WO2023186131A1 - 一种编码抗vegf-a和ang-2双特异性抗体的aav载体 - Google Patents

一种编码抗vegf-a和ang-2双特异性抗体的aav载体 Download PDF

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WO2023186131A1
WO2023186131A1 PCT/CN2023/085610 CN2023085610W WO2023186131A1 WO 2023186131 A1 WO2023186131 A1 WO 2023186131A1 CN 2023085610 W CN2023085610 W CN 2023085610W WO 2023186131 A1 WO2023186131 A1 WO 2023186131A1
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vegf
ang
seq
sequence
bispecific antibody
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才源
马珍
周佩佩
张明亮
赵锦
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合肥星眸生物科技有限公司
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Definitions

  • the present invention relates to the fields of immunology and gene delivery. More specifically, the present application relates to compositions, systems, and methods for producing proteins of interest, such as antibodies.
  • Age-related macular degeneration is a group of macular diseases induced by various factors and related to age. Its common feature is the occurrence of lesions in the macular retina, retinal pigment epithelium and choroid, which leads to visual impairment in patients. Functional impairment and progressive decrease in central vision. The prevalence of AMD in our country and even around the world continues to rise and increases with age. It is one of the main causes of irreversible visual impairment in people over 50 years old. As the population ages, the number of AMD patients is expected to reach 288 million by 2040. There is no unified standard for the clinical classification of AMD. At present, both domestic and foreign countries tend to stage first and then classify.
  • Neovascular age-related macular degeneration also known as wet age-related macular degeneration (wAMD)
  • CNV choroidal neovascularization
  • DME diabetic macular edema
  • VEGF Vascular endothelial growth factor
  • drugs targeting VEGF have made great clinical progress and breakthroughs, they can significantly reduce the degree of vascular leakage and edema, improve vision, and no serious complications have been found.
  • these protein drugs injected into the body are rapidly eliminated through metabolism, requiring multiple intraocular injections to maintain therapeutic effects. Long-term treatment increases the patient's economic burden. Repeated injections will increase the patient's pain and the possibility of adverse reactions. There will be a certain degree of vision loss when switching from regular dosing to low-frequency dosing. Some patients are prone to relapse after treatment. .
  • In response to the limitations of existing therapies for wAMD and DME there is a need in the field for more economical, longer-lasting, and more effective treatment strategies.
  • the present invention aims to provide an anti-VEGF-A and anti-ANG-2 gene therapy.
  • the invention provides a bispecific antibody, which includes an anti-VEGF-A and ANG-2 binding domain, which is preferably a single-chain antibody tandem molecule (tandem scFv, ta-scFv).
  • the chain variable region (VH) and light chain variable region (VL) are arranged in the following order from N-terminus to C-terminus:
  • the VH anti-VEGF-A includes CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2; CDR3 shown in SEQ ID NO:3;
  • the VL anti-VEGF-A includes CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5; CDR3 shown in SEQ ID NO:6 or SEQ ID NO:47;
  • the VH anti-ANG-2 includes CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8; CDR3 shown in SEQ ID NO:9;
  • the VL anti-ANG-2 includes CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11; CDR3 shown in SEQ ID NO:12.
  • the VH anti-VEGF-A includes the sequence shown in SEQ ID NO:13 or has a sequence identity of 70%, 80%, 90%, 95%, or 99% with SEQ ID NO:13 sequence;
  • the VL anti-VEGF-A includes the sequence shown in SEQ ID NO:14 or has a sequence identity of 70%, 80%, 90%, 95%, or 99% with SEQ ID NO:14 or SEQ ID NO:48 sequence;
  • the VH anti-ANG-2 includes the sequence shown in SEQ ID NO: 15 or a sequence with a sequence identity of 70%, 80%, 90%, 95%, or 99% to SEQ ID NO: 15;
  • the VL anti-ANG-2 includes the sequence shown in SEQ ID NO: 16 or a sequence with a sequence identity of 70%, 80%, 90%, 95%, or 99% to SEQ ID NO: 16.
  • the antibody includes the sequence shown in SEQ ID NO: 17, 18, 19, 49, 51 or 53 or has a sequence identity of SEQ ID NO: 17, 18, 19, 49, 51 or 53. 70%, 80%, 90%, 95%, or 99% of the sequence.
  • the N-terminus of the bispecific antibody construct includes a signal peptide sequence or a tag sequence, preferably the signal peptide sequence is a CD5-sp signal peptide, which includes as shown in SEQ ID NO: 23 sequence.
  • the antibody includes the sequence shown in SEQ ID NO: 20, 21, 22, 50, 52 or 54 or has a sequence identity of SEQ ID NO: 20, 21, 22, 50, 52 or 54. 70%, 80%, 90%, 95%, or 99% of the sequence.
  • the heavy chain variable region (VH) and the light chain variable region (VL) are operably linked by (G 4 S) n , where n is an integer greater than 1, preferably 1 to Any integer between 4, such as G 4 S, (G 4 S) 2 , (G 4 S) 3 or (G 4 S) 4 , for example,
  • VL anti-ANG-2- (G 4 S) m -VH anti-VEGF-A- (G 4 S) m+X -VL anti-VEGF-A- (G 4 S) m -VH anti-ANG-2 , where m ⁇ 1 , _ _ _ _ _ _ _ _
  • the present invention provides a nucleic acid sequence encoding the aforementioned bispecific antibody.
