity-determining region Sequence ID 211: Amino acid sequence of a nanobody Sequence ID 212: Amino acid sequence of the complementarity-determining region Sequence ID 213: Amino acid sequence of a nanobody Sequence ID 214: Amino acid sequence of a nanobody Sequence ID 215: Amino acid sequence of the complementarity-determining region Sequence ID 216: Amino acid sequence of the complementarity-determining region Sequence ID 217: Amino acid sequence of the complementarity-determining region Sequence ID 218: Amino acid sequence of a nanobody Sequence ID 219: Amino acid sequence of the complementarity-determining region Sequence ID 220: Amino acid sequence of the complementarity-determining region Sequence ID 221: Amino acid sequence of the complementarity-determining region Sequence ID 222: Amino acid sequence of a nanobody Sequence ID 223: Amino acid sequence of the complementarity-determining region Sequence ID 224: Amino acid sequence of the complementarity-determining region Sequence ID 225: Amino acid sequence of the complementarity-determining region Sequence ID 226: Amino acid sequence of a nanobody Sequence ID 227: Amino acid sequence of the complementarity-determining region Sequence ID 228: Amino acid sequence of the complementarity-determining region Sequence ID 229: Amino acid sequence of the complementarity-determining region Sequence ID 230: Amino acid sequence of a nanobody Sequence ID 231: Amino acid sequence of the complementarity-determining region Sequence ID 232: Amino acid sequence of the complementarity-determining region Sequence ID 233: Amino acid sequence of a nanobody Sequence ID 234: Amino acid sequence of the complementarity-determining region Sequence ID 235: Amino acid sequence of the complementarity-determining region Sequence ID 236: Amino acid sequence of the complementarity-determining region Sequence ID 237: Amino acid sequence of a nanobody Sequence ID 238: Amino acid sequence of the complementarity-determining region Sequence ID 239: Amino acid sequence of the complementarity-determining region Sequence ID 240: Amino acid sequence of the complementarity-determining region Sequence ID 241: Amino acid sequence of a nanobody Sequence ID 242: Amino acid sequence of the complementarity-determining region Sequence ID 243: Amino acid sequence of the complementarity-determining region Sequence ID 244: Amino acid sequence of the complementarity-determining region Sequence ID 245: Amino acid sequence of a nanobody Sequence ID 246: Amino acid sequence of the complementarity-determining region Sequence ID 247: Amino acid sequence of the complementarity-determining region Sequence ID 248: Amino acid sequence of the complementarity-determining region Sequence ID 249: Amino acid sequence of a nanobody Sequence ID 250: Amino acid sequence of the complementarity-determining region Sequence ID 251: Amino acid sequence of the complementarity-determining region Sequence ID 252: Amino acid sequence of the complementarity-determining region Sequence ID 253: Amino acid sequence of a nanobody Sequence ID 254: Amino acid sequence of the complementarity-determining region Sequence ID 255: Amino acid sequence of the complementarity-determining region Sequence ID 256: Amino acid sequence of the complementarity-determining region Sequence ID 257: Amino acid sequence of a nanobody Sequence ID 258: Amino acid sequence of the complementarity-determining region Sequence ID 259: Amino acid sequence of the complementarity-determining region Sequence ID 260: Amino acid sequence of the complementarity-determining region Sequence ID 261: Amino acid sequence of a nanobody Sequence ID 262: Amino acid sequence of the complementarity-determining region Sequence ID 263: Amino acid sequence of the complementarity-determining region Sequence ID 264: Amino acid sequence of the complementarity-determining region Sequence ID 265: Amino acid sequence of a nanobody Sequence ID 266: Amino acid sequence of the complementarity-determining region Sequence ID 267: Amino acid sequence of a nanobody Sequence ID 268: Amino acid sequence of a nanobody Sequence ID 269: Amino acid sequence of a nanobody Sequence ID 270: Amino acid sequence of the complementarity-determining region Sequence ID 271: Amino acid sequence of a nanobody Sequence ID 272: Amino acid sequence of a nanobody Sequence ID 273: Amino acid sequence of a nanobody Sequence ID 274: Amino acid sequence of the complementarity-determining region Sequence ID 275: Amino acid sequence of the complementarity-determining region Sequence ID 276: Amino acid sequence of the complementarity-determining region Sequence ID 277: Amino acid sequence of a nanobody Sequence ID 278: Amino acid sequence of a nanobody Sequence ID 279: Amino acid sequence of a nanobody Sequence ID 280: Amino acid sequence of the complementarity-determining region Sequence ID 281: Amino acid sequence of a nanobody Sequence ID 282: Amino acid sequence of the complementarity-determining region Sequence ID 283: Amino acid sequence of a nanobody Sequence ID 284: Amino acid sequence of a nanobody Sequence ID 285: Amino acid sequence of the complementarity-determining region Sequence ID 286: Amino acid sequence of a nanobody Sequence ID 287: Amino acid sequence of the complementarity-determining region Sequence ID 289: Amino acid sequence of the complementarity-determining region Sequence ID 290: Amino acid sequence of a nanobody Sequence ID 291: Amino acid sequence of the complementarity-determining region Sequence ID 292: Amino acid sequence of a nanobody Sequence ID 293: Amino acid sequence of the complementarity-determining region Sequence ID 294: Amino acid sequence of the complementarity-determining region Sequence ID 295: Amino acid sequence of a nanobody Sequence ID 296: Amino acid sequence of the complementarity-determining region Sequence ID 297: Amino acid sequence of the complementarity-determining region Sequence ID 298: Amino acid sequence of the complementarity-determining region Sequence ID 299: Amino acid sequence of a nanobody Xaa, ranked 39th, could be any natural amino acid. Sequence ID 300: Amino acid sequence of the complementarity-determining region

