AAV vector encoding anti-VEGF-A and anti-Ang-2 bispecific antibody

Abstract

Provided is a bispecific antibody, the nucleic acid encoding it, the AAV virus vector containing the nucleic acid, and virus particle.Furthermore, provided is a pharmaceutical composition comprising the bispecific antibody, the nucleic acid encoding it, the AAV virus vector, or virus particle.The bispecific antibody comprises the binding domain specific for VEGF-A and ANG-2, and can be used to treat wet age-related macular degeneration or diabetic macular edema in subjects.

Description

[Technical field]

[0001] Technical Field The present invention relates to the fields of immunology and gene delivery. More particularly, the present application relates to compositions, systems and methods for producing proteins of interest, such as antibodies. [Background technology]

[0002] background Age-related macular degeneration (AMD) is an age-related macular disease caused by various factors. Its common feature is the lesion of the macular retina and retinal pigment epithelium and choroid, which causes visual impairment and progressive loss of central vision in patients. The prevalence of AMD in China and the world increases with age, and AMD is one of the leading causes of irreversible visual impairment in people over 50 years old. With the aging of the population, it is expected that the number of AMD patients will reach 288 million by 2040. There is no unified standard for the clinical classification of AMD. Currently, both at home and abroad, the disease is first classified into stages, and in the later stages, it tends to be classified into two types: dry (atrophic) and neovascular (exudative, wet). Among them, neovascular age-related macular degeneration (nAMD), also known as wet age-related macular degeneration (wAMD), is the main clinical disease type that causes visual impairment, and is mainly characterized by the appearance of choroidal neovascularization (CNV) in the macula, and bleeding and exudation in the macula. Diabetic macular edema (DME) is currently one of the important causes of blindness in developed Western countries, but in China, with the improving standard of living and aging population, the prevalence of DME is gradually increasing, severely affecting patients' visual function and quality of life.

[0003] Vascular endothelial growth factor (VEGF) is one of the most important factors in the development of wAMD and DME. It acts specifically on vascular endothelial cells, promotes the proliferation of vascular endothelial cells, induces the formation of new blood vessels, and increases vascular leakage. Currently, it has been found that inhibiting the expression of VEGF induces vascular reconstruction and promotes the regression of immature new blood vessels. Therefore, VEGF is a promising therapeutic target for wAMD and DME. Since the emergence of anti-VEGF drugs, represented by ranibizumab in 2006, anti-VEGF drugs have been widely used to treat wAMD and DME. In the last decade, the simultaneous application of multiple anti-VEGF therapeutic drugs has significantly reduced irreversible vision loss due to wAMD and DME.

[0004] Although the progress of VEGF-targeting drugs in clinical practice has been remarkable, significantly reducing the degree of vascular leakage and edema, improving visual acuity, and no serious complications have been found, these protein drugs injected into the body are rapidly eliminated by metabolism, so multiple intraocular injections are required to maintain the therapeutic effect. Long-term administration increases the economic burden on patients, repeated injections increase the patient's pain and the possibility of side effects, some visual loss occurs when switching from regular to less frequent administration, and some patients are prone to recurrence after treatment. In response to the limitations of existing treatments for wAMD and DME, the art is in need of more economical, more durable, and more effective treatment strategies.

[0005] The present invention aims to provide anti-VEGF-A and anti-ANG-2 gene therapy. Summary of the Invention

[0006] The present invention provides a bispecific antibody comprising anti-VEGF-A and anti-ANG-2 binding domains, which is preferably a tandem single chain antibody molecule (tandem scFv, ta-scFv), in which the heavy chain variable region (VH) and the light chain variable region (VL) of the bispecific antibody are arranged from the N-terminus to the C-terminus in the following order: 1) VH anti-VEGF-A -VL anti-VEGF-A -VHanti-ANG-2 -VL anti-ANG-2 ; 2) VL anti-ANG-2 -VH anti-VEGF-A -VL anti-VEGF-A -VH anti-ANG-2 ;or, 3) VL anti-VEGF-A -VH anti-ANG-2 -VL anti-ANG-2 -VH anti-VEGF-A ; Here, VH anti-VEGF-A comprises a CDR1 set forth in SEQ ID NO:1, a CDR2 set forth in SEQ ID NO:2, and a CDR3 set forth in SEQ ID NO:3; VL anti-VEGF-A comprises a CDR1 set forth in SEQ ID NO:4, a CDR2 set forth in SEQ ID NO:5, and a CDR3 set forth in SEQ ID NO:6 or SEQ ID NO:47; VH anti-ANG-2 comprises a CDR1 as set forth in SEQ ID NO:7, a CDR2 as set forth in SEQ ID NO:8, and a CDR3 as set forth in SEQ ID NO:9; VL anti-ANG-2 comprises a CDR1 as set forth in SEQ ID NO:10, a CDR2 as set forth in SEQ ID NO:11, and a CDR3 as set forth in SEQ ID NO:12.

[0007] In one embodiment, the VH anti-VEGF-A comprises the sequence shown in SEQ ID NO:13, or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO:13; VL anti-VEGF-A comprises the sequence shown in SEQ ID NO:14, or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO:14 or SEQ ID NO:48; VH anti-ANG-2 comprises the sequence shown in SEQ ID NO:15, or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO:15; VL anti-ANG-2 comprises the sequence shown in SEQ ID NO:16, or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO:16.

[0008] In one embodiment, the antibody comprises a sequence as set forth in SEQ ID NO: 17, 18, 19, 49, 51 or 53, or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO: 17, 18, 19, 49, 51 or 53.

[0009] In one embodiment, the N-terminus of the bispecific antibody construct comprises a signal peptide or tag sequence, preferably the signal peptide sequence comprises the CD5A polypeptide comprising the sequence shown in SEQ ID NO:23. -sp It is a signal peptide.