  • the present invention also provides a vector including a nucleic acid sequence encoding the aforementioned bispecific antibody.
  • the vector is preferably an AAV virus.
  • the AAV viral vector further includes: 5'ITR and 3'ITR, a promoter and polyA sequence.
  • the invention provides an AAV virus particle, including any of the above AAV viral vectors and capsid proteins, preferably the serotypes of the capsid proteins are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.
  • the present invention provides a pharmaceutical composition, including at least one of the bispecific antibody as described above, a nucleic acid sequence, a vector such as an AAV viral vector and an AAV virus particle as described above, and a pharmaceutically acceptable carrier.
  • the present invention provides bispecific antibodies, nucleic acid sequences, vectors such as AAV viral vectors, AAV viral particles as described above, or pharmaceutical compositions as described above for use in the treatment or prevention of cancer, intraocular Use in medicines for neovascular syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration, or diabetic macular edema.
  • vectors such as AAV viral vectors, AAV viral particles as described above, or pharmaceutical compositions as described above for use in the treatment or prevention of cancer, intraocular Use in medicines for neovascular syndrome, rheumatoid arthritis, psoriasis, proliferative retinopathy, age-related macular degeneration, or diabetic macular edema.
  • the age-related macular degeneration is wet age-related macular degeneration.
  • the drug is administered by intravitreal or subretinal injection.
  • Figure 1 shows the structural schematic diagram of XMVA01, XMVA04 and XMVA09.
  • Figure 2 shows the vector information of pAAV9-XMVA09 and ssAAV-XMVA09.
  • Figure 3 shows the expression of VEGF165 neutralizing protein after cells were transfected with pAAV9-XMVA01, pAAV9-XMVA04 and pAAV9-XMVA09 vectors.
  • Figure 4 shows the effect of expressing XMVA01, XMVA04 and XMVA09 in cells on the proliferation of human retinal microvascular endothelial cells (HRMEC) under ECGS stimulation.
  • HRMEC retinal microvascular endothelial cells
  • Figures 5 and 6 show the effects of expressing XMVA01, XMVA04 and XMVA09 in cells on the lumen formation of HRMECs.
  • Figure 7 shows a schematic structural diagram of XMVA10.
  • Figure 8 shows a schematic structural diagram of XMVA11.
  • Figure 9 shows a schematic structural diagram of XMVA13.
  • Figure 10 shows a schematic structural diagram of XMVA14.
  • Figure 11 shows a schematic structural diagram of XMVA15.
  • Figure 12 shows fluorescein angiography (FFA) images of laser-induced wAMD model mice after injection of AAV-XMVA09.
  • Figure 13 shows the level 3 spot percentage and average spot leakage score in laser-induced wAMD model mice after injection of AAV-XMVA09.
  • Figure 14 shows the changes in the number of level IV spots and fluorescence leakage area in the laser-induced wAMD model rhesus monkey control group.
  • Figure 15 shows the changes in the number of grade IV spots and fluorescence leakage area in rhesus monkeys injected with AAV-XMVA09 in the laser-induced wAMD model.
  • Figure 16 shows the changes in retinal thickness of level IV spots in the laser-induced wAMD model rhesus monkey control group.
  • Figure 17 shows the changes in retinal thickness of grade IV spots in laser-induced wAMD model rhesus monkeys injected with AAV-XMVA09.
  • Figure 18 shows the permeability changes of HRMECs induced by high glucose.
  • Figure 19 shows the regulatory effect of XMVA09 on the permeability of HRMECs based on the VE-cadherin index.
  • Figure 20 shows the judgment of XMVA09’s effect on HRMECs based on biotin-avidin system indicators. permeability control effect.
  • Figure 21 shows the monitoring analysis of fasting blood glucose and body weight of diabetic model mice.
  • Figure 22 shows images of retinal blood vessel leakage in diabetic model mice.
  • Figure 23 shows changes in retinal blood vessel leakage area in diabetic model mice after injection of AAV-XMVA09.
  • the antibodies in the present invention are multispecific and can be humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies; the antibody form in the present invention is An scFv is spliced into an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the antibody can bind to two different antigens, such as VEGF-A and ANG-2.
  • VL Antibody light chain variable region
  • VH antibody heavy chain variable region
  • CDRs complementarity-determining regions
  • FWR interspersed regions
  • the CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering.
  • each VH and VL generally includes 3 CDRs and 4 FWRs arranged from amino terminus to carboxyl terminus in the following order: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4.
  • the CDRs of the antibodies and antigen-binding fragments disclosed herein are defined or identified by Kabat numbering.
  • vector refers to a vector that includes a recombinant polynucleotide that includes expression control sequences operably linked to the nucleotide sequence to be expressed.
  • VH amino acid sequence and VL amino acid sequence of anti-VEGF are added to the N-terminus, using (G 4 S) 3 and G respectively.
  • (G 4 S) 3 peptide linkers are connected to form a structure of 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 open reading frame, nucleotide sequence designed according to human codon preference , and introduced a BamHI restriction site at the 5' end and an EcoR V restriction site at the 3' end.
  • Example 2 Determination of expression of XMVA01, XMVA04, and XMVA09 in cells
  • pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 plasmids were transfected into HEK293T cells respectively, the supernatant was collected, and the protein expression in the supernatant was detected by ELISA.
  • the specific operation is as follows: resuspend the 293T cells in complete culture medium of DMEM containing 10% fetal bovine serum, inoculate them into a culture dish, and culture them in a 37°C, 5% CO2 incubator until the confluence of the cells reaches 70%-90%.
