Medicine for improving thyroid-associated ophthalmopathy and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a medicine for improving thyroid-related eye diseases and application of the medicine. The single-chain antibody provided by the invention has small molecular weight and good physiological activity, and can effectively inhibit the activity and function of IGF1R mediated by IGF1, thereby inhibiting cell proliferation. According to the medicine prepared from the recombinant adeno-associated virus, the treatment cost can be remarkably reduced, side effects caused by repeated injection of antibody medicines are avoided, the safety is very high, and a new clinical treatment method and strategy are provided for inhibiting pathological phenotypes of thyroid-associated ophthalmopathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a drug for improving thyroid-related eye diseases and an application thereof. Background Art

[0002] Thyroid-associated ophthalmopathy (TAO; thyroid eye disease, TED), also known as Graves' ophthalmopathy (GO), is an organ-specific autoimmune disease closely related to thyroid disease. It ranks first in the incidence of orbital diseases in adults and is the most common extrathyroidal manifestation of diffuse toxic goiter (GD), accounting for 25% to 40% of GD cases. It also occurs in 2% of patients with chronic lymphocytic thyroiditis, a small number of patients with hypothyroidism, and individuals with normal thyroid function. The clinical manifestations of TAO are complex and can cause unilateral or bilateral eyelid retraction, proptosis, diplopia, restrictive strabismus, exposure keratopathy, and dysthyroid optic neuropathy (DON), severely impacting patients' quality of life.

[0003] Thyroid eye disease (TED) is an autoimmune disease caused by hyperthyroidism. It is caused by autoantigens activating a signaling complex mediated by the IGF-1R (Insulin-like growth factor 1 receptor) on cells within the orbit, leading to the body's own immune system attacking tissues around and behind the eyes. IGF-1R is a tetrameric transmembrane receptor tyrosine kinase found on the surface of human cells and serves as the cell surface receptor for the hormone insulin-like growth factor 1 (IGF-1). Studies have shown that IGF-1R is widely expressed in normal human tissues and is highly expressed in TED. IGF-1R activation stimulates cell proliferation, survival, transformation, metastasis, and angiogenesis, while inhibition of IGF-1R can alleviate and improve TED symptoms.

[0004] Tetumumab is a fully humanized monoclonal antibody that binds to the extracellular subunit domain of IGF-IR. It has been used as a treatment for a variety of solid tumors and lymphomas and was first approved in the United States in 2020 for the treatment of acute and chronic TED. For most indications, maintenance therapy requires repeated infusions, for example, eight total infusions, with the first recommended dose being 10 mg / kg, followed by doubling the dose to 20 mg / kg and seven additional infusions, each three weeks apart. In clinical treatment, infusion times typically exceed 60 to 90 minutes, placing a significant treatment burden on patients.

[0005] Gene therapy drugs have become the focus of ophthalmic therapeutic drugs in recent years because they can reduce the frequency of treatment and reduce the burden of drugs. rAAV vectors have become the most widely used viruses in clinical trials of gene therapy in the world due to their diverse types, extremely low immunogenicity, high safety, wide range of host cells, strong diffusion ability, and long time of gene expression in vivo. At present, rAAV vectors have been used to mediate gene fragments encoding monoclonal antibodies (mAbs) to obtain long-term expression in the body, thereby reducing the number of administrations of traditional monoclonal antibody injections. Studies have shown that AAV-delivered single-chain antibodies against VEGF can significantly inhibit fundus angiogenesis. They are not truncated forms of a complete monoclonal antibody and have been proven to be effective in in vitro and in vivo experiments. The significant reduction in the frequency of administration through rAAV vector delivery provides a reference for the present invention. Although the variable region of a complete monoclonal antibody is the key region for specific binding to the antibody, the biological activity of the scFv fragment of the IGF1R antibody is still unclear. The single-chain antibody provided by the present invention has a small molecular weight and good biological activity, and can effectively inhibit the activity and function of IGF1R mediated by IGF1, thereby inhibiting cell proliferation. The present invention utilizes recombinant adeno-associated virus to carry the scFv fragment of the anti-IGF1R antibody to bind to IGF1R, inhibit cell proliferation, avoid the side effects caused by repeated injections of antibody drugs, and provide a new clinical treatment method and strategy for inhibiting the pathological phenotype of thyroid-related eye disease. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a single-chain antibody and its encoding nucleic acid for use in the preparation of a drug for improving thyroid-related eye diseases.

[0007] In the IGF1R single-chain antibody provided by the present invention:

[0008] The three CDR regions of the light chain are selected from CDR-L1 with an amino acid sequence as set forth in SEQ ID NO: 1; CDR-L2 with an amino acid sequence as set forth in SEQ ID NO: 2; and CDR-L3 with an amino acid sequence as set forth in SEQ ID NO: 3; or sequences thereof having at least 80% (preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity;

[0009] And the heavy chain comprises three CDR regions, wherein the amino acid sequence of at least one of the CDR regions has the amino acid sequence of CDR-H1 as shown in SEQ ID NO:4; the amino acid sequence of CDR-H2 as shown in SEQ ID NO:5 or SEQ ID NO:6; and the amino acid sequence of CDR-H3 as shown in SEQ ID NO:8 or a sequence having at least 80% (preferably 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity thereto.

[0010] In some specific embodiments, the IGF1R single-chain antibody comprises a light chain variable region, a linker, and a heavy chain variable region; wherein the amino acid sequence of the light chain variable region is as shown in 21 bp to 128 bp of SEQ ID NO: 7;

[0011] The amino acid sequence of the heavy chain variable region is shown from bp 149 to bp 266 of SEQ ID NO: 7.