[0010] In one embodiment, the antibody comprises a sequence as shown in SEQ ID NO: 20, 21, 22, 50, 52 or 54, or a sequence that is 70%, 80%, 90%, 95% or 99% identical to the sequence of SEQ ID NO: 20, 21, 22, 50, 52 or 54.

[0011] In one embodiment, the heavy chain variable region (VH) and the light chain variable region (VL) are (G4S) n (wherein n is an integer of 1 or more, preferably any integer from 1 to 4), for example, G4S, (G4S)2, (G4S)3 or (G4S)4 are operably linked, for example, 1) VH anti-VEGF-A -(G4S) m+X -VL anti-VEGF-A -(G4S) m -VH anti-ANG-2 -(G4S) m+X -VL anti-ANG-2 where m≧1 and X≧2, preferably VH anti-VEGF-A -(G4S)3-VL anti-VEGF-A -G4S-VH anti-ANG-2 -(G4S)3-VL anti-ANG-2 ; 2) VL anti-ANG-2 -(G4S) m -VH anti-VEGF-A -(G4S) m+X -VL anti-VEGF-A -(G4S) m -VH anti-ANG-2 , where m≧1 and X≧2, preferably VL anti-ANG-2 -G4S-VH anti-VEGF-A-(G4S)3-VL anti-VEGF-A -G4S-VH anti-ANG-2 ;or 3) VL anti-VEGF-A -(G4S) m -VH anti-ANG-2 -(G4S) m+X -VL anti-ANG-2 -(G4S) m -VH anti-VEGF-A , where m≧1 and X≧2, preferably VL anti-VEGF-A -G4S-VH anti-ANG-2 -(G4S)3-VL anti-ANG-2 -G4S-VH anti-VEGF-A .

[0012] Furthermore, the present invention provides nucleic acid sequences encoding the bispecific antibodies as described above.

[0013] The present invention also provides a vector comprising a nucleic acid sequence encoding the bispecific antibody.

[0014] In one embodiment, the vector is preferably an AAV virus.

[0015] In one embodiment, the AAV viral vector further comprises 5' and 3' ITRs, a promoter, and a polyA sequence.

[0016] In another aspect, the invention provides an AAV viral particle comprising any of the AAV viral vectors as described above and a capsid protein, preferably the capsid protein is of the AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 serotype.

[0017] The present invention provides pharmaceutical compositions comprising at least one of a bispecific antibody, a nucleic acid sequence, a vector, such as the AAV viral vectors and AAV viral particles described above, and a pharma- ceutically acceptable carrier.

[0018] The invention provides the use of a bispecific antibody, a nucleic acid sequence, a vector such as an AAV viral vector as described above, an AAV particle as described above, or a pharmaceutical composition as described above in the manufacture of a medicament for treating or preventing cancer, intraocular neovascular syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration or diabetic macular edema.

[0019] In one embodiment, the age-related macular degeneration is wet age-related macular degeneration.

[0020] In certain embodiments, the medicament is administered by intravitreal or subretinal injection. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic structural diagram of XMVA01, XMVA04 and XMVA09. [Diagram 2] FIG. 2 shows vector information for pAAV9-XMVA09 and ssAAV-XMVA09. [Diagram 3] FIG. 3 shows the expression levels of VEGF165-neutralizing proteins in cells following transfection with pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 vectors. [Figure 4] FIG. 4 shows the effect of intracellular expression of XMVA01, XMVA04 and XMVA09 on the proliferation of human retinal microvascular endothelial cells (HRMEC) under ECGS stimulation. [Diagram 5] Figure 5 shows the effect of intracellular expression of XMVA01, XMVA04 and XMVA09 on tube formation in HRMEC. [Figure 6] Figure 6 shows the effect of intracellular expression of XMVA01, XMVA04 and XMVA09 on tube formation in HRMEC. [Figure 7] FIG. 7 shows a schematic diagram of the structure of XMVA10. [Figure 8] FIG. 8 shows a schematic structural diagram of XMVA11. [Figure 9]FIG. 9 shows a schematic structural diagram of XMVA13. [Figure 10] FIG. 10 shows a schematic diagram of the structure of XMVA14. [Figure 11] FIG. 11 shows a schematic diagram of the structure of XMVA15. [Figure 12] FIG. 12 shows fluorescein fundus angiography (FFA) images of laser-induced wAMD model mice after injection of AAV-XMVA09. [Figure 13] FIG. 13 shows the percentage of grade 3 optic plaques and the mean leaky optic plaque score after AAV-XMVA09 injection in laser-induced wAMD model mice. [Figure 14] FIG. 14 shows changes in grade IV light plaque count and fluorescence leakage area in a laser-induced wAMD model rhesus monkey control group. [Figure 15] FIG. 15 shows the changes in grade IV light dot count and fluorescent leakage area in laser-induced wAMD model rhesus monkeys injected with AAV-XMVA09. [Figure 16] FIG. 16 shows the change in retinal thickness of grade IV light spots in a laser-induced wAMD model and a control group of rhesus monkeys. [Figure 17] FIG. 17 shows the change in retinal thickness at grade IV optic macula in a laser-induced wAMD model rhesus monkey injected with AAV-XMVA09. [Figure 18] FIG. 18 shows the permeability changes of high glucose-induced HRMEC. [Figure 19] FIG. 19 shows the regulatory effect of XMVA09 on the permeability of HRMEC, based on the VE-cadherin indicator. [Figure 20] FIG. 20 shows the regulatory effect of XMVA09 on the permeability of HRMEC based on the biotin-avidin system indicator. [Figure 21] FIG. 21 shows the fasting blood glucose and body weight monitoring analysis in diabetic model mice. [Figure 22] FIG. 22 shows images of retinal vascular leakage in a diabetic mouse model. [Figure 23]FIG. 23 shows changes in retinal vascular leakage areas after AAV-XMVA09 injection in diabetic model mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will be described in detail below according to the embodiments with the accompanying drawings. The above and other aspects of the present invention will become apparent in the following detailed description. The scope of the present invention is not limited to the following examples.