  • Lipofectamine 2000 (Invitrogen) transfection reagent the plasmid was transfected according to its instructions. After continuing to culture for 72 hours, the supernatant was collected and stored at -80°C for later use.
  • the specific detection method of ELISA is as follows: Coat the enzyme plate (Thermo) with 0.1 ⁇ g/mL VEGF165 (proteintech) protein, 100 ⁇ L per well, incubate at 4°C overnight, and wash the enzyme plate three times with PBS containing 0.05% Tween (Sangon) , 3 minutes each time. Block with 200 ⁇ L/well of PBS containing 2% BSA (Maike Biotech) for 1 hour at room temperature, and wash three times again.
  • Example 3 Effects of expressing XMVA01, XMVA04, and XMVA09 in cells on the proliferation of human retinal microvascular endothelial cells (HRMEC)
  • HRMECs were conditioned with complete medium (Science Cell) in ECM containing 1% ECGS The cell density is 4 ⁇ 10 4 /mL, and 100 ⁇ L/well is seeded in a flat-bottom 96-well plate; the cell supernatant collected after transfecting HEK293T cells with pAAV9-XMVA01, pAAV9-XMVA04, and pAAV9-XMVA09 plasmids is 100 ⁇ L/well. Add into the well, use the cell supernatant without transfected plasmid as the control group, and culture it in a 37°C, 5% CO2 incubator for 72 hours; follow the Cell Counting Kit-8 (CCK-8, Biosharp) instructions.
  • Example 4 Effects of expressing XMVA01, XMVA04, and XMVA09 in cells on the lumen formation of HRMECs
  • VH amino acid sequence and VL amino acid sequence of the anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of the anti-ANG-2, and the secretion signal peptide CD5-sp nucleotide sequence are added to the N-terminus, using G 4 S, (G 4 S) 3 and G 4 S peptide linkers are connected to form the structure CD5-sp-VL anti - ANG-2 -G 4 S-VH anti-VEGF-A -(G 4 S) 3 -VL anti-VEGF-A -G 4
  • the S-VH anti-ANG-2 open reading frame was designed with a nucleotide sequence based on human codon preference, and a BamHI cleavage site was introduced at the 5' end and an EcoR V cleavage site was introduced at the 3' end, named XMVA10 (see Figure 7). Among them, anti-VEGF The VH amino acid sequence, VL amino acid sequence, and anti-ANG-2 VH amino acid sequence
  • VH amino acid sequence and VL amino acid sequence of anti-VEGF are added to the N-terminus, using G 4 S and (G 4 S respectively.
  • G 4 S peptide linkers are connected to form the structure 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 open reading frame, the nucleotide sequence is designed according to human codon preference, and a BamHI cleavage site is introduced at the 5' end and an EcoR V cleavage site is introduced at the 3' end, named XMVA11 (See Figure 8).
  • the VH amino acid sequence and VL amino acid sequence of anti-VEGF, and the VH amino acid sequence and VL amino acid sequence of anti-ANG-2 are the same as XMVA09.
  • VH amino acid sequence and VL amino acid sequence of anti-VEGF, the VH amino acid sequence and VL amino acid sequence of anti-ANG-2, and the secretion signal peptide CD5-sp nucleotide sequence are added to the N terminus, using (G 4 S) 3 and G 4 S and (G 4 S) 3 peptide linkers are connected to form the structure 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 open reading frame, the nucleotide sequence is designed according to human codon preference, and a BamHI cleavage site is introduced at the 5' end and an EcoR V enzyme is introduced at the 3' end The cleavage site was named XMVA13 (see Figure 9).
  • VH amino acid sequence and VL amino acid sequence of the anti-VEGF, the VH amino acid sequence and the VL amino acid sequence of the anti-ANG-2, and the secretion signal peptide CD5-sp nucleotide sequence are added to the N-terminus, using G 4 S, (G 4 S) 3 and G 4 S peptide linkers are connected to form the structure CD5-sp-VL anti - ANG-2 -G 4 S-VH anti-VEGF-A -(G 4 S) 3 -VL anti-VEGF-A -G 4
  • the S-VH anti-ANG-2 open reading frame was designed with a nucleotide sequence based on human codon preference, and a BamHI cleavage site was introduced at the 5' end and an EcoR V cleavage site was introduced at the 3' end, named XMVA14 (see Figure 10).
  • VH amino acid sequence and VL amino acid sequence of anti-VEGF, the VH amino acid sequence and VL amino acid sequence of anti-ANG-2, plus the secretion signal peptide CD5-sp nucleotide sequence at the N-terminus They were connected with G 4 S, (G 4 S) 3 and G 4 S peptide linkers respectively to form the structure 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 open reading frame, the nucleotide sequence is designed according to human codon preference, and a BamHI restriction site is introduced at the 5' end and a BamHI restriction site is introduced at the 3' end
  • the EcoR V restriction site is named XMVA15 (see Figure 11).
  • the VH amino acid sequence and VL amino acid sequence of anti-VEGF, and the VH amino acid sequence and VL amino acid sequence of anti-ANG-2 are
  • the XMVA10, XMVA11, XMVA13, XMVA14 and XMVA15 vectors were constructed through conventional molecular biology operations such as ligation, transformation and clone screening and identification, and high-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MN) for later use.