[0555] Sequence ID 301: Amino acid sequence of the complementarity-determining region Sequence ID 302: Amino acid sequence of the complementarity-determining region Sequence ID 303: Amino acid sequence of a nanobody The third-ranked Xaa could be any natural amino acid. Sequence ID 304: Amino acid sequence of the complementarity-determining region Sequence ID 305: Amino acid sequence of the complementarity-determining region Sequence ID 306: Amino acid sequence of the complementarity-determining region Sequence ID 307: Amino acid sequence of a nanobody Xaa, ranked 39th, could be any natural amino acid. Sequence ID 308: Amino acid sequence of the complementarity-determining region Sequence ID 309: Amino acid sequence of the complementarity-determining region Sequence ID 310: Amino acid sequence of the complementarity-determining region Sequence ID 311: Amino acid sequence of a nanobody The third-ranked Xaa could be any natural amino acid. Sequence ID 312: Amino acid sequence of the complementarity-determining region Sequence ID 313: Amino acid sequence of the complementarity-determining region Sequence ID 314: Amino acid sequence of the complementarity-determining region Sequence ID 315: Amino acid sequence of a nanobody Sequence ID 316: Amino acid sequence of the complementarity-determining region Sequence ID 317: Amino acid sequence of the complementarity-determining region Sequence ID 318: Amino acid sequence of the complementarity-determining region Sequence ID 319: Amino acid sequence of a nanobody Sequence ID 320: Amino acid sequence of the complementarity-determining region Sequence ID 321: Amino acid sequence of the complementarity-determining region Sequence ID 322: Amino acid sequence of the complementarity-determining region Sequence ID 323: Amino acid sequence of a nanobody Sequence ID 324: Amino acid sequence of the complementarity-determining region Sequence ID 325: Amino acid sequence of the complementarity-determining region Sequence ID 326: Amino acid sequence of a nanobody Sequence ID 327: Amino acid sequence of the complementarity-determining region Sequence ID 328: Amino acid sequence of the complementarity-determining region Sequence ID 329: Amino acid sequence of a nanobody Sequence ID 331: Amino acid sequence of a nanobody Sequence ID 332: Amino acid sequence of the complementarity-determining region Sequence ID 333: Amino acid sequence of a nanobody Sequence ID 334: Amino acid sequence of a nanobody Sequence ID 336: Amino acid sequence of the complementarity-determining region Sequence ID 337: Amino acid sequence of the complementarity-determining region Sequence ID 338: Amino acid sequence of a nanobody Sequence ID 339: Amino acid sequence of the complementarity-determining region Sequence ID 340: Amino acid sequence of a nanobody Sequence ID 341: Amino acid sequence of a nanobody Sequence ID 342: Amino acid sequence of a nanobody Sequence ID 343: Amino acid sequence of the complementarity-determining region Sequence ID 344: Amino acid sequence of the complementarity-determining region Sequence ID 345: Amino acid sequence of the complementarity-determining region Sequence ID 346: Amino acid sequence of a nanobody Sequence ID 347: Amino acid sequence of the complementarity-determining region Sequence ID 348: Amino acid sequence of the complementarity-determining region Sequence ID 349: Amino acid sequence of the complementarity-determining region Sequence ID 350: Amino acid sequence of a nanobody Sequence ID 351: Amino acid sequence of the complementarity-determining region Sequence ID 352: Amino acid sequence of the complementarity-determining region Sequence ID 353: Amino acid sequence of the complementarity-determining region Sequence ID 354: Amino acid sequence of a nanobody Sequence ID 355: Amino acid sequence of a nanobody Sequence ID 356: Amino acid sequence of the complementarity-determining region Sequence ID 357: Amino acid sequence of a nanobody Sequence ID 358: Amino acid sequence of the complementarity-determining region Sequence ID 359: Amino acid sequence of the complementarity-determining region Sequence ID 360: Amino acid sequence of the complementarity-determining region Sequence ID 361: Amino acid sequence of a nanobody Sequence ID 362: Amino acid sequence of the complementarity-determining region Sequence ID 363: Amino acid sequence of a nanobody Sequence ID 364: Amino acid sequence of a nanobody Sequence ID 365: Amino acid sequence of the complementarity-determining region Sequence ID 366: Amino acid sequence of the complementarity-determining region Sequence ID 367: Amino acid sequence of a nanobody Sequence ID 368: Amino acid sequence of the complementarity-determining region Sequence ID 369: Amino acid sequence of the complementarity-determining region Sequence ID 370: Amino acid sequence of the complementarity-determining region Sequence ID 371: Amino acid sequence of a nanobody Sequence ID 372: Amino acid sequence of the complementarity-determining region Sequence ID 373: Amino acid sequence of a nanobody Sequence ID 374: Amino acid sequence of the complementarity-determining region Sequence ID 375: Amino acid sequence of the complementarity-determining region Sequence ID 376: Amino acid sequence of a nanobody Sequence ID 377: Amino acid sequence of the complementarity-determining region Sequence ID 378: Amino acid sequence of the complementarity-determining region Sequence ID 379: Amino acid sequence of the complementarity-determining region Sequence ID 380: Amino acid sequence of a nanobody Sequence ID 381: Amino acid sequence of the complementarity-determining region Sequence ID 382: Amino acid sequence of the complementarity-determining region Sequence ID 383: Amino acid sequence of the complementarity-determining region Sequence ID 384: Amino acid sequence of a nanobody Sequence ID 385: Amino acid sequence of the complementarity-determining region Sequence ID 386: Amino acid sequence of a nanobody Sequence ID 387: Amino acid sequence of the complementarity-determining region Sequence ID 388: Amino acid sequence of the complementarity-determining region Sequence ID 389: Amino acid sequence of the complementarity-determining region Sequence ID 390: Amino acid sequence of a nanobody Sequence ID 391: Amino acid sequence of the complementarity-determining region Sequence ID 392: Amino acid sequence of a nanobody Sequence ID 393: Amino acid sequence of a nanobody Sequence ID 394: Amino acid sequence of the complementarity-determining region Sequence ID 395: Amino acid sequence of the complementarity-determining region Sequence ID 396: Amino acid sequence of the complementarity-determining region Sequence ID 397: Amino acid sequence of a nanobody Sequence ID 398: Amino acid sequence of the complementarity-determining region Sequence ID 399: Amino acid sequence of the complementarity-determining region Sequence ID 400: Amino acid sequence of the complementarity-determining region