[0012] In a specific embodiment, the IGF1R single-chain antibody includes a secretory signal peptide, a light chain variable region, a linker and a heavy chain variable region from the N-terminus to the C-terminus. The addition of the secretory signal peptide of the present invention improves the gene expression level and more effectively inhibits cell proliferation. The expression of anti-antiIGF1RscFv is delivered through the AAV adeno-associated virus vector, which can effectively inhibit cell proliferation in the long term and reduce the frequency of medication. At the same time, it can also effectively improve the patient's treatment compliance and reduce the risk of treatment. In the present invention, the amino acid sequence of the secretory signal peptide is shown in SEQ ID NO: 7, 1bp-20bp. Experiments show that compared with other secretory signal peptides, this signal peptide is more conducive to improving the expression level of the IGF1R single-chain antibody. In the present invention, the linker is used to connect the two variable regions of the single-chain antibody, which can be (G4S) n , wherein n=an integer from 2 to 10. In some preferred embodiments, the linker is (G4S)4.

[0013] Furthermore, the present invention also provides a nucleic acid encoding the IGF1R single-chain antibody as described above.

[0014] The nucleic acids of the present invention include nucleic acids encoding light chain variable regions and nucleic acids encoding heavy chain variable regions. The nucleic acids may be DNA, RNA, cDNA or PNA, which may be single-stranded or double-stranded. The nucleic acids may include nucleotide sequences having different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). The nucleic acids may be linear or circular in topology. The nucleic acids may be, for example, part of a vector (e.g., an expression or cloning vector), or a combination of one or more fragments. The nucleic acids may be obtained directly from natural sources, or may be prepared with the assistance of recombination, enzymatic methods or chemical techniques. The RNA form may be mRNA obtained by gene transcription, etc.

[0015] The nucleic acid encoding the IGF1R single-chain antibody of the present invention comprises

[0016] (1) the nucleic acid sequence encoding CDR-L1 is as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence having at least 80% sequence identity thereto;

[0017] (2) a nucleic acid encoding CDR-L2 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a sequence having at least 80% sequence identity thereto;

[0018] (3) the nucleic acid encoding CDR-L3 is as shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, or a sequence having at least 80% sequence identity thereto;

[0019] (4) the nucleic acid encoding CDR-H1 is as shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or a sequence having at least 80% sequence identity thereto;

[0020] (5) the nucleic acid encoding CDR-H2 is as shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, or a sequence having at least 80% sequence identity thereto;

[0021] (6) The nucleic acid encoding CDR-H3 is as shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, or a sequence having at least 80% sequence identity thereto.

[0022] In some embodiments, the nucleic acid sequence encoding the light chain variable region is shown as SEQ ID NO:29 or SEQ ID NO:30; the nucleic acid sequence encoding the heavy chain variable region is shown as SEQ ID NO:31 or SEQ ID NO:32.

[0023] More specifically, the nucleic acid sequence encoding the IGF1R single-chain antibody (including the signal peptide and antibody amino acid sequence) of the present invention is shown in SEQ ID NO: 38 or SEQ ID NO: 39.

[0024] In a preferred embodiment of the present invention, the nucleic acid is DNA, which has been codon-optimized.

[0025] Compared with unoptimized nucleic acid sequences or other optimized solutions, the nucleic acid used in the present invention can be better expressed in the eye, thereby exerting a more effective therapeutic effect.

[0026] Furthermore, the present invention also provides an expression unit, which includes a promoter and a nucleic acid as described above. The expression unit may also include a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator. The regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc. In an embodiment of the present invention, the promoter is a chicken β-actin promoter (CBA). Compared with other promoters, CBA is more conducive to increasing the expression level of the encoded IGF1R single-chain antibody, thereby improving the effect of inhibiting cell proliferation.

[0027] The present invention also provides a recombinant vector comprising the nucleic acid or expression unit as described above.

[0028] In some embodiments, the recombinant vector comprises, in sequence, a 5' terminal inverted repeat sequence, a chicken β-actin CBA promoter sequence, a Kozak sequence, the nucleic acid of the IGF1R single-chain antibody of the present invention, bGH, SV40, hGH, a polyA signal sequence, and a 3' terminal inverted repeat sequence, wherein the chicken β-actin CBA promoter sequence is shown in SEQ ID NO: 40, and the Kozak sequence nucleic acid sequence is shown in SEQ ID NO: 33.

[0029] The vector backbone of the present invention may be derived from plants, animals, bacteria, fungi, phages, or viruses, but the present invention is not limited thereto. The viral vectors include adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, or lentiviral vectors. The present invention uses an adeno-associated virus (AAV) vector as a backbone for the expression of single-chain antibodies. In some specific embodiments, the backbone vector of the recombinant vector is pAAV2.

[0030] In some embodiments, the ITRs of the recombinant vector are of AAV2 and AAV8 serotypes, and the capsid backbone vector is one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh9, AAVrh10, and AAVrh74.

[0031] The present invention also provides a plasmid combination, which includes the recombinant vector as described above and a helper plasmid for virus packaging. In the present invention, the helper plasmid for virus packaging includes a Helper plasmid and a Rep-Cap2 plasmid.

[0032] The present invention also provides a host, the genome of which is integrated with the aforementioned nucleic acid; or is transfected or transformed with the aforementioned recombinant vector or the aforementioned plasmid combination.

[0033] The host described in the present invention is used for the preservation or amplification of the nucleic acid described in the present invention, and can also be used for the construction and expression of adeno-associated viruses, which is not limited by the present invention. In the present invention, the host is a prokaryotic organism or an animal cell. The prokaryotic organism includes but is not limited to Escherichia coli, and the animal cell is selected from at least one of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9 and FD-CHOS. In some specific embodiments of the present invention, HEK293T cells are used as the host for virus packaging.

[0034] The present invention also provides a recombinant adeno-associated virus obtained by culturing the host of the present invention.

[0035] The steps of constructing the recombinant adeno-associated virus described in the present invention include: constructing an AAV2-antiIGF1RscFv recombinant plasmid; verifying the expression of antiIGF1R by in vitro cell plasmid transfection; packaging the AAV2-antiIGF1RscFv virus; and harvesting the recombinant adeno-associated virus by in vitro cell virus transfection.