  • MN endotoxin-free plasmid extraction kit
  • Example 2 the plasmids were transfected into HEK293T cells, the supernatant was collected, and the protein expression in the supernatant was detected by ELISA. According to the method described in Example 3, the effect of expressing the above vector in cells on the proliferation of HRMECs was evaluated. According to the method described in Example 4, the effect of expressing the above plasmid in cells on the lumen formation of HRMECs was evaluated. Recombinant AAV viruses were prepared and characterized according to the method described in Example 5.
  • Example 6 Preparation and identification of recombinant AAV viruses
  • An endotoxin-free plasmid extraction kit MN
  • MN endotoxin-free plasmid extraction kit
  • AAV virus including a helper plasmid (phhelper), AAV Cap and Rep protein expression plasmids, and a plasmid expressing the target gene ( ssAAV-XMVA09) and PEI transfection promoter to form a transfection complex according to the mass ratio of 2:1:1, transfect HEK293T cells, and perform AAV-XMVA09 virus packaging.
  • the supernatant was collected twice to obtain AAV virus particles containing the target gene.
  • Example 7 Inhibitory effect of XMVA09 on laser-induced CNV in wAMD model mice
  • High-energy laser photocoagulation of the retina is a commonly used CNV animal model at home and abroad, and it is also the standard animal model in most current treatment evaluation experiments. It selectively destroys the photoreceptor outer segment disc membrane, Bruch's membrane, retinal pigment epithelium and part of the anterior choriocapillaris network, followed by a damage repair reaction, including the invasion and growth of fibroblasts, retinal pigment epithelial cells and vascular endothelial cells, and finally in the light New blood vessels form in the coagulated area.
  • mice CNV model 1-2 drops of compound tropicamide eye drops were dropped into each eye of the mouse to dilate the pupils, and 5% chloral hydrate was injected intramuscularly for anesthesia. After anesthesia, carbomer eye drops were instilled in both eyes, a fundus laser lens was placed, and photocoagulation was performed around the optic papilla at a distance of about 1.5-2 PD from the optic disc to avoid blood vessels.
  • the laser parameters are: wavelength 532nm, power 80mW, spot diameter 50 ⁇ m, exposure time 100ms. After photocoagulation, the animal's eyes are coated with erythromycin eye ointment.
  • mice were subjected to fluorescein fundus angiography (FFA): intraperitoneal injection of fluorescein sodium injection (15 mg/mL, 10 mL/kg), early (within 1.5 minutes), late (3 Minutes later) collect several clear pictures with both eyes, rate the degree of fluorescence leakage of the effective spot, and calculate the percentage of level 3 leakage spots and the average score of spot leakage.
  • FFA fluorescein fundus angiography
  • An effective spot refers to a spot that does not have severe retinal hemorrhage nearby and can be completely displayed in FFA.
  • Spot fluorescence leakage rating standards are: Level 0 (no fluorescence leakage), Level 1 (mild fluorescence leakage, leakage area is 1 to 50% of the laser spot size), Level 2 (moderate fluorescence leakage, leakage The leakage area is 50-100% of the laser spot size), level 3 (severe fluorescence leakage, the leakage area is larger than the laser spot size).
  • the percentage of light spots at each level (%) the total number of light spots at the corresponding level ⁇ the total number of 4 types of light spots ⁇ 100%.
  • the average score of light spot leakage [(number of level 0 light spots ⁇ 0) + (number of level 1 light spots ⁇ 1) + (number of level 2 light spots ⁇ 2) + (number of level 3 light spots ⁇ 3)] ⁇ the total number of 4 types of light spots.
  • Example 8 Inhibitory effect of XMVA09 on CNV in laser-induced wAMD model rhesus monkeys
  • Group XMVA09 AAV-XMVA09 (50 ⁇ L/eye) was injected into the vitreous cavity of both eyes 21 days before laser modeling, and CNV was induced by fundus laser in both eyes on day 0.
  • Control group CNV was induced by fundus laser in both eyes on day 0, and PBS (50 ⁇ L/eye) was injected into both eyes on day 21.
  • the animal's head is fixed in front of the ophthalmic laser photocoagulation instrument.
  • laser photocoagulation is performed at a distance from the optic disc around the center of the macula, with 9 points in each eye.
  • the laser parameters are: wavelength 532nm, power 650mW-700mW, spot diameter 50 ⁇ m, exposure time 0.1 seconds.
  • FP and FFA measure the fluorescein leakage area.
  • the specific inspection methods are as follows: (1) The animals are anesthetized by intramuscular injection of ketamine hydrochloride injection (20 mg/kg) and dexmedetomidine hydrochloride injection (0.03 mg/kg).
  • grading standards for fluorescein leakage from laser spots are: Level I (spot without high fluorescence), Level II (spot with high fluorescence but no leakage), Level III (spot with high fluorescence and slight fluorescein leakage, but no leakage). beyond the edge of the spot), Level IV (high fluorescence in the spot, significant fluorescein leakage, and leakage beyond the edge of the spot).
  • OCT measures changes in retinal thickness.
  • the specific examination methods are as follows: (1) After the animal completes the FP and FFA examination, place it in front of the OCT instrument and adjust the animal's eyes to look straight at the scanning lens. (2) Use the follow-up mode to perform multi-layer linear scanning with the macula as the center, and the scanning area covers all laser points. (3) Select the layer where the retinal thickness changes most obviously and the retinal boundary is clear after modeling, and use the measurement software that comes with the instrument to measure the maximum retinal thickness in this area as an OCT efficacy evaluation index.