[0556] Sequence ID 401: Amino acid sequence of a nanobody Sequence ID 402: Amino acid sequence of the complementarity-determining region Sequence ID 403: Amino acid sequence of the complementarity-determining region Sequence ID 404: Amino acid sequence of the complementarity-determining region Sequence ID 405: Amino acid sequence of a nanobody Sequence ID 406: Amino acid sequence of the complementarity-determining region Sequence ID 407: Amino acid sequence of the complementarity-determining region Sequence ID 408: Amino acid sequence of the complementarity-determining region Sequence ID 409: Type 2 chitin-binding domain (ChtBD2) The Xaa in positions 2-12 could be any natural amino acid. The Xaa at positions 43-47 could be any natural amino acid. The Xaa at positions 54-62 could be any natural amino acid. The Xaa at positions 87-98 could be any natural amino acid. The Xaa at positions 116-121 could be any natural amino acid. Sequence ID 410: Type 2 chitin-binding domain (ChtBD2) Sequence ID 411: Amino acid sequence of a nanobody Sequence ID 412: Amino acid sequence of the complementarity-determining region Sequence ID 413: Amino acid sequence of the complementarity-determining region Sequence ID 414: Amino acid sequence of the complementarity-determining region Sequence ID 415: Amino acid sequence of a nanobody Sequence ID 416: Amino acid sequence of a nanobody Sequence ID 417: Amino acid sequence of a nanobody Sequence ID 418: Amino acid sequence of the complementarity-determining region Sequence ID 419: Amino acid sequence of the complementarity-determining region Sequence ID 420: Amino acid sequence of the complementarity-determining region Sequence ID 421: Amino acid sequence of a nanobody Sequence ID 422: Amino acid sequence of the complementarity-determining region Sequence ID 423: Amino acid sequence of the complementarity-determining region Sequence ID 424: Amino acid sequence of the complementarity-determining region Sequence ID 425: Amino acid sequence of a nanobody Sequence ID 426: Amino acid sequence of the complementarity-determining region Sequence ID 427: Amino acid sequence of the complementarity-determining region Sequence ID 428: Amino acid sequence of the complementarity-determining region Sequence ID 429: Amino acid sequence of a nanobody Sequence ID 430: Amino acid sequence of the complementarity-determining region Sequence ID 431: Amino acid sequence of a nanobody Sequence ID 432: Amino acid sequence of the complementarity-determining region Sequence ID 433: Amino acid sequence of the complementarity-determining region Sequence ID 434: Amino acid sequence of the complementarity-determining region Sequence ID 435: Amino acid sequence of a nanobody Sequence ID 436: Amino acid sequence of the complementarity-determining region Sequence ID 437: Amino acid sequence of the complementarity-determining region Sequence ID 438: Amino acid sequence of a nanobody Sequence ID 439: Amino acid sequence of the complementarity-determining region Sequence ID 440: Amino acid sequence of the complementarity-determining region Sequence ID 441: Amino acid sequence of the complementarity-determining region Sequence ID 442: Amino acid sequence of a nanobody Sequence ID 443: Amino acid sequence of the complementarity-determining region Sequence ID 444: Amino acid sequence of the complementarity-determining region Sequence ID 445: Amino acid sequence of the complementarity-determining region Sequence ID 446: Amino acid sequence of a nanobody Sequence ID 447: Amino acid sequence of the complementarity-determining region Sequence ID 448: Amino acid sequence of the complementarity-determining region Sequence ID 449: Amino acid