[0036] More specifically, the construction method includes:

[0037] S1, the construction of recombinant plasmid pAAV2-antiIGF1RscFv, the steps are as follows, (1) gene synthesis antiIGF1RscFv gene fragment; (2) the application BglII and BsrGI restriction endonuclease double enzyme cutting vector: AAV2-GFP, 37℃ enzyme cutting overnight after application of gel recovery purification kit to recover the vector; (3) the vector and the insert are connected in a molar ratio of 1:3 for homologous recombination, and the transformation plate is cultured at 37℃ overnight; (4) pick single colony overnight shaking, use plasmid extraction kit to extract plasmid, use enzyme cutting plasmid identification, after successful identification, the plasmid is sent to the company for sequencing, and finally the successful sequence alignment represents the successful construction of the plasmid;

[0038] S2, pAAV2-antiIGF1RscFv plasmid transfection cell: 293T cells are subcultured into 10cm dish, and then transfection is carried out after the cells grow stably; the transfection method is as follows: taking 1.5mL EP tube, marking: A tube: adding 10μg AAV2-antiIGF1RscFv plasmid, adding 480μL Opti-MEM mixing; B tube: adding 20μg PEI, adding 480μL Opti-MEM mixing; adding the liquid in A tube into B tube, mixing, and standing at room temperature for 15min, then gently mixing the liquid and adding it into 10cm dish, and placing it into 37℃ incubator for 2-3 days to collect cells for qPCR, Western blot and ELISA detection;

[0039] S3, pAAV2-antiIGF1RscFv in vitro expression antiIGF1R protein;

[0040] S4, virus packaging and purification.

[0041] Thereafter, it also includes verifying AAV2-antiIGF1RscFv virus expression antiIGF1R protein, and detecting the inhibition of AAV2-antiIGF1RscFv virus on cell function;

[0042] Further, the present application also provides the use of any one of I) to VII) in preparing a drug for improving thyroid-related eye disease.

[0043] I) the IGF1R single-chain antibody as described above;

[0044] II) the nucleic acid as described above;

[0045] III) the expression unit as described above;

[0046] IV) the recombinant vector as described above;

[0047] V) the plasmid combination as described above;

[0048] VI) a host as described above;

[0049] VII) Recombinant adeno-associated virus as described above.

[0050] The present invention also provides a drug for improving thyroid-related eye diseases, which comprises the recombinant adeno-associated virus of the present invention.

[0051] The drug described in the present invention is a new gene therapy drug for inhibiting thyroid-related eye diseases based on AAV vectors. The drug is carried by an AAV2 vector carrying the antiIGF1RscFv gene expression frame and inserted with a new secretion signal peptide sequence, which further improves its gene expression level and can inhibit cell proliferation.

[0052] The drug of the present invention comprises the recombinant adeno-associated virus as described above and also comprises pharmaceutically acceptable excipients. In some embodiments, the titer of the recombinant adeno-associated virus is 1×10 7 GC~1×10 13 GC / eye. Pharmaceutically acceptable excipients include, but are not limited to, buffers, osmotic pressure regulators, antimicrobial agents, or preservatives. The drugs described herein also include other drugs that treat thyroid-related eye diseases. In some embodiments, the drugs described herein are in the form of injections, including liquid injections or powder injections, which are not limited in the present invention. The injections are periocular or retroorbital injections.

[0053] The present invention provides an IGF1R single-chain antibody, expression unit, recombinant vector, host, and recombinant adeno-associated virus vector, as well as a preparation method, application, and medicine. Compared with the prior art, the present invention has at least one of the following advantages:

[0054] 1. The single-chain antibody provided by the present invention has a small molecular weight and good biological activity, can more effectively inhibit cell proliferation and differentiation, and improve the effect of inhibiting thyroid-related eye diseases.

[0055] 2. The encoding nucleic acid provided by the present invention has been codon-optimized to increase the gene expression level. The novel gene therapy drug for inhibiting thyroid-related eye disease based on the AAV vector carries the antiIGF1RscFv gene expression frame by inserting a new secretion signal peptide sequence, which further increases its gene expression level and secretion amount. At the same time, it can inhibit cell proliferation. The expression of the anti-IGF1R protein antiIGF1RscFv is delivered by the adeno-associated virus vector, which can effectively inhibit cell proliferation in the long term and reduce the frequency of medication. At the same time, it can also effectively improve patient compliance with treatment and reduce treatment risks and trauma.

[0056] 3. The present invention constructs an AAV2-antiIGF1RscFv recombinant plasmid; verifies the expression of antiIGF1R by in vitro cell plasmid transfection; packages the AAV2-antiIGF1RscFv virus; verifies the expression of antiIGF1R protein by in vitro cell virus transfection, and detects the inhibitory effect of the AAV2-antiIGF1RscFv virus on cell function. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the construction of recombinant adeno-associated virus AAV2-antiIGF1RscFv (A), AAV2-antiIGF1RFab (B) and AAV2-antiIGF1RFull length (C) plasmids in the examples of the present invention.

[0058] Figure 2 The expression of antiIGF1R was verified by length transfection of AAV2-antiIGF1RscFv, AAV2-antiIGF1RFab and AAV2-antiIGF1RFull in the examples of the present invention; and the protein level expression of antiIGF1R in cells was verified by Western Blot.

[0059] Figure 3 The plasmid transfection in the examples of the present invention was used to verify the binding of antiIGF1R expressed by AAV2-antiIGF1RscFv, AAV2-antiIGF1RFab and AAV2-antiIGF1RFull length to IGF1R; Western Blot was used to compare the differences in the inhibition of IGF1-mediated IGF1R phosphorylation by antiIGF1R proteins expressed by different forms of antibody plasmids.

[0060] Figure 4 The codon-optimized plasmid transfection in the embodiment of the present invention was used to verify the expression of antiIGF1RscFv in the AAV2-antiIGF1RscFv plasmid; the protein expression level of antiIGF1RscFv in cells and culture supernatant was verified by Western Blot.