  • Immunofluorescence staining steps Discard the culture supernatant and leave 200 ⁇ L of culture medium. Add 200 ⁇ L of 4% PFA to start gradient fixation and fix at room temperature for 20 minutes. After discarding the supernatant, directly add 200 ⁇ L of PFA and continue fixation at room temperature for 10 minutes.
  • Example 10 Determining the regulatory effect of XMVA09 protein on HRMECs permeability based on VE-cadherin index
  • Example 11 Determining the regulatory effect of XMVA09 protein on the permeability of HRMECs based on biotin-avidin system indicators
  • the supernatant of cells transfected with ssAAV-XMVA09 plasmid expressing HEK293T was incubated with high sugar (30mM glucose), and HRMECs were treated for 17 hours.
  • the control group was a non-modeling group without high sugar treatment; stained with FITC-avidin , and then fixed with 4% PFA, started staining with AF594-phalloidin (Invitrogen A12381), then added an anti-fluorescence quencher containing DAPI, and took photos and analysis under a fluorescence microscope.
  • the results are shown in Figure 20.
  • the protein expressed by ssAAV-XMVA09 has a significant inhibitory effect on the increase in permeability of HRMECs treated with high glucose.
  • Drug induction is currently one of the methods for establishing diabetic animal models, which can better simulate the clinical pathological manifestations and clinical characteristics of diabetes.
  • Streptozotocin (STZ) is the most commonly used drug to induce diabetes. It mainly causes hyperglycemia by destroying pancreatic beta cells, which in turn causes the loss of capillary pericytes, thinning of the vascular layer, and destruction of the blood-retina barrier, further leading to vascular leakage. Leak formation is a good model for studying DME.
  • mice 80 male C57BL/6J mice of SPF grade 6 to 8 weeks old (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.), weighing 20-25g, were randomly divided into blank group, DME modeling control group, and DME Build a model set. Before modeling, fasting blood glucose and body weight of mice in the three groups were measured: fasted for 6 hours (since After drinking water), blood was collected from the tip of the tail for blood glucose measurement and weighed. This was the 0th day of modeling. Modeling was started on the first day. The mice were fasted (drinking water freely) for 6 hours. The blank group was not treated.
  • the DME modeling control group was injected with a single intraperitoneal injection of 50mM sodium citrate solution (Sigama) 60mg/kg for 5 consecutive days.
  • the model group received a single intraperitoneal injection of STZ (Sigama) 60 mg/kg for 5 consecutive days. After the injection, they could eat and drink freely. Blood glucose and body weight were measured and recorded regularly. The modeling lasted for 135 days.
  • mice from each of the three groups of mice in Example 12 were injected into the tail vein with Evans blue, the retinas were dissected and separated, and vascular leakage was observed under a fluorescence microscope.
  • the specific operation is as follows: 50 ⁇ L of Evans blue (Sigama) 50 mg/mL is injected into the tail vein per mouse. After blood circulation for 1.5 hours, the mice are euthanized. Both eyes are removed, fixed in 4% paraformaldehyde (Biosharp) for 45 minutes, and peeled off. The retina of the eyeball was spread out, observed under a fluorescence microscope and photographed.
  • the results are shown in Figure 22.
  • the retinal vascular leakage in the DME modeling group was significantly increased compared with the DME modeling control group and the blank group. There was no significant difference between the retinas of the DME modeling control group and the blank group.
  • the diabetic mouse model has a DME phenotype. .
  • Example 14 Inhibitory effect of XMVA09 on retinal vascular leakage in diabetic mouse model
  • mice in the DME modeling group in Example 12 were injected and administered.
  • the specific dosage regimen is shown in Table 6 below:
  • the Evans blue tail vein injection method was used for the XMVA09 group, the control group, and the DME modeling control group in Example 12, and the retina was dissected and separated, and the vascular leakage was observed under a fluorescence microscope.
  • GraphPad Prism 8.0 software was used for data processing and statistical analysis. The statistical level was set at 5% or p ⁇ 0.05, and the mean and standard error (Mean ⁇ SEM) of each analysis index were calculated. If p ⁇ 0.05, the difference was statistically significant.