[0023] The antibody in the present invention is multispecific and can be a humanized antibody, a single chain antibody, a chimeric antibody, a synthetic antibody, a recombinant antibody, a heteroantibody, a mutated antibody, and a grafted antibody; the antibody format in the present invention is an scFv spliced ​​by an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), and the antibody can bind to two different antigens, such as VEGF-A and ANG-2.

[0024] The antibody light chain variable region (VL) and the antibody heavy chain variable region (VH) are further divided into hypervariable regions called complementarity determining regions (CDRs) and conserved regions called framework regions (FWRs). The CDRs of the antibodies and antigen-binding fragments described herein are defined or identified by Kabat numbering. In one embodiment, each VH and VL generally comprises three CDRs and four FWRs arranged in the following order from amino-terminus to carboxy-terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. The CDRs of the antibodies and antigen-binding fragments described herein are defined or identified by Kabat numbering.

[0025] As used herein, the term "vector" refers to a vector that contains a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.

[0026] All test methods in the following examples are preferred methods unless otherwise specified. EXAMPLES

[0027] Example 1. Construction of a plasmid vector Construction of AAV vector plasmid expressing anti-VEGF-A / anti-ANG-2 genes The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 were linked with (G4S)3, G4S, and (G4S)3 peptide linkers, respectively, to form CD5 -sp -VH anti-VEGF-A -(G4S)3-VL anti-VEGF-A -G4S-VH anti-ANG-2 -(G4S)3-VL anti-ANG-2 To form an open reading frame having the structure -sp The nucleotide sequences were designed according to human codon preference, a BamH I cleavage site was introduced at the 5' end, and an EcoR V cleavage site was introduced at the 3' end, and constructed according to the respective sequences in Table 1 and Table 2, and named XMVA01, XMVA04, and XMVA09 (full-length genes were synthesized by Nanjing GenScript Biotech Corporation). A schematic diagram of their structures is shown in Figure 1.

[0028] The above constructs and pAAV9 neo The -CAG plasmid was double digested with BamHI / EcoRV, and by routine molecular biology operations such as ligation, transformation, cloning screening and identification, pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 vectors were constructed, respectively; the information of pAAV9-XMVA09 vector is shown in Figure 2A. An endotoxin-free plasmid extraction kit (MN) was used to obtain high-quality plasmid DNA for later use.

[0029] [Table 1-1] [Table 1-2] [Table 1-3]

[0030] [Table 2-1] [Table 2-2] [Table 2-3]

[0031] Example 2. Measuring the expression of XMVA01, XMVA04, and XMVA09 in cells HEK293T cells were transfected with pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 plasmids, respectively, the supernatants were collected, and the expression of proteins in the supernatants was detected by ELISA. The specific operations are as follows: 293T cells were suspended in complete culture medium of DMEM containing 10% fetal bovine serum, inoculated into culture dishes, and cultured at 37°C in a 5% CO2 incubator until the cells reached 70-90% confluence, after which the plasmids were introduced using Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's instructions. After 72 hours of culture, the supernatants were collected and stored at -80°C for later use.

[0032] The specific detection method by ELISA was as follows: 100 μL per well of 0.1 μg / mL VEGF165 (proteintech) protein was coated on an ELISA plate (Thermo) and incubated overnight at 4°C, after which the ELISA plate was washed three times with PBS containing 0.05% Tween (Sangon) for 3 min each time. The plate was blocked with 200 μL / well of PBS containing 2% BSA (MikeBio) for 1 h at room temperature and washed three times again. Conbercept (10 mg / mL) standard and cell supernatants collected after transfecting HEK293T cells with pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 plasmids were added to the ELISA plate. The standard solution was serially diluted 8-fold from 312.5ng / mL, the supernatants to be tested were diluted 1:10 or 1:50, the data were averaged, and the cell supernatants without plasmid were used as the control group. The plates were incubated at 37°C for 1 hour and washed three times. A 1:5000 dilution of horseradish peroxidase-labeled goat anti-human IgG antibody (Jackson Immuno Research) was added to each well and incubated at 37°C for 1 hour, followed by washing three times. 100μL / well of TMB color development solution (Beyotime) was added to each well and reacted at room temperature in the dark for 6 minutes, after which 100μL / well of stop solution (Beyotime) was added to the wells to terminate the reaction. The OD value at 450nm was detected with a microplate reader, and the content of the test sample was calculated. The results are shown in Figure 3. Cells transfected with pAAV9-XMVA01 vector, pAAV9-XMVA04 vector and pAAV9-XMVA09 vector could effectively express VEGF165 neutralizing protein with significant difference compared with the control group.

[0033] Example 3. Effect of intracellular expression of XMVA01, XMVA04 and XMVA09 on proliferation of human retinal microvascular endothelial cells (HRMEC) The cell density of HRMEC was 4 × 10 in ECM complete medium (Science Cell) containing 1% ECGS. 4The solution was adjusted to 100 μL / mL and inoculated into a flat-bottom 96-well plate at 100 μL / well; the pAAV9-XMVA01 plasmid, pAAV9-XMVA04 plasmid, and pAAV9-XMVA09 plasmid were transfected into HEK293T cells, respectively, and the collected cell supernatant was added to each well at 100 μL, and the cell supernatant not transfected with the plasmid was used as the control group, and the cells were cultured at 37 °C in a 5% CO2 incubator for 72 hours. According to the instructions of the Cell Counting Kit-8 (CCK-8, Biosharp), 20 μL / well of CCK-8 solution was added to the test group and the control group, respectively. After 4 hours of incubation, the OD value was detected at a wavelength of 450 nm using a microplate reader. The results are shown in Figure 4. The protein expressed by pAAV9-XMVA09 could effectively suppress the proliferation of HRMEC under ECGS stimulation, and the inhibitory effect was significantly superior to that of pAAV9-XMVA01 and pAAV9-XMVA04.