[0061] Figure 5 The plasmid transfection in the examples of the present invention was used to verify the binding effect of antiIGF1RscFv in the AAV2-antiIGF1RscFv plasmid on IGF1R; Western Blot was used to compare the differences in the inhibition of IGF1-mediated IGF1R phosphorylation by antiIGF1RscFv proteins expressed by different plasmids.

[0062] Figure 6 The figures are the results of cesium chloride density gradient centrifugation of adeno-associated virus and the detection of virus purity and titer in the embodiments of the present invention; wherein A is the first centrifugation on the cesium chloride density gradient; B is the second centrifugation on the cesium chloride density gradient; C is the silver staining verification of the virus capsid; and D is the agarose verification of the virus genome.

[0063] Figure 7 In the examples of the present invention, 293T cells were infected with the packaged AAV2-antiIGF1RscFv virus to verify the expression of the antiIGF1RscFv protein in the cell culture supernatant; wherein, A is Western Blot verification of the expression of antiIGF1RscFv in the cells and supernatant; B is ELISA verification of the expression of secreted antiIGF1RscFv in the supernatant.

[0064] Figure 8 In the examples of the present invention, AAV2-antiIGF1RscFv virus was used to infect cells to verify the binding of antiIGF1RscFv in AAV2-antiIGF1RscFv to IGF1R; the effect of antiIGF1RscFv protein in inhibiting IGF1-mediated IGF1R and AKT phosphorylation was detected by Western Blot.

[0065] Figure 9 For the cell proliferation experiment in the examples of the present invention, IGF1, Teprotumumab, AAV2-antiIGF1RscFv, and empty vector were added to the supernatant of primary orbital fibroblasts to verify the inhibitory effect of antiIGF1RscFv secreted in the supernatant on cell proliferation. DETAILED DESCRIPTION

[0066] The present invention provides drugs and applications for improving thyroid-related eye diseases. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0067] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0068] In the present invention, the CDR region amino acid sequence and FR region amino acid sequence of the IGF1 R single-chain antibody are shown in Table 1:

[0069] Table 1

[0070] name sequence FR-L1 EIVLTQSPATLSLSPGE CDR-L1 RATLSCRASQSVSSYLA(SEQ ID NO:1) FR-L2 WYQQKPGQAPRLLIY CDR-L2 DASKRAT (SEQ ID NO: 2) FR-L3 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC CDR-L3 QQRSKWPPWT (SEQ ID NO: 3) FR-L4 FGQGTKVESK FR-H1 QVELVESGGGVVQPGRSQRLSCAASGFTFS CDR-H1 SYGMH (SEQ ID NO: 4) FR-H2 WVRQAPGKGLEWVA CDR-H2 IIWFDGSSTYYADSVRG(SEQ ID NO:5) FR-H3 RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAR CDR-H3 ELGRRYFDL (SEQ ID NO: 6) FR-H4 WGRGTLVSVSS

[0071] Below in conjunction with embodiment, the present invention is further elaborated:

[0072] Example 1

[0073] Construction of recombinant plasmids pAAV2-antiIGF1RscFv, pAAV2-antiIGF1RFab, and pAAV2-antiIGF1RFulllength (genome schematic diagram as shown in Figure 1 ), the steps are as follows:

[0074] (1) Gene synthesis obtained the nucleic acid sequence of the antiIGF1RscFv insert encoding the full-length IGF1R single-chain antibody + secretion signal peptide shown in SEQ ID NO:38, the nucleic acid sequence of the antiIGF1RFab insert encoding the IGF1R antibody Fab segment + secretion signal peptide shown in SEQ ID NO:41, and the nucleic acid sequence of the antiIGF1R Full length insert encoding the full-length IGF1R antibody + secretion signal peptide shown in SEQ ID NO:42.

[0075] (2) Use BglII and BsrGI restriction endonucleases to double-digest the vector: AAV2-GFP. After digestion at 37℃ overnight, use a gel recovery and purification kit to recover the vector.

[0076] (3) The vector and the insert were homologously recombined at a molar ratio of 1:3, and the transformation plate was cultured at 37°C overnight.

[0077] (4) Pick a single clone of overnight shake bacteria (add kanamycin), use a plasmid extraction kit to extract the plasmid, apply enzyme digestion to identify the plasmid, and send the plasmid to the company for sequencing after successful identification. Finally, a successful sequence alignment indicates that the pAAV2-antiIGF1RscFv-1 plasmid (referred to as pSY001 plasmid), pAAV2-antiIGF1RFab plasmid (referred to as pSY017 plasmid) and pAAV2-antiIGF1RFull length plasmid (referred to as pSY018 plasmid) are successfully constructed.

[0078] Example 2

[0079] pAAV2-antiIGF1RscFv-1 (pSY001), pAAV2-antiIGF1RFab (pSY017) and pAAV2-antiIGF1RFull length (pSY018) plasmids were transfected into cells respectively. 293T cells were transferred to 10cm dishes and transfected after the cells grew stably. Transfection method: Take a 1.5mL EP tube and mark it.

[0080] Tube A: add 10 μg AAV2-antiIGF1R scFv plasmid, add 480 μL Opti-MEM and mix; Tube B: add 20 μg PEI, add 480 μL Opti-MEM and mix;

[0081] Add the liquid in Tube A to Tube B, mix and stand at room temperature for 15 min, then gently mix the liquid and add it to a 10 cm dish, and incubate in a 37 °C incubator for 3 days to collect the cells for subsequent experiments.