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Abstract

提供了一种双特异性抗体、其编码核酸、包含该核酸的AAV病毒载体和病毒颗粒。还提供了包含该双特异性抗体、编码核酸、AAV病毒载体或病毒颗粒的药物组合物。该双特异性抗体包含特异于VEGF-A和ANG-2的结合结构域，可用于治疗受试者的湿性年龄相关性黄斑变性或糖尿病性黄斑水肿。

Description

一种编码抗VEGF-A和ANG-2双特异性抗体的AAV载体 发明领域
本发明涉及免疫学和基因递送领域。更具体地，本申请涉及用于生产感兴趣的蛋白诸如抗体的组合物、系统和方法。
背景技术
年龄相关性黄斑变性(age-related macular degeneration，AMD)是由各种因素诱导并与年龄相关的一组黄斑疾病，其共同特点是黄斑部视网膜及视网膜色素上皮和脉络膜发生病变，并导致患者视功能障碍和中心视力进行性下降。我国乃至世界AMD的患病率不断上升且随着年龄增加而升高，是50岁以上人群不可逆视力损伤的主要原因之一。随着人口的老龄化，预计到2040年AMD患者将达2.88亿人。AMD临床分型没有统一标准，国内外目前均倾向于先分期再分型，晚期分成2种类型，分别为干性型(萎缩型)和新生血管性型(渗出型、湿性型)，其中，新生血管性年龄相关性黄斑变性(neovascular age-related macular degeneration，nAMD)也称湿性年龄相关性黄斑变性(wet age-related macular degeneration，wAMD)，是导致视力丧失的主要临床类型，其主要特点是脉络膜新生血管(choroidal neovascularization，CNV)出现在黄斑区，从而导致黄斑区发生出血、渗出。糖尿病性视网膜病变(diabetic macular edema，DME)目前是西方发达国家重要致盲的原因之一，随着我国人民生活水平的提高，人口老龄化加剧，DME的发病率已逐渐增高，严重影响患者的视功能和生存质量。
血管内皮生长因子(vascular endothelial growth factor，VEGF)是wAMD和DME的发病机制中最重要的因素之一，它能特异地作用于血管内皮细胞，促使血管内皮细胞的增殖，诱导新血管的生成以及血管渗漏的增加。目前研究发现，阻断VEGF表达可诱导血管重塑的发生，同时促使尚未成熟的新生血管回退，因此VEGF是一个有前景的wAMD和DME的治疗靶点。自2006年以雷珠单抗(Ranibizumab)为代表的抗VEGF药物应用以来，抗VEGF药物已被广泛应用于治疗wAMD和DME。在近10 年间，由于同时应用多种抗VEGF治疗方法，因wAMD和DME导致的不可逆盲已有大幅减少。
尽管靶向VEGF的药物在临床上取得了长足的进步和突破，它可以显著降低血管渗漏和水肿的程度，改善视力，并且未发现严重的并发症。但是，这些注射入体内的蛋白质药物会随着代谢而迅速清除，因此需要多次眼内注射以维持治疗效果。长期治疗增加了患者的经济负担，重复注射会增加患者的痛苦和发生不良反应的可能性，在从常规给药转变为低频给药时会出现一定程度的视力下降，一些患者在治疗后容易复发。针对wAMD和DME现有疗法的局限性，在本领域上出现了对更加经济且持续时间更长、效果更优的治疗策略的需求。
本发明旨在提供一种抗VEGF-A和抗ANG-2基因疗法。
发明内容
本发明提供了一种双特异性抗体，其包括抗VEGF-A和ANG-2的结合结构域，其优选为单链抗体串联分子(tandem scFv，ta-scFv)，所述双特异性抗体重链可变区(VH)和轻链可变区(VL)从N-末端到C-末端按照如下顺序排列：
1)VH抗VEGF-A-VL抗VEGF-A-VH抗ANG-2-VL抗ANG-2；
2)VL抗ANG-2-VH抗VEGF-A-VL抗VEGF-A-VH抗ANG-2；或
3)VL抗VEGF-A-VH抗ANG-2-VL抗ANG-2-VH抗VEGF-A；
其中，所述VH抗VEGF-A包括SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2；SEQ ID NO:3所示的CDR3；
所述VL抗VEGF-A包括SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2；SEQ ID NO:6或SEQ ID NO:47所示的CDR3；
所述VH抗ANG-2包括SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2；SEQ ID NO:9所示的CDR3；
所述VL抗ANG-2包括SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2；SEQ ID NO:12所示的CDR3。
在一个实施例中，所述VH抗VEGF-A包括SEQ ID NO:13所示的序列或与SEQ ID NO:13序列同一性为70％、80％、90％、95％、或99％的序列；
所述VL抗VEGF-A包括SEQ ID NO:14所示的序列或与SEQ ID NO:14或SEQ ID NO:48序列同一性为70％、80％、90％、95％、或99％的序列；
所述VH抗ANG-2包括SEQ ID NO:15所示的序列或与SEQ ID NO:15序列同一性为70％、80％、90％、95％、或99％的序列；
所述VL抗ANG-2包括SEQ ID NO:16所示的序列或与SEQ ID NO:16序列同一性为70％、80％、90％、95％、或99％的序列。
在一个实施例中，所述抗体包括SEQ ID NO:17、18、19、49、51或53所示的序列或与SEQ ID NO:17、18、19、49、51或53序列同一性为70％、80％、90％、95％、或99％的序列。
在一个实施例中，所述双特异性抗体构建体的N-末端包含信号肽序列或标签序列，优选所述信号肽序列为CD5-sp信号肽，其包含如SEQ ID NO：23所示的序列。
在一个实施例中，所述抗体包括SEQ ID NO:20、21、22、50、52或54所示的序列或与SEQ ID NO:20、21、22、50、52或54序列同一性为70％、80％、90％、95％、或99％的序列。
在一个实施例中，重链可变区(VH)和轻链可变区(VL)之间可操作性地由(G4S)n链接，其中n为大于1的整数，优选为1至4间任意整数，例如G4S、(G4S)2、(G4S)3或(G4S)4，例如，