[0034] Example 4. Effect of intracellular expression of XMVA01, XMVA04 and XMVA09 on tube formation in HRMEC Thawed Matrigel (Corning) was evenly spread at 50 μL / well on a flat-bottom 96-well plate and incubated at 37 °C in a 5% CO2 incubator for 1 h; pAAV9-XMVA01 plasmid, pAAV9-XMVA04 plasmid, and pAAV9-XMVA09 plasmid were transfected into HEK293T cells, respectively, and the collected cell supernatant was used to treat HRMECs, and the cell supernatant without plasmid transfection was used as a control. After 48 h of culture, the cells were resuspended in basal medium containing ECM without growth factors and serum, and the cell density was adjusted to 2 × 10 5The supernatant was adjusted to 100 μL / mL and seeded at 100 μL / well into a 96-well plate containing Matrigel, cultured at 37 °C in a 5% CO2 incubator, and observed once every 2 hours; after 4 to 8 hours, the cells were observed and photographed under a microscope, and the effects of the tested supernatant on the formation of HRMEC tubes were recorded. The results are shown in Figure 5. The protein expressed by pAAV9-XMVA09 could effectively inhibit the formation of HRMEC tubes, and its inhibitory effect was significantly better than that of pAAV9-XMVA01 and pAAV9-XMVA04.

[0035] Example 5. Construction of other plasmid vectors Scheme 1 The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 are linked by a G4S peptide linker, a (G4S)3 peptide linker, and a G4S peptide linker, respectively. -sp A nucleotide sequence was added to the N-terminus of CD5 -sp -VL anti-ANG-2 -G4S-VH anti-VEGF-A -(G4S)3-VL anti-VEGF-A -G4S-VH anti-ANG-2 The nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site at the 5' end and an EcoR V cleavage site at the 3' end, and was named XMVA10 (Figure 7). The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 were identical to those of XMVA09.

[0036] Scheme 2 The VH and VL amino acid sequences of the anti-VEGF antibody and the VH and VL amino acid sequences of the anti-ANG-2 antibody are, respectively, a G4S peptide linker, a (G4S)3 peptide linker, and a G4S peptide linker linked to the secretory signal peptide CD5 -sp A nucleotide sequence was added to the N-terminus of CD5 -sp -VL anti-VEGF-A -G4S-VHanti-ANG-2 -(G4S)3-VL anti-ANG-2 -G4S-VH anti-VEGF-A The nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site at the 5' end and an EcoR V cleavage site at the 3' end, and was named XMVA11 (Figure 8). The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 were identical to those of XMVA09.

[0037] Scheme 3 The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 are linked by a (G4S)3 peptide linker, a G4S peptide linker, and a (G4S)3 peptide linker, respectively. -sp A nucleotide sequence was added to the N-terminus of CD5 -sp -VH anti-VEGF-A -(G4S)3-VL anti-VEGF-A -G4S-VH anti-ANG-2 -(G4S)3-VL anti-ANG-2 The nucleotide sequence was designed according to human codon preferences, with a BamH I cleavage site at the 5' end and an EcoR V cleavage site at the 3' end, and was designated XMVA13 (Figure 9).

[0038] Scheme 4 The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 are linked by a G4S peptide linker, a (G4S)3 peptide linker, and a G4S peptide linker, respectively, and the secretory signal peptide CD5 is attached to the N-terminus. -sp Adding a nucleotide sequence to CD5 -sp -VL anti-ANG-2 -G4S-VH anti-VEGF-A -(G4S)3-VL anti-VEGF-A -G4S-VH anti-ANG-2The nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site at the 5' end and an EcoR V cleavage site at the 3' end, and was named XMVA14 (Figure 10). The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 were identical to those of XMVA13.

[0039] Scheme 5 The VH and VL amino acid sequences of the anti-VEGF antibody and the VH and VL amino acid sequences of the anti-ANG-2 antibody are respectively linked by a G4S peptide linker, a (G4S)3 peptide linker, and a G4S peptide linker. -sp -VL anti-VEGF-A -G4S-VH anti-ANG-2 -(G4S)3-VL anti-ANG-2 -G4S-VH anti-VEGF-A To form an open reading frame having the structure -sp a nucleotide sequence was added to the N-terminus; this nucleotide sequence was designed according to human codon preference, with a BamH I cleavage site at the 5' end and an EcoR V cleavage site at the 3' end, and was named XMVA15 (Figure 11). The VH and VL amino acid sequences of anti-VEGF and anti-ANG-2 were identical to those of XMVA13.

[0040] XMVA10 vector, XMVA11 vector, XMVA13 vector, XMVA14 vector and XMVA15 vector were constructed by routine molecular biology operations such as ligation, transformation, cloning screening and identification, and high-quality plasmid DNA was obtained for later use using an endotoxin-free plasmid extraction kit (MN).

[0041] The plasmids were transfected into HEK293T cells, the supernatants were collected, and the protein expression in the supernatants was detected by ELISA according to the method described in Example 2. The effect of the above vectors expressed in cells on the proliferation of HRMEC was detected according to the method described in Example 3. The effect of the above plasmids expressed in cells on HRMEC tube formation was detected according to the method described in Example 4. Recombinant AAV viruses were prepared and identified according to the method described in Example 5.