[0082] Example 3

[0083] pAAV2-antiIGF1R in vitro expression of antiIGF1R protein

[0084] (1) Western blot detection of antiIGF1R protein expression

[0085] Prepare a 10% SDS-PAGE gel, add about 500 ml of 1x running buffer to the electrophoresis tank, then add the samples to the sample wells in order, turn on the power supply, and run the electrophoresis in constant voltage mode (80V for the concentrated gel and 120V for the separation gel). When the gel runs to the bottom, stop the electrophoresis. Activate the PVDF membrane by immersing it in methanol, discard the concentrated gel portion, and immerse it in the transfer solution in the order of blackboard-fiber pad-thick filter paper-PAGE gel-PVDF membrane-thick filter paper-fiber pad-whiteboard, and perform wet transfer in constant current mode (200 mA, 120 min). After the transfer is complete, block with 5% skim milk. Wash with 1x TBST for 10 min on a horizontal shaker. Completely immerse the PVDF membrane with antibody (1:5000 dilution of antibody), place it on a horizontal shaker, and incubate at room temperature for 60 min. Wash the membrane with 1x TBST 5 times for 5 min each time, then image in a pre-cooled chemiluminescence instrument.

[0086] As shown in Figure 2 Western blot verified that antiIGF1R protein expression could be detected in cells transfected with the constructed pAAV2-antiIGF1R scFv-1 (pSY001), pAAV2-antiIGF1R Fab (pSY017), and pAAV2-antiIGF1R Full length (pSY018) plasmids.

[0087] Example 4

[0088] pAAV2-antiIGF1RscFv in vitro expresses antiIGF1R protein to inhibit IGF1R phosphorylation under IGF1 stimulation. pAAV2-antiIGF1RscFv-1 (pSY001), pAAV2-antiIGF1RFab (pSY017), and pAAV2-antiIGF1RFull length (pSY018) plasmids were transfected into cells respectively. 293T cells were transferred to 10cm dishes and transfected after the cells grew stably. Transfection method: Take a 1.5mL EP tube and mark it.

[0089] Tube A: Add 10 μg AAV2-antiIGF1RscFv plasmid and mix thoroughly in 480 μL Opti-MEM; Tube B: Add 20 μg PEI and mix thoroughly in 480 μL Opti-MEM;

[0090] The liquid in tube A was added to tube B, mixed and allowed to stand at room temperature for 15 minutes. After 15 minutes, the liquid was gently mixed and added to a 10 cm dish. The dish was cultured in a 37°C incubator for 3 days. After treatment with IGF1 (10 ng / mL) for 2 hours, the cells were collected for Western blot analysis.

[0091] Western blot was used to detect the expression of IGF1R and phosphorylated IGF1R proteins.

[0092] Prepare a 10% SDS-PAGE gel and add approximately 500 ml of 1× electrophoresis buffer to the electrophoresis tank. Then, load the samples sequentially into the sample wells. Connect the power supply and run electrophoresis in constant voltage mode (80 V for stacking gel, 120 V for separating gel). Stop the electrophoresis when the gel reaches the bottom. Activate the PVDF membrane by immersing it in methanol. Discard the stacking gel and transfer the membrane in transfer buffer in the order of blackboard, fiber pad, thick filter paper, PAGE gel, PVDF membrane, thick filter paper, fiber pad, and whiteboard. Perform wet transfer in constant current mode (200 mA, 120 min). After transfer, block the membrane with 5% skim milk powder. Completely submerge the PVDF membrane in primary antibody (1:1000 dilution) and incubate on a horizontal shaker at room temperature for 120 min. Wash the membrane three times with 1× TBST for 10 min each time to completely remove any residual primary antibody. Then, apply diluted secondary antibody (1:5000 dilution) and incubate at room temperature for 60 min. The membrane was washed five times with 1× TBST for 5 min each time and then imaged in a pre-cooled chemiluminescence instrument.

[0093] like Figure 3As shown, Western blot results showed that IGF1 stimulation could enhance the phosphorylation level of IGF1R, and teixumab could inhibit the IGF1-mediated changes in the phosphorylation level of IGF1R. Similar to teixumab, pAAV2-antiIGF1RscFv-1 (pSY001), pAAV2-antiIGF1RFab (pSY017) and pAAV2-antiIGF1RFulllength (pSY018) could all inhibit the phosphorylation level of IGF1R.

[0094] Example 5: Anti-IGF1R protein expression in vitro after anti-IGF1R scFv codon optimization

[0095] (1) Plasmid construction

[0096] The nucleic acid sequence of the antiIGF1RscFv insert fragment, which includes the codon-optimized antiIGF1RscFv fragment and the secretion signal peptide, was obtained by gene synthesis, and the recombinant plasmid pAAV2-antiIGF1RscFv was constructed according to the method of Example 1. The nucleic acid sequence of the antiIGF1RscFv insert fragment in the pAAV2-antiIGF1RscFv-11 plasmid (hereinafter referred to as the pSY011 plasmid) is shown in SEQ ID NO: 39.

[0097] (2) Cell transfection

[0098] The pSY001 and pSY011 plasmids were transfected into cells separately. 293T cells were plated onto 10 cm dishes and transfected after stable cell growth. The cells were then harvested for subsequent experiments.

[0099] (3) Western blot detection of anti-IGF1R protein expression

[0100] like Figure 4 As shown, Western blot verified that the expression of antiIGF1RscFv protein could be detected in the culture supernatant and cells of cells transfected with the constructed pSY011 plasmid, and the expression level of antiIGF1RscFv protein of the pSY011 plasmid was the highest after codon optimization.

[0101] Example 6

[0102] pAAV2-antiIGF1RscFv in vitro expresses antiIGF1R protein to inhibit IGF1R phosphorylation under IGF1 stimulation. pAAV2-antiIGF1RscFv-1 (pSY001) and pAAV2-antiIGF1RscFv-11 (pSY011) are transfected into cells. 293T cells are transferred to 10cm dishes and transfected after the cells have grown stably. Transfection method: take a 1.5mL EP tube and mark it.

[0103] Tube A: Add 10 μg AAV2-antiIGF1RscFv plasmid and mix thoroughly in 480 μL Opti-MEM; Tube B: Add 20 μg PEI and mix thoroughly in 480 μL Opti-MEM;

[0104] The liquid in tube A was added to tube B, mixed and allowed to stand at room temperature for 15 minutes. After 15 minutes, the liquid was gently mixed and added to a 10 cm dish. The dish was cultured in a 37°C incubator for 3 days. After treatment with IGF1 (10 ng / mL) for 2 hours, the cells were collected for Western blot analysis.