1)VH抗VEGF-A-(G4S)m+X-VL抗VEGF-A-(G4S)m-VH抗ANG-2-(G4S)m+X-VL抗 ANG-2；其中m≥1，X≥2，优选为VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2；
2)VL抗ANG-2-(G4S)m-VH抗VEGF-A-(G4S)m+X-VL抗VEGF-A-(G4S)m-VH抗ANG-2，其中m≥1，X≥2，优选为VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗 VEGF-A-G4S-VH抗ANG-2；或
3)VL抗VEGF-A-(G4S)m-VH抗ANG-2-(G4S)m+X-VL抗ANG-2-(G4S)m-VH抗VEGF-A， 其中m≥1，X≥2，优选为VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S-VH抗VEGF-A。
进一步，本发明提供了一种核酸序列，其编码前述双特异性抗体。
本发明还提供了一种载体，包括编码前述双特异性抗体的核酸序列。
在一个实施例中，所述载体优选为AAV病毒。
在一个实施例中，AAV病毒载体进一步包括：5’ITR和3’ITR、启动子和polyA序列。
又一方面，本发明提供了一种AAV病毒颗粒，包括如前任意的AAV病毒载体和衣壳蛋白，优选其中所述衣壳蛋白的血清型为AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、或AAV9。
本发明提供了一种药物组合物，包括如前所述的双特异性抗体、核酸序列、载体诸如AAV病毒载体和如前所述的AAV病毒颗粒中的至少一种，以及药学上可接受的载体。
本发明提供如前所述双特异性抗体、核酸序列、载体诸如AAV病毒载体、如前所述的AAV病毒颗粒、或如前所述的药物组合物在制备用于治疗或预防癌症、眼内新生血管综合征、类风湿性关节炎、银屑病、增殖性视网膜病变、年龄相关性黄斑变性或糖尿病性黄斑水肿的药物中的用途。
在一个实施例中，所述年龄相关性黄斑变性为湿性年龄相关性黄斑变性。
在一个具体实施例中，所述药物通过玻璃体腔或视网膜下腔注射施用。
附图说明
图1示出了XMVA01、XMVA04和XMVA09的结构示意图。
图2示出了pAAV9-XMVA09、ssAAV-XMVA09的载体信息。
图3示出了pAAV9-XMVA01、pAAV9-XMVA04和pAAV9-XMVA09载体转染细胞后VEGF165中和蛋白的表达量。
图4示出了在细胞中表达XMVA01、XMVA04和XMVA09对人视网膜微血管内皮细胞(HRMEC)在ECGS刺激下增殖的影响。
图5、6示出了在细胞中表达XMVA01、XMVA04和XMVA09对HRMECs管腔形成的影响。
图7示出了XMVA10的结构示意图。
图8示出了XMVA11的结构示意图。
图9示出了XMVA13的结构示意图。
图10示出了XMVA14的结构示意图。
图11示出了XMVA15的结构示意图。
图12示出了激光诱导wAMD模型小鼠注射AAV-XMVA09后的荧光素眼底血管造影(FFA)图像。
图13示出了激光诱导wAMD模型小鼠注射AAV-XMVA09后的3级光斑百分比及光斑渗漏平均分。
图14示出了激光诱导wAMD模型恒河猴对照组的IV级光斑数量和荧光渗漏面积变化。
图15示出了激光诱导wAMD模型恒河猴注射AAV-XMVA09的IV级光斑数量和荧光渗漏面积变化。
图16示出了激光诱导wAMD模型恒河猴对照组的IV级光斑视网膜厚度变化。
图17示出了激光诱导wAMD模型恒河猴注射AAV-XMVA09的IV级光斑视网膜厚度变化。
图18示出高糖诱导HRMECs的通透性变化。
图19示出基于VE-cadherin指标判断XMVA09对HRMECs通透性的调控效应。
图20示出基于生物素-亲和素系统指标判断XMVA09对HRMECs通 透性的调控效应。
图21示出糖尿病模型小鼠空腹血糖和体重的监测分析。
图22示出糖尿病模型小鼠视网膜血管渗漏图像。
图23示出糖尿病模型小鼠注射AAV-XMVA09后的视网膜血管渗漏面积变化。
发明详述
以下根据实施例，并且结合附图，详细描述本发明。从下文的详细描述中，本发明的上述方面和本发明的其他方面将是明显的。本发明的范围不局限于下列实施例。
本发明中的抗体是多特异性的、可以为人源化的、单链的、嵌合的、合成的、重组的、杂合的、突变的、以及嫁接的抗体；本发明中的抗体形式为抗体轻链可变区(VL)和抗体重链可变区(VH)拼接而成的scFv，该抗体可以结合两种不同的抗原，例如VEGF-A和ANG-2。
抗体轻链可变区(VL)和抗体重链可变区(VH)，VH和VL区可以进一步细分成：称为互补决定区(CDR)的高变区，以及穿插分布的称为框架区(FWR)的更保守区域。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。在一个实施方案中，每个VH和VL一般包括从氨基端到羧基端按以下顺序排列的3个CDR和4个FWR：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4。本发明公开的抗体和抗原结合片段的CDR由Kabat编号所定义或识别。
本文使用的“载体”是指包括重组多核苷酸的载体，所述重组多核苷酸包括可操作地连接至待表达的核苷酸序列的表达控制序列。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
实施例
实施例1：质粒载体的构建
表达anti-VEGF-A/ANG-2基因的AAV载体质粒的构建
抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用(G4S)3、G4S、(G4S)3肽接头连接，组成了结构为CD5-sp-VH抗VEGF-A- (G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，根据表1、表2中各自的序列进行构建，分别命名为XMVA01、XMVA04和XMVA09(由南京金斯瑞生物科技公司合成全基因)，其结构示意图见图1；
用BamH I/EcoR V双酶切上述构建体和pAAV9neo-CAG质粒，通过连接、转化及克隆筛选鉴定等常规分子生物学操作分别构建了pAAV9-XMVA01、pAAV9-XMVA04和pAAV9-XMVA09载体；pAAV9-XMVA09载体信息见图2A。用无内毒素的质粒提取试剂盒(MN)获得高质量的质粒DNA备用。
表1 XMVA09的序列信息



表2.XMVA01和XMVA04序列信息