[0042] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0043] Example 6. Preparation and identification of recombinant AAV viruses The XMVA09 construct and ssAAV plasmid were double-digested with BamH I / EcoR V, and ssAAV-XMVA09 vector was constructed by conventional molecular biology operations such as ligation, transformation, cloning screening, and identification, and the vector information is shown in Figure 2B. High-quality plasmid DNA for later use was obtained using an endotoxin-free plasmid extraction kit (MN), and a three-plasmid packaging system with a mass ratio of 2:1:1, consisting of a helper plasmid (phelper), an AAV Cap and Rep protein expression plasmid, and a plasmid expressing the target gene (ssAAV-XMVA09), was used to form a transfection complex with a PEI transfection promoter and transfected into HEK293T cells to package AAV-XMVA09, thereby preparing recombinant AAV virus. The supernatant was collected twice, on the 3rd and 7th days after transfection, to obtain AAV particles containing the target gene. Purified AAV virus was obtained by density gradient centrifugation (Beckman ultracentrifuge) using varying gradients of iodoxanol (15%, 25%, 40%, and 60%). AAV quality was confirmed by transmission electron microscopy, and AAV virus titer was quantified by qPCR.

[0044] Example 7. Inhibitory effect of XMVA09 on laser-induced CNV in wAMD model mice The animal model of CNV induced by retinal high-energy laser photocoagulation is a commonly used animal model at home and abroad, and is currently the standard animal model in most treatment evaluation studies. High-energy laser photocoagulation selectively destroys the photoreceptor outer segment disk membrane, Bruch's membrane, retinal pigment epithelium and part of the anterior choriocapillary network, followed by a damage repair reaction involving the infiltration and proliferation of fibroblasts, retinal pigment epithelial cells and vascular endothelial cells, and finally the formation of neovascularization in the photocoagulated area.

[0045] Thirty SPF grade male C57 BL / 6J mice, aged 6-8 weeks and weighing 22-25 g (purchased from Beijing Vital River Laboratories Experimental Animal Technology Co., Ltd.) were used. The specific administration scheme is shown in Table 4 below:

[0046] [Table 4]

[0047] Since the expression of the target gene stabilizes after a certain period of time after AAV injection, intravitreal injection was performed on the first day, and on the 22nd day, animals that were successfully injected were subjected to bilateral fundus laser photocoagulation to induce a CNV model.

[0048] Establishment of mouse CNV model: The pupils of both eyes of the mouse were dilated with 1-2 drops of topicamide eye drops, and 5% chloral hydrate was injected intramuscularly for anesthesia. After anesthesia, carbomer eye drops were instilled into both eyes, a fundus laser scope was attached, and photocoagulation was performed with the optic disc at a distance of about 1.5-2PD from the optic disc as the center, avoiding blood vessels. The laser parameters were as follows: wavelength 532nm, energy 80mW, spot size 50μm, irradiation time 100ms. Erythromycin eye ointment was applied to both eyes of the animals after photocoagulation.

[0049] On day 29, the mice were subjected to fluorescein fundus angiography (FFA) detection: fluorescein sodium injection (15 mg / mL, 10 mL / kg) was intraperitoneally injected, and several clear photos of both eyes were taken early (within 1.5 minutes) and late (3 minutes) after sodium fluorescein injection to evaluate the degree of fluorescence leakage of the effective light spot, and the percentage of grade 3 leakage light spot and the average point of leakage light spot were calculated. The results are shown in Figure 12 and Figure 13. In laser-induced wAMD model mice, a single intravitreal injection of AAV-XMVA09 significantly suppressed the formation of CNV.

[0050] An effective light spot means a light spot that is completely visible on FFA without severe retinal hemorrhage nearby. The grading criteria for the fluorescence leakage spot are as follows: grade 0 (no fluorescence leakage), grade 1 (mild fluorescence leakage, 1-50% of the laser spot diameter), grade 2 (moderate fluorescence leakage, 50-100% of the laser spot diameter), grade 3 (severe fluorescence leakage, leakage area larger than the laser spot diameter).

[0051] The percentage of light spots of each grade = total number of light spots of the corresponding grade ÷ total number of light spots of four types × 100%.

[0052] Average number of leakage light points = [(number of light points of grade 0 x 0) + (number of light points of grade 1 x 1) + (number of light points of grade 2 x 2) + (number of light points of grade 3 x 3)] ÷ total number of light points of four types.

[0053] Example 8. Inhibitory effect of XMVA09 on CNV in rhesus monkeys with laser-induced wAMD model Seven male rhesus monkeys (purchased from Ya'an Primed Bio-tech Co., Ltd.) of general grade (qualified by quarantine before testing) aged 5-6 years were used. The specific administration scheme is shown in Table 5 below:

[0054] [Table 5]

[0055] XMVA09 group: AAV-XMVA09 (50 μL / eye) was injected intravitreally into both eyes on day 21 before laser modeling, and CNV was induced in both eyes by fundus laser on day 0.

[0056] Control group: CNV was induced in both eyes by fundus laser on day 0, and PBS (50 μL / eye) was intravitreally injected into both eyes on day 21.