[0105] Western blot was used to detect the expression of IGF1R and phosphorylated IGF1R proteins.

[0106] Prepare a 10% SDS-PAGE gel and add approximately 500 ml of 1× electrophoresis buffer to the electrophoresis tank. Then, load the samples sequentially into the sample wells. Connect the power supply and run electrophoresis in constant voltage mode (80 V for stacking gel, 120 V for separating gel). Stop the electrophoresis when the gel reaches the bottom. Activate the PVDF membrane by immersing it in methanol. Discard the stacking gel and transfer the membrane in transfer buffer in the order of blackboard, fiber pad, thick filter paper, PAGE gel, PVDF membrane, thick filter paper, fiber pad, and whiteboard. Perform wet transfer in constant current mode (200 mA, 120 min). After transfer, block the membrane with 5% skim milk powder. Completely submerge the PVDF membrane in primary antibody (1:1000 dilution) and incubate on a horizontal shaker at room temperature for 120 min. Wash the membrane three times with 1× TBST for 10 min each time to completely remove any residual primary antibody. Then, apply diluted secondary antibody (1:5000 dilution) and incubate at room temperature for 60 min. The membrane was washed five times with 1× TBST for 5 min each time and then imaged in a pre-cooled chemiluminescence instrument.

[0107] like Figure 5As shown in the figure, Western blot results showed that IGF1 stimulation enhanced the phosphorylation level of IGF1R, and pAAV2-antiIGF1RscFv-1(pSY001) could inhibit IGF1-mediated changes in IGF1R phosphorylation level. Compared with pAAV2-antiIGF1RscFv-1(pSY001), pAAV2-antiIGF1RscFv-11(pSY011) more significantly inhibited the phosphorylation level of IGF1R.

[0108] Example 7

[0109] Virus packaging and purification

[0110] 1. Transfection

[0111] (1) When the density of 293T cells in the cell factory reaches 80%, transfect the plasmid and add 700 μg of plasmid to the cell factory to prepare:

[0112] A: Take a 50 ml centrifuge tube and add the following plasmids: Helper plasmid, Rep-Cap2 plasmid, and pAAV2-antiIGF1RscFv-11 (pSY011) plasmid in equal molar ratios;

[0113] B: The mass ratio of PEI to plasmid is 2:1.

[0114] (2) Add B to A dropwise, mix thoroughly by inverting, and let stand at room temperature for 15 minutes.

[0115] (3) Take out the cell factory, turn the centrifuge tube upside down twice before adding MIX, add it dropwise into the culture medium, and place it in the incubator for culture.

[0116] (4) On the second day after transfection, discard the supernatant and replace it with an equal amount of serum-free culture medium.

[0117] 2. Collect viruses

[0118] (1) Collect the virus on the third day after changing the medium.

[0119] (2) Shake the cell factory vigorously to make the cells float, add them to a 50 ml centrifuge tube, centrifuge at 1500 g, 4°C for 10 min, and discard the supernatant to retain the cells at the bottom.

[0120] (3) Add appropriate amount of Lysis buffer to the centrifuge tube, mix the cells and vortex.

[0121] (4) Pour liquid nitrogen into a suitable container, quickly freeze the centrifuge tube in liquid nitrogen for 15 minutes, place it in 37°C until completely thawed, and vortex for 5 minutes. Place it in liquid nitrogen again, and repeat this process three times.

[0122] (5) Centrifuge at 1500g, 4℃ for 30 min.

[0123] 3. PEG precipitation

[0124] (1) Take the supernatant and transfer it to a new 50ml centrifuge tube. Add PEG8000, mix thoroughly by inversion, and let it precipitate overnight at 4℃.

[0125] (2) The next day, centrifuge at 1500g at 4°C for 30 minutes.

[0126] (3) Discard the supernatant.

[0127] (4) Dissolve the precipitate in 2 ml of phosphate solution.

[0128] 4. Cesium Chloride Density Gradient Centrifugation

[0129] After precipitation, the virus was concentrated by cesium chloride density gradient centrifugation, and finally the titer and purity of the virus were tested by qPCR, silver staining and genome identification. Figure 6 The results are from cesium chloride density gradient centrifugation and silver staining of the virus. Figure 6 A in the middle shows a clear virus band obtained after the first cesium chloride density gradient centrifugation for 24 hours. The band below is the genome band within the complete virus, and the empty virus shell (a) does not contain the genome; the virus band was extracted and silver stained to detect the virus purity. Figure 6 The results of agarose gel electrophoresis of viral genome in B are shown, and the band positions are correct. Figure 6 As can be seen in Figure C, the stained proteins are all viral capsid proteins. The above results indicate that a virus with low impurities and high purity has been prepared.

[0130] Example 8

[0131] 1. AAV2-antiIGF1RscFv infection of cells

[0132] 293T cells were transferred to 10 cm dishes and virus infection was performed when the cells grew to about 80%.

[0133] The MOI was detected by experiment = 1×10 5 (denoted as 1e5) has a good infection efficiency, and the MOI is 1×10 5 The AAV2-antiIGF1RscFv / empty vector was evenly added dropwise to the culture dish for infection, and the cells were cultured in a 37°C incubator for 3 days, and the cells and supernatant were collected for subsequent experimental detection.