sequence of the complementarity-determining region Sequence ID 450: Amino acid sequence of a nanobody Sequence ID 451: Amino acid sequence of the complementarity-determining region Sequence ID 452: Amino acid sequence of the complementarity-determining region Sequence ID 453: Amino acid sequence of a nanobody Sequence ID 454: Amino acid sequence of the complementarity-determining region Sequence ID 455: Amino acid sequence of the complementarity-determining region Sequence ID 456: Amino acid sequence of the complementarity-determining region Sequence ID 457: Amino acid sequence of a nanobody Sequence ID 458: Amino acid sequence of the complementarity-determining region Sequence ID 459: Amino acid sequence of the complementarity-determining region Sequence ID 460: Amino acid sequence of the complementarity-determining region Sequence ID 461: Amino acid sequence of a nanobody Sequence ID 462: Amino acid sequence of the complementarity-determining region Sequence ID 463: Amino acid sequence of the complementarity-determining region Sequence ID 464: Amino acid sequence of the complementarity-determining region Sequence ID 465: Amino acid sequence of a nanobody Sequence ID 466: Amino acid sequence of the complementarity-determining region Sequence ID 467: Amino acid sequence of the complementarity-determining region Sequence ID 468: Amino acid sequence of the complementarity-determining region Sequence ID 469: Amino acid sequence of a nanobody Sequence ID 470: Amino acid sequence of the complementarity-determining region Sequence ID 471: Amino acid sequence of the complementarity-determining region Sequence ID 472: Amino acid sequence of the complementarity-determining region Sequence ID 473: Amino acid sequence of a nanobody Sequence ID 474: Amino acid sequence of the complementarity-determining region Sequence ID 475: Amino acid sequence of the complementarity-determining region Sequence ID 476: Amino acid sequence of the complementarity-determining region Sequence ID 477: Amino acid sequence of a nanobody Sequence ID 478: Amino acid sequence of the complementarity-determining region Sequence ID 479: Amino acid sequence of the complementarity-determining region Sequence ID 480: Amino acid sequence of the complementarity-determining region Sequence ID 481: Amino acid sequence of a nanobody Sequence ID 482: Amino acid sequence of the complementarity-determining region Sequence ID 483: Amino acid sequence of the complementarity-determining region Sequence ID 484: Amino acid sequence of a nanobody Sequence ID 485: Amino acid sequence of the complementarity-determining region Sequence ID 486: Amino acid sequence of the complementarity-determining region Sequence ID 487: Amino acid sequence of the complementarity-determining region Sequence ID 488: Amino acid sequence of a nanobody Sequence ID 489: Amino acid sequence of the complementarity-determining region Sequence ID 490: Amino acid sequence of the complementarity-determining region Sequence ID 491: Amino acid sequence of the complementarity-determining region Sequence ID 492: Amino acid sequence of a nanobody Sequence ID 493: Amino acid sequence of the complementarity-determining region Sequence ID 494: Amino acid sequence of the complementarity-determining region Sequence ID 495: Amino acid sequence of the complementarity-determining region Sequence ID 496: Amino acid sequence of a nanobody Sequence ID 497: Amino acid sequence of the complementarity-determining region Sequence ID 498: Amino acid sequence of the complementarity-determining region Sequence ID 499: Amino acid sequence of a nanobody Sequence ID 500: Amino acid sequence of the complementarity-determining region