[0057] All animals underwent optical coherence tomography (OCT) examination before laser modeling and fundus photography (FP), and FFA and OCT examinations were performed on the 19th and 77th days after laser modeling, with the number of grade IV light spots, leakage area, and retinal thickness as the main efficacy evaluation indexes. Establishment of rhesus monkey CNV model: Animals were anesthetized with intramuscular injection of ketamine hydrochloride (20 mg / kg) and dexmedetomidine hydrochloride (0.03 mg / kg), and the pupils of both eyes were dilated with instillation of Midori (tropicamide combination eye drops). After the pupils of both eyes became 6 mm or more, the animal's head was fixed in front of an ophthalmic laser photocoagulation device, and after observing the retinal structure with an ophthalmoscope, laser photocoagulation was performed at 9 points per eye at the optic disc distance centered on the center of the macula. The laser parameters were wavelength 532 nm, energy 650 mW-700 mW, spot size 50 μm, and exposure time 0.1 seconds. The fluorescein leakage area was measured by FP and FFA, and the specific test method is as follows: (1) The animals were anesthetized by intramuscular injection of ketamine hydrochloride (20 mg / kg) and dexmedetomidine hydrochloride (0.03 mg / kg). (2) The pupils of both eyes were dilated with 1 to 2 drops of tropicamide-containing eye drops. (3) The pupils of the animals after pupil dilation were observed, and if the pupils were 6 mm or more under light irradiation, color photographs of the fundus centered on the macula were taken with a fundus camera. (4) Then, 10% sodium fluorescein injection (Fluorescein, Alcon) was rapidly injected at a volume of 0.075 mL / kg into the saphenous vein of the lower limb. After fluorescein injection, FFA photographs centered on the macula were taken with a fundus camera in the early (within 1 min), middle (within 5 min), and late (within 10 min) phases. (6) The area of ​​fluorescein leakage from grade IV spots during the late phase (10 min) of FFA imaging was automatically measured using Image J software (version 1.53e, NIH, Wayne, Raband, USA) and used as an evaluation index for the efficacy of FFA.The grading criteria for fluorescein leakage from the laser spot were: Grade I (no high fluorescence in the light spot), Grade II (high fluorescence in the light spot, no leakage), Grade III (high fluorescence in the light spot, slight fluorescein leakage, but leakage did not extend beyond the edge of the light spot), and Grade IV (high fluorescence in the light spot, significant fluorescein leakage, leakage beyond the edge of the light spot).

[0058] The change in retinal thickness was measured by OCT. The specific test method is as follows: (1) After the FP and FFA tests, the animal was placed in front of the OCT device and adjusted so that the animal's eyes looked directly at the scanning lens. (2) Multi-layer linear scanning was performed by adopting a follow-up mode centered on the macula so that the scanning area covered all laser points. (3) The layer with the most obvious change in retinal thickness after modeling and a clear retinal boundary was selected, and the maximum retinal thickness of that area was measured using the measurement software installed in the device, which was used as an evaluation index of the effectiveness of OCT.

[0059] The results are shown in Figures 14, 15, 16 and 17. FFA examinations on days 19 and 77 after laser modeling in animals from each group showed different fluorescence leakage at the laser spots in the fundus. Compared with the control group, a single intravitreal injection of AAV-XMVA09 showed obvious ameliorative effects on the number of light spots, leakage area and retinal thickness of grade IV CNV in the laser-induced wAMD rhesus monkey model.

[0060] Example 9. High glucose-induced HRMEC permeability increase model 24-well plates were pretreated with 0.1% gelatin for 1-2 hours at 37°C and then dried for a few minutes; HRMECs were then resuspended in ECM (Sciencell) complete medium containing 10% FBS + 1% PS + 1% ECGS and plated at 3 × 10 5The cells were seeded uniformly on a well plate according to the standard of 1000 / well. The next day, starvation treatment was started in low serum medium containing 0.5% FBS for 7 hours, and then the HRMECs were exposed to a 30 mM high glucose environment overnight. The control group was not subjected to high glucose treatment. The next day, the cells were immunofluorescently co-stained with VE-cadherin for membrane proteins and DAPI for nuclei, and observed and photographed under a fluorescent microscope. The results are shown in Figure 18. The continuity of the cell membrane of HRMECs treated with high glucose was destroyed, resulting in cell-cell expansion and increased permeability.

[0061] Immunofluorescence staining procedure: Discard the culture supernatant, make the remaining volume 200μL, add 200μL of 4% PFA, and start gradient fixation at room temperature for 20 minutes. After discarding the supernatant, add 200μL of PFA directly and continue fixation at room temperature for 10 minutes. After discarding the fixative, wash with PBS three times, 5 minutes each time. VE-cadherin (Santa Cruz Biotechnology Inc) was diluted 1:150 in PBS, 300μL / well was added, incubated at room temperature for 2 hours, discard the supernatant, and wash three times with PBS. Discard the supernatant, and wash three times with PBS (5 minutes each time). Donkey anti-mouse IgG (H+L) high-adsorption secondary antibody, AlexaFluorTM488 (Invitrogen) was diluted 1:200 in PBS, 200μL / well was added, and incubated at room temperature for 2 hours. Discard the supernatant, and wash three times with PBS (5 minutes each time). Finally, 100 μL / well of DAPI-containing mounting medium (Beyotime) was added, and the cells were observed and photographed under a fluorescent microscope.

[0062] Example 10. Determination of the regulatory effect of XMVA09 protein on the permeability of HRMEC based on the VE-cadherin indicator 24-well plates were precoated with 0.1% gelatin overnight at 4°C or for 2 hours at 37°C. The coating solution was discarded, and 3 × 10 HRMEC cells were plated. 5The cells were seeded 100x per well. The next day, when the monolayer cells reached a certain confluence, starvation treatment was started for 7 hours in low serum medium containing 0.5% FBS. The supernatant of untransfected HEK293T cells and the supernatant of HEK293T cells transfected with ssAAV-XMVA09 plasmid were co-cultured with high glucose (30 mM glucose), respectively. HRMEC were treated overnight, and the control group was a non-modeling group without high glucose treatment. Immunofluorescence staining of VE-cadherin was performed, the detailed procedure was the same as in Example 9, and photo analysis was performed under a fluorescent microscope. The results are shown in Figure 19. The protein expressed by ssAAV-XMVA09 showed a significant inhibitory effect on the increase in permeability of HRMEC after high glucose treatment.