[0134] 2. AAV2-antiIGF1RscFv in vitro expression of antiIGF1R protein

[0135] (1) Western blot detection of anti-IGF1R protein expression

[0136] Prepare a 10% SDS-PAGE gel and add approximately 500 ml of 1× electrophoresis buffer to the electrophoresis tank. Then, load the samples sequentially into the sample wells. Connect the power supply and run electrophoresis in constant voltage mode (80 V for stacking gel, 120 V for separating gel). Stop the electrophoresis when the gel reaches the bottom. Activate the PVDF membrane by immersing it in methanol. Discard the stacking gel and transfer the membrane in transfer buffer in the order of blackboard, fiber pad, thick filter paper, PAGE gel, PVDF membrane, thick filter paper, fiber pad, and whiteboard. Perform wet transfer in constant current mode (200 mA, 120 min). After transfer, block with 5% skim milk powder. Wash with 1× TBST for 10 min on a horizontal shaker. Completely submerge the PVDF membrane in antibody (1:5000 dilution) and incubate on a horizontal shaker at room temperature for 60 min. Wash the membrane five times with 1× TBST for 5 min each, then image in a pre-chilled chemiluminescence analyzer.

[0137] like Figure 7 As shown in A, Western blot verified that the expression of antiIGF1RscFv could be detected in the culture supernatant and cells of cells infected with AAV2-antiIGF1RscFv-11 (AAV2-SY011) virus.

[0138] (2) ELISA detection of anti-IGF1R protein expression

[0139] Antibody expression and VEGF binding were analyzed by ELISA. 96-well ELISA plates (Fisher, Loughborough, UK) were coated with 1 μg / mL hIGF1R and incubated overnight at 4°C. The plates were then blocked by incubation in 2% BSA at 37°C for 1 hour. Tetumumab was serially diluted as a standard. The culture supernatant was serially diluted in triplicate and added to each well and incubated at 37°C for 90 minutes. Anti-kappa light chain antibody was diluted 1:5000 in blocking buffer (PBS) and incubated at room temperature (RT) for 1 hour. After each step, the plates were washed five times with PBS + 0.05% Tween 20. TMB colorimetric solution was added and the reaction was allowed to proceed at room temperature in the dark. The reaction was terminated with 2.5 M H2SO4 and read on a microplate reader at 450 nm.

[0140] like Figure 7 As shown in B, ELISA verified that the secretion of antiIGF1RscFv could be detected in the culture supernatant of cells infected with AAV2-antiIGF1RscFv-11 (AAV2-SY011) virus.

[0141] Example 9

[0142] 1. AAV2-antiIGF1RscFv infection of cells

[0143] 293T cells were transferred to 10 cm dishes and virus infection was performed when the cells grew to about 80%.

[0144] The MOI was detected by experiment = 1×10 5 (denoted as 1e5) has a good infection efficiency, and the MOI is 1×10 5 The AAV2-antiIGF1RscFv / empty vector was evenly added dropwise to the culture dish for infection, and the cells were cultured in a 37°C incubator for 3 days, and the cells and supernatant were collected for subsequent experimental detection.

[0145] 2. Western blot was used to detect the expression of IGF1R and phosphorylated IGF1R protein, AKT and phosphorylated AKT protein.

[0146] Prepare a 10% SDS-PAGE gel and add approximately 500 ml of 1× electrophoresis buffer to the electrophoresis tank. Then, load the samples sequentially into the sample wells. Connect the power supply and run electrophoresis in constant voltage mode (80 V for stacking gel, 120 V for separating gel). Stop the electrophoresis when the gel reaches the bottom. Activate the PVDF membrane by immersing it in methanol. Discard the stacking gel and transfer the membrane in transfer buffer in the order of blackboard, fiber pad, thick filter paper, PAGE gel, PVDF membrane, thick filter paper, fiber pad, and whiteboard. Perform wet transfer in constant current mode (200 mA, 120 min). After transfer, block the membrane with 5% skim milk powder. Completely submerge the PVDF membrane in primary antibody (1:1000 dilution) and incubate on a horizontal shaker at room temperature for 120 min. Wash the membrane three times with 1× TBST for 10 min each time to completely remove any residual primary antibody. Then, apply diluted secondary antibody (1:5000 dilution) and incubate at room temperature for 60 min. The membrane was washed five times with 1× TBST for 5 min each time and then imaged in a pre-cooled chemiluminescence instrument.

[0147] like Figure 8 As shown, Western blot results showed that IGF1 stimulation could enhance the phosphorylation level of IGF1R, and teixumab could inhibit the IGF1-mediated changes in the phosphorylation levels of IGF1R and AKT. Similar to teixumab, AAV2-antiIGF1RscFv-11 (pSY011) could inhibit the phosphorylation levels of IGF1R and AKT.

[0148] Example 10

[0149] Inhibitory effect of AAV2-antiIGF1RscFv virus on the proliferation of primary orbital fibroblasts

[0150] Primary orbital fibroblasts in logarithmic growth phase were obtained, digested with 0.25% trypsin, and resuspended in DMEM medium containing 10% FBS. The cell suspension density was adjusted to 3×10 4 cells / ml, 100 μl per well was seeded into a 96-well plate, and the cells were cultured in DMEM medium without FBS in a 5% CO2, 37°C incubator and starved for 12 h.

[0151] The original cell culture medium was removed and 100 μl of DMEM medium without FBS was added as a blank control group (denoted as Ctrl).

[0152] On the basis of the blank control, 100 μl IGF1 (concentration of 500 ng / mL) was added as the positive control group.

[0153] On the basis of the positive control, Teprotumumab was used as the drug intervention group at a concentration of 500 ng / ml;

[0154] Dilute the empty vector with DMEM without FBS as the negative control group;

[0155] AAV2-antiIGF1RscFv-11 (AAV2-SY011) was used as the experimental group in DMEM without FBS; 3 parallel wells were set up in each group.

[0156] Each group was cultured for another 72 h in a 5% CO2, 37°C incubator. 10 μl of CCK8 was added to each well and cultured for another 2 h in a 5% CO2, 37°C incubator. The OD value of each well was measured at a wavelength of 450 nm using a microplate reader. The experiment was repeated three times.