实施例2：XMVA01、XMVA04、XMVA09在细胞中的表达测定
将pAAV9-XMVA01、pAAV9-XMVA04、pAAV9-XMVA09质粒分别转染HEK293T细胞，收集上清，用ELISA检测上清中蛋白的表达。具体操作如下：用含10％胎牛血清的DMEM的完全培养基重悬293T细胞后，接种至培养皿中，37℃、5％CO2培养箱培养至细胞的汇合度达到70％-90％时，采用Lipofectamine 2000(Invitrogen)促转试剂，按照其说明书转染质粒。继续培养72小时后，收集上清液，-80℃备用。
ELISA具体检测方法如下：以0.1μg/mL VEGF165(proteintech)蛋白包被酶标板(Thermo)，每孔100μL，4℃过夜孵育，用含有0.05％Tween(Sangon)的PBS洗涤酶标板3次，每次3分钟。用含有2％BSA(麦客生物)的PBS 200μL/孔室温封闭1小时，再次洗涤3次。在酶标板中分别加入康柏西普(10mg/mL)标准品和pAAV9-XMVA01、pAAV9-XMVA04、pAAV9-XMVA09质粒转染HEK293T细胞后收集的细胞上清，标准品从第一孔以312.5ng/mL浓度开始连续8个稀释度进行倍比稀释，待测上清以1：10或1：50进行倍比稀释，数据取平均值，以未转染质粒的细胞上清液作为对照组(Control组)，37℃孵育1小时，洗涤3次。每孔加入1：5000稀释的辣根过氧化氢酶标记的羊抗人IgG抗体(JacksonImmunoResearch)，37℃孵育1小时，再洗涤3次。每孔加入TMB显色液(Beyotime)100μL/孔，室温避光反应6分钟，向上述孔中加入100μL/孔终止液(Beyotime)终止反应。用酶标仪在450nm处测定OD值，计算待测样品的含量，结果见图3。pAAV9-XMVA01、pAAV9-XMVA04和pAAV9-XMVA09载体转染细胞后能有效表达VEGF165中和蛋白，和对照组相比，具有显著差异。
实施例3：在细胞中表达XMVA01、XMVA04、XMVA09对人视网膜微血管内皮细胞(HRMEC)增殖的影响
用含1％ECGS的ECM的完全培养基(Science Cell)调整HRMECs 的细胞密度为4×104/mL，100μL/孔接种于平底96孔板中；将pAAV9-XMVA01、pAAV9-XMVA04、pAAV9-XMVA09质粒分别转染HEK293T细胞后收集的细胞上清以100μL/孔加入孔内，以未转染质粒的细胞上清作为对照组(control组)，37℃、5％CO2培养箱中培养72小时；按照Cell Counting Kit-8(CCK-8，Biosharp)说明书分别在实验组和对照组中加入20μL/孔的CCK-8的溶液，孵育4小时后，用酶标仪在450nm波长下检测OD值。结果见图4，pAAV9-XMVA09所表达蛋白能有效抑制ECGS刺激下的HRMECs的增殖，且抑制效果显著优于pAAV9-XMVA01和pAAV9-XMVA04。
实施例4：在细胞中表达XMVA01、XMVA04、XMVA09对HRMECs管腔形成的影响
将解冻后的Matrigel(Corning)基质胶均一地铺在平底96孔板中，50μL/孔，37℃、5％CO2培养箱孵育1小时；以pAAV9-XMVA01、pAAV9-XMVA04、pAAV9n-XMVA09质粒分别转染HEK293T细胞后收集的细胞上清处理HRMECs，以未转染质粒的细胞上清作为对照组(control组)，培养48小时后用不含生长因子和血清的ECM的基础培养基重悬，并调整细胞密度为2×105/mL，100μL/孔接种在含基质胶的96孔板内；于37℃、5％CO2培养箱中培养，每间隔2小时观察一次，4-8小时后，在显微镜下观察、拍照，记录经待测上清处理后对HRMECs管腔的形成的影响。结果见图5，pAAV9-XMVA09所表达的蛋白能有效抑制HRMECs管腔的形成，且抑制效果显著优于pAAV9-XMVA01和pAAV9-XMVA04。
实施例5：其他质粒载体的构建
方案1
抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G4S、(G4S)3、G4S肽接头连接，组成了结构为CD5-sp-VL抗 ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA10(见图7)。其中抗VEGF的 VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA09相同。
方案2
抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G4S、(G4S)3、G4S肽接头连接，组成了结构为CD5-sp-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S-VH抗VEGF-A的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA11(见图8)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA09相同。
方案3
抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用(G4S)3、G4S、(G4S)3肽接头连接，组成了结构为CD5-sp-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA13(见图9)。
方案4
抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列,分别用G4S、(G4S)3、G4S肽接头连接，组成了结构为CD5-sp-VL抗 ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA14(见图10)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA13相同。
方案5
抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列，在N端加上分泌信号肽CD5-sp核苷酸序列, 分别用G4S、(G4S)3、G4S肽接头连接，组成了结构为CD5-sp-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S-VH抗VEGF-A的开放阅读框，根据人密码子偏好设计核苷酸序列，并在5’端引入BamH I酶切位点，在3’端引入EcoR V