[0557] Sequence ID 501: Amino acid sequence of the complementarity-determining region Sequence ID 502: Amino acid sequence of the complementarity-determining region Sequence ID 503: Amino acid sequence of a nanobody Sequence ID 504: Amino acid sequence of the complementarity-determining region Sequence ID 505: Amino acid sequence of the complementarity-determining region Sequence ID 506: Amino acid sequence of the complementarity-determining region Sequence ID 507: Amino acid sequence of a nanobody Sequence ID 508: Amino acid sequence of the complementarity-determining region Sequence ID 509: Amino acid sequence of the complementarity-determining region Sequence ID 510: Amino acid sequence of the complementarity-determining region Sequence ID 511: Amino acid sequence of a nanobody Sequence ID 512: Amino acid sequence of the complementarity-determining region Sequence ID 513: Amino acid sequence of the complementarity-determining region Sequence ID 514: Amino acid sequence of the complementarity-determining region Sequence ID 515: Amino acid sequence of a nanobody Sequence ID 516: Amino acid sequence of a nanobody Sequence ID 517: Amino acid sequence of the complementarity-determining region Sequence ID 518: Amino acid sequence of a nanobody Sequence ID 519: Amino acid sequence of a nanobody Sequence ID 520: Amino acid sequence of a nanobody Sequence ID 521: Amino acid sequence of the complementarity-determining region Sequence ID 522: Amino acid sequence of the complementarity-determining region Sequence ID 523: Amino acid sequence of the complementarity-determining region Sequence ID 524: Amino acid sequence of a nanobody Sequence ID 525: Amino acid sequence of a nanobody Sequence ID 526: Amino acid sequence of the complementarity-determining region Sequence ID 527: Amino acid sequence of a nanobody Sequence ID 528: Amino acid sequence of a nanobody Sequence ID 529: Amino acid sequence of a nanobody Sequence ID 530: Amino acid sequence of a nanobody Sequence ID 531: Amino acid sequence of the complementarity-determining region Sequence ID 532: Amino acid sequence of the complementarity-determining region Sequence ID 533: Amino acid sequence of the complementarity-determining region Sequence ID 534: Amino acid sequence of a nanobody Sequence ID 535: Amino acid sequence of a nanobody Sequence ID 536: Amino acid sequence of a nanobody Sequence ID 537: Amino acid sequence of a nanobody Sequence ID 538: Amino acid sequence of a nanobody Sequence ID 539: Amino acid sequence of the complementarity-determining region Sequence ID 540: Amino acid sequence of the complementarity-determining region Sequence ID 541: Amino acid sequence of the complementarity-determining region Sequence ID 542: Amino acid sequence of a nanobody Sequence ID 543: Amino acid sequence of a nanobody Sequence ID 544: Amino acid sequence of a nanobody Sequence ID 545: Amino acid sequence of the complementarity-determining region Sequence ID 546: Amino acid sequence of a nanobody Sequence ID 547: Amino acid sequence of the complementarity-determining region Sequence ID 548: Amino acid sequence of the complementarity-determining region Sequence ID 549: Amino acid sequence of the complementarity-determining region Sequence ID 550: Amino acid sequence of a nanobody Sequence ID 551: Amino acid sequence of a nanobody Sequence ID 552: Amino acid sequence of the complementarity-determining region Sequence ID 553: Amino acid sequence of the complementarity-determining region Sequence ID 554: Amino acid sequence of the complementarity-determining region Sequence ID 555: Amino acid sequence of a nanobody Sequence ID 556: Amino acid sequence of the complementarity-determining region Sequence ID 557: Amino acid sequence of the complementarity-determining region Sequence ID 558: Amino acid sequence of the complementarity-determining region Sequence ID 559: Amino acid sequence of a nanobody Sequence ID 560: Amino acid sequence of a nanobody Sequence ID 561: Amino acid sequence of the complementarity-determining region Sequence ID 562: Amino acid sequence of the complementarity-determining region Sequence ID 563: Amino acid sequence of the complementarity-determining region Sequence ID 564: Amino acid sequence of a nanobody Sequence ID 565: Amino acid sequence of the complementarity-determining region Sequence ID 566: Amino acid sequence of the complementarity-determining region Sequence ID 567: Amino acid sequence of the complementarity-determining region Sequence ID 568: Amino acid sequence of a nanobody Sequence ID 569: Amino acid sequence of the complementarity-determining region Sequence ID 570: Amino acid sequence of the complementarity-determining region Sequence ID 571: Amino acid sequence of the complementarity-determining region Sequence ID 572: Amino acid sequence of a nanobody Sequence ID 573: Amino acid sequence of the complementarity-determining region Sequence ID 574: Amino acid sequence of the complementarity-determining region Sequence ID 575: Amino acid sequence of the complementarity-determining region Sequence ID 576: Amino acid sequence of a nanobody Sequence ID 577: Amino acid sequence of the complementarity-determining region Sequence ID 578: Amino acid sequence of the complementarity-determining region Sequence ID 579: Amino acid sequence of a nanobody Sequence ID 580: Amino acid sequence of the complementarity-determining region Sequence ID 581: Amino acid sequence of the complementarity-determining region Sequence ID 582: Amino acid sequence of the complementarity-determining region Sequence ID 583: Amino acid sequence of a nanobody Sequence ID 584: Amino acid sequence of the complementarity-determining region Sequence ID 585: Amino acid sequence of the complementarity-determining region Sequence ID 586: Amino acid sequence of the complementarity-determining region Sequence ID 587: Amino acid sequence of a nanobody Sequence ID 588: Amino acid sequence of the complementarity-determining region Sequence ID 589: Amino acid sequence of the complementarity-determining region Sequence ID 590: Amino acid sequence of the complementarity-determining region Sequence ID 591: Amino acid sequence of a nanobody Sequence ID 592: Amino acid sequence of the complementarity-determining region Sequence ID 593: Amino acid sequence of a nanobody Sequence ID 594: Amino acid sequence of a nanobody Sequence ID 595: Amino acid sequence of the complementarity-determining region Sequence ID 596: Amino acid sequence of a nanobody