[0157] The pathological manifestations of thyroid-related eye disease include the proliferation of fibroblasts. In order to explore whether viruses have an inhibitory effect on their proliferation function, primary orbital fibroblasts were evenly plated in 96-well plates, and then different viruses and drugs were used to interfere with cell proliferation, such as Figure 9 As shown in the figure, compared with the negative control group (empty vector), the cell proliferation in the experimental group with AAV2-antiIGF1RscFv-11 (AAV2-SY011) virus was significantly reduced, indicating that AAV2-antiIGF1RscFv-11 has a certain inhibitory effect on the proliferation of primary orbital fibroblasts.

[0158] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An IGF1R single-chain antibody, characterized in that The three CDR regions of the light chain are selected from CDR-L1 with an amino acid sequence as shown in SEQ ID NO: 1; CDR-L2 with an amino acid sequence as shown in SEQ ID NO: 2; CDR-L3 with an amino acid sequence as shown in SEQ ID NO: 3; or sequences having at least 80% sequence identity thereto; The three CDR regions of its heavy chain are respectively selected from CDR-H1 with an amino acid sequence as shown in SEQ ID NO: 5; CDR-H2 with an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 5; CDR-H3 with an amino acid sequence as shown in SEQ ID NO: 6; or sequences having at least 80% sequence identity therewith.

2. The IGF1R single-chain antibody according to claim 1, characterized in that The IGF1R single-chain antibody comprises a light chain variable region, a linker and a heavy chain variable region; wherein, the amino acid sequence of the light chain variable region is as shown in bp 21 to bp 128 of SEQ ID NO: 7, or a sequence having at least 80% sequence identity thereto; The amino acid sequence of the heavy chain variable region is shown as bp 149 to bp 266 of SEQ ID NO: 7, or a sequence having at least 80% sequence identity thereto.

3. The IGF1R single-chain antibody according to claim 2, characterized in that The IGF1R single-chain antibody includes a secretion signal peptide, a light chain variable region, a linker and a heavy chain variable region from the N-terminus to the C-terminus; The amino acid sequence of the secretory signal peptide is shown in SEQ ID NO: 7, 1 bp to 20 bp; The linker is (G4S) n , n=an integer of 2 to 10, preferably n=4.

4. A nucleic acid encoding the IGF1R single-chain antibody according to any one of claims 1 to 3, characterized in that: include: (1) the nucleic acid sequence encoding CDR-L1 is as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence having at least 80% sequence identity thereto; (2) a nucleic acid encoding CDR-L2 as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a sequence having at least 80% sequence identity thereto; (3) the nucleic acid encoding CDR-L3 is as shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, or a sequence having at least 80% sequence identity thereto; (4) the nucleic acid encoding CDR-H1 is as shown in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or a sequence having at least 80% sequence identity thereto; (5) the nucleic acid encoding CDR-H2 is as shown in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, or a sequence having at least 80% sequence identity thereto; (6) The nucleic acid encoding CDR-H3 is as shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, or a sequence having at least 80% sequence identity thereto.

5. The nucleic acid according to claim 4, characterized in that The nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO: 29 or SEQ ID NO: 30, or a sequence having at least 80% sequence identity thereto; The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 31 or SEQ ID NO: 32, or a sequence having at least 80% sequence identity thereto.

6. The nucleic acid according to claim 4, characterized in that The nucleic acid sequence encoding the secretory signal peptide is shown as SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37, or a sequence having at least 80% sequence identity thereto.

7. The nucleic acid according to any one of claims 4 to 6, characterized in that The nucleic acid sequence encoding the IGF1R single-chain antibody containing a secretory signal peptide is shown in SEQ ID NO: 38 or SEQ ID NO: 39, or a sequence having at least 80% sequence identity thereto.

8. An expression unit, characterized in that The invention comprises a promoter and the nucleic acid according to any one of claims 4 to 7; the promoter is preferably a chicken β-actin CBA promoter, a CMV promoter, an EF-1α promoter, a CAG promoter or other pan-promoters, a muscle-specific promoter or other tissue-specific promoters.

9. A recombinant vector, characterized in that Comprising the nucleic acid of claim 4 or the expression unit of claim 8.

10. The recombinant vector according to claim 9, characterized in that The invention sequentially comprises a 5' terminal inverted repeat sequence, a chicken β-actin CBA promoter sequence, a Kozak sequence, the nucleic acid according to any one of claims 4 to 7, bGH, SV40, hGH, a polyA signal sequence and a 3' terminal inverted repeat sequence, wherein the chicken β-actin CBA promoter sequence is shown in SEQ ID NO: 40, and the Kozak sequence nucleic acid sequence is shown in SEQ ID NO:

33.

11. A plasmid combination, characterized in that The invention comprises the recombinant vector according to claim 9 or 10, and a helper plasmid for virus packaging, wherein the helper plasmid for virus packaging comprises a Helper plasmid and a Rep-Cap2 plasmid.

12. A host, characterized in that (I) a nucleic acid according to any one of claims 4 to 7 is integrated; or (II), transfecting or transforming the recombinant vector according to any one of claims 8 to 10; The host is preferably a prokaryotic organism or an animal cell; the prokaryotic organism is not limited to Escherichia coli; the animal cell is not limited to one or more of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9 or FD-CHOS.

13. A recombinant adeno-associated virus obtained by culturing the host according to claim 12.

14. Use of any one of the following I) to VII) in the preparation of a medicament for improving thyroid-related eye disease; I) The IGF1R single-chain antibody according to any one of claims 1 to 3; II) the nucleic acid according to any one of claims 4 to 7; III) the expression unit according to claim 8 or 9; IV) the recombinant vector according to any one of claims 10 to 12; V) the host of claims 13-14; VI) The recombinant adeno-associated virus according to claim 15.

15. A drug for improving thyroid-related eye disease, characterized in that: The recombinant adeno-associated virus according to claim 13 preferably further comprises a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient includes but is not limited to one or more of a buffer, an osmotic pressure regulator, an antibacterial agent or a preservative.

16. The drug according to claim 15, wherein The dosage form of the drug includes but is not limited to injection or eye drops; The injection includes injection solution or injection powder; Preferably, the injection is one or more of periocular injection and retrobulbar injection.

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