[0063] Example 11. Determination of the regulatory effect of XMVA09 protein on the permeability of HRMEC based on the biotin-avidin system indicator Gelatin EZ-Link (商標) Biotinylated gelatin was obtained by treatment with NHS-LC-LC-biotin. Biotinylated gelatin was used to coat cell culture plates overnight at 4 °C or for 2 h at 37 °C. HRMECs were directly seeded on the culture plates, with an inoculum of cells of 3 × 10 5 The following day, starvation treatment was started for 7 hours in low serum medium containing 0.5% FBS. Then, the supernatant of untransfected HEK293T cells and the supernatant of HEK293T cells transfected with ssAAV-XMVA09 plasmid were co-cultured with high glucose (30 mM glucose), and HRMEC were treated for 17 hours. The control group was a non-modeling group without high glucose treatment. After staining with FITC-avidin and fixing with 4% PFA, staining was started with AF594-phalloidin (Invitrogen A12381), anti-fluorescence quenching agent containing DAPI was added, and photographic analysis was performed with a fluorescent microscope. The results are shown in Figure 20. The protein expressed by ssAAV-XMVA09 showed a significant inhibitory effect on the increase in permeability of HRMEC after high glucose treatment.

[0064] Example 12. Construction of diabetic model mice Drug induction is one of the current methods for establishing diabetic model animals that can better simulate the clinicopathological symptoms and clinical characteristics of diabetes. Streptozotocin (STZ) is the most commonly used drug for inducing diabetes, which causes hyperglycemia mainly by destroying pancreatic β cells, resulting in loss of capillary pericytes, thinning of the vascular layer, and destruction of the blood-retinal barrier, further causing vascular leakage, making it a suitable model for testing DME.

[0065] Establishment of diabetic mouse model: Eighty SPF grade 6-8 week old male C57 BL / 6J mice (purchased from Zhejiang Vital River Laboratories Experimental Animal Technology Co., Ltd.) weighing 20-25g were randomly divided into a blank group, a DME model control group, and a DME model group. Before modeling, the fasting blood glucose levels and body weights of the three groups of mice were measured. On modeling day 0, after 6 hours of fasting (with free access to water), blood was collected from the tip of the tail for blood glucose measurement, and the mice were weighed. Modeling began on day 1, when the mice were fasted for 6 hours (with free access to water). The blank group was not administered, the DME model control group was intraperitoneally administered 60 mg / kg of 50 mM sodium citrate solution (Sigma) in a single dose for 5 consecutive days, and the DME model group was intraperitoneally administered 60 mg / kg of STZ (Sigma) in a single dose for 5 consecutive days. The mice were allowed to eat and drink freely, and blood glucose levels and body weights were measured and recorded regularly. Modeling continued for 135 days.

[0066] The results are shown in Figure 21. On the 19th day after modeling, the state in which the fasting blood glucose level of the mouse exceeded 16.7 mmol / L continued for about 3 weeks, indicating that the diabetic model mouse was successfully constructed.

[0067] Example 13. Phenotype verification of DME in diabetic model mice On the 81st day after modeling, Evans Blue was injected into some of the mice from each of the three groups in Example 12 via the tail vein, the retinas were peeled off and separated, and vascular leakage was observed under a fluorescent microscope. The specific procedures were as follows: 50 mg / mL of Evans Blue (Sigma) was injected into the tail vein at 50 μL / mouse, and blood was allowed to circulate for 1.5 hours, after which the mice were euthanized, and both eyes were enucleated and fixed with 4% paraformaldehyde (Biosharp) for 45 minutes, and the retinas of the eyeballs were peeled off and spread out, and observed and photographed under a fluorescent microscope.

[0068] The results are shown in Figure 22. The retinal vascular leakage in the DME model group was obviously increased compared with that in the DME model control group and the blank group, and there was no obvious difference between the retina of the DME model control group and that of the blank group, indicating that the diabetic model mice had a DME phenotype.

[0069] Example 14. Inhibitory effect of XMVA09 on retinal vascular leakage in diabetic mouse models The DME model mice of Example 12 were administered with injection on the 95th day after modeling. The specific administration scheme is shown in Table 6 below:

[0070] [Table 6]

[0071] On the 29th day after injection, the mice in the XMVA09 group, the control group, and the DME model control group of Example 12 were injected with Evans blue via tail vein injection, and the retinas were dissected and observed for vascular leakage under a fluorescent microscope.

[0072] The results are shown in Figure 23. The rate of retinal vascular leakage in the DME model control group was significantly lower than that in the control group, and the rate of retinal vascular leakage in the XMVA09 group was significantly lower than that in the control group, indicating that a single intravitreal injection of AAV-XMVA09 had a significant inhibitory effect on retinal vascular leakage in diabetic model mice.

[0073] statistical analysis GraphPad Prism 8.0 software was used for data processing and statistical analysis. The statistical level was 5% or p ≤ 0.05, and the mean and standard error (mean ± SEM) of each analytical index were calculated. p ≤ 0.05 indicates a statistically significant difference.

Claims

1. A bispecific antibody comprising anti-VEGF-A and anti-ANG-2 binding domains, wherein the heavy chain variable region (VH) and light chain variable region (VL) of the bispecific antibody are arranged in the following order from the N-terminus to the C-terminus: 1)VH ａｎｔｉ－ＶＥＧＦ－Ａ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -VH ａｎｔｉ－ＡＮＧ－２ -VL ａｎｔｉ－ＡＮＧ－２ ; 2) VL ａｎｔｉ－ＡＮＧ－２ -VH ａｎｔｉ－ＶＥＧＦ－Ａ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -VH ａｎｔｉ－ＡＮＧ－２ ;or 3)VL ａｎｔｉ－ＶＥＧＦ－Ａ -VH ａｎｔｉ－ＡＮＧ－２ -VL ａｎｔｉ－ＡＮＧ－２ -VH ａｎｔｉ－ＶＥＧＦ－Ａ ; Here, VH ａｎｔｉ－ＶＥＧＦ－Ａ This includes CDR1 shown in Sequence ID 1, CDR2 shown in Sequence ID 2, and CDR3 shown in Sequence ID 3; VL ａｎｔｉ－ＶＥＧＦ－Ａ This includes CDR1 shown in Sequence ID 4, CDR2 shown in Sequence ID 5, and CDR3 shown in Sequence ID 6 or Sequence ID 47; VH ａｎｔｉ－ＡＮＧ－２ This includes CDR1 shown in Sequence ID 7, CDR2 shown in Sequence ID 8, and CDR3 shown in Sequence ID 9; VL ａｎｔｉ－ＡＮＧ－２ This includes CDR1 shown in sequence number 10, CDR2 shown in sequence number 11, and CDR3 shown in sequence number 12.