Claims

1. An anti-chitin deacetylase nanobody comprising complementarity-determining regions (CDRs) represented by SEQ ID NOs: 351, 347 and 356, SEQ ID NOs: 387-389, or SEQ ID NOs: 402-404, which are continuously arranged on the nanobody in the direction from N to C, wherein the nanobody has insect control activity, and the insect is selected from the group consisting of Helicoverpa armigera, Myzus persicae, Bemisia tabaci and Aphis gossypii.

2. The nanobody according to claim 1, further comprising a heterotoxin portion.

3. The nanobody according to claim 1 or 2, formulated for delivery by spraying, irrigation and / or fumigation.

4. The nanobody according to any one of claims 1 to 3, wherein the amino acid sequence of the nanobody includes sequence number 355, 386, or 401.

5. A polynucleotide encoding a nanobody according to any one of claims 1 to 4.

6. A host cell comprising a nanobody according to any one of claims 1 to 4, or a polynucleotide encoding the same.

7. A method for producing an anti-chitin deacetylase nanobody, comprising introducing the polynucleotide described in Claim 5 into a host cell in vitro.

8. A method for controlling insects, comprising contacting an insect with a nanobody according to any one of claims 1 to 4, or a polynucleotide encoding the same, or a host cell expressing the same.

9. A plant comprising a nanobody according to any one of claims 1 to 4, or a polynucleotide encoding the same.

10. An industrial product comprising a nanobody according to any one of claims 1 to 4.

Images