2. VH ａｎｔｉ－ＶＥＧＦ－Ａ However, it includes the sequence shown in sequence number 13, or a sequence that is 70%, 80%, 90%, 95%, or 99% identical to the sequence in sequence number 13; VL ａｎｔｉ－ＶＥＧＦ－Ａ However, it includes the sequence shown in sequence number 14 or sequence number 48, or a sequence that is 70%, 80%, 90%, 95%, or 99% identical to the sequence in sequence number 14 or sequence number 48; VH ａｎｔｉ－ＡＮＧ－２ However, it includes the sequence shown in sequence number 15, or a sequence that is 70%, 80%, 90%, 95%, or 99% identical to the sequence in sequence number 15; VL ａｎｔｉ－ＡＮＧ－２ However, it includes the sequence shown in sequence number 16, or a sequence that is 70%, 80%, 90%, 95%, or 99% identical to the sequence in sequence number 16. The bispecific antibody according to claim 1.

3. The bispecific antibody according to claim 1, wherein the antibody comprises the sequence shown in SEQ ID NO: 17, 18, 19, 49, 51, or 53, or a sequence that is 70%, 80%, 90%, 95%, or 99% identical to the sequence in SEQ ID NO: 17, 18, 19, 49, 51, or 53.

4. The N-terminus of the bispecific antibody contains a signal peptide sequence or a tag sequence, preferably the signal peptide sequence being CD5 as shown in SEQ ID NO:

23. －ｓｐ A bispecific antibody according to claim 1, which is a signal peptide.

5. The bispecific antibody according to claim 4, wherein the antibody comprises the sequence shown in SEQ ID NO: 20, 21, 22, 50, 52, or 54, or a sequence that is 70%, 80%, 90%, 95%, or 99% identical to the sequence in SEQ ID NO: 20, 21, 22, 50, 52, or 54.

6. The heavy chain variable region (VH) and the light chain variable region (VL) are (G ４ S) ｎ The bispecific antibody according to claim 1, wherein the molecules are linked by a sign, where n is an integer from 1 to 4.

7. The structure of a bispecific antibody is VH ａｎｔｉ－ＶＥＧＦ－Ａ -(G ４ S) ３ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -G ４ S-VH ａｎｔｉ－ＡＮＧ－２ -(G ４ S) ３ -VL ａｎｔｉ－ＡＮＧ－２ ; VL ａｎｔｉ－ＡＮＧ－２ -G ４ S-VH ａｎｔｉ－ＶＥＧＦ－Ａ -(G ４ S) ３ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -G ４ S-VH ａｎｔｉ－ＡＮＧ－２ ; VL ａｎｔｉ－ＶＥＧＦ－Ａ -G ４ S-VH ａｎｔｉ－ＡＮＧ－２ -(G ４ S) ３ -VL ａｎｔｉ－ＡＮＧ－２ -G ４ S-VH ａｎｔｉ－ＶＥＧＦ－Ａ ; CD5 －ｓｐ -VH ａｎｔｉ－ＶＥＧＦ－Ａ -(G ４ S) ３ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -G ４ S-VH ａｎｔｉ－ＡＮＧ－２ -(G ４ S) ３ -VL ａｎｔｉ－ＡＮＧ－２ ; CD5 －ｓｐ -VL ａｎｔｉ－ＡＮＧ－２ -G ４ S-VH ａｎｔｉ－ＶＥＧＦ－Ａ - (G ４ S) ３ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -G ４ S-VH ａｎｔｉ－ＡＮＧ－２ ;or CD5 －ｓｐ -VL ａｎｔｉ－ＶＥＧＦ－Ａ -G ４ S-VH ａｎｔｉ－ＡＮＧ－２ -(G ４ S) ３ -VL ａｎｔｉ－ＡＮＧ－２ -G ４ S-VH ａｎｔｉ－ＶＥＧＦ－Ａ The bispecific antibody according to claim 1.

8. A nucleic acid encoding a bispecific antibody as described in claim 1.

9. A vector comprising a nucleic acid sequence encoding the bispecific antibody described in claim 1, preferably an AAV virus vector.

10. The AAV virus vector according to claim 9, further comprising a 5'-ITR and a 3'-ITR, a promoter, and a polyA sequence.

11. AAV virus particles comprising the AAV virus vector and capsid protein described in claim 9.

12. The AAV virus particle according to claim 11, wherein the serotype of the capsid protein is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.

13. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 7, a nucleic acid according to claim 8, a vector according to claim 9 or 10, at least one AAV virus particle according to claim 11 or 12, and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for treating or preventing cancer, intraocular neovascularization syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration, or diabetic macular edema, comprising at least one of a bispecific antibody according to any one of claims 1 to 7, a nucleic acid according to claim 8, a vector according to claim 9 or 10, or an AAV virus particle according to claim 11 or 12.

15. The pharmaceutical composition according to claim 14, wherein the age-related macular degeneration is wet age-related macular degeneration.

16. The pharmaceutical composition according to claim 14, which is administered by intravitreous injection, subretinal injection, or choroidal injection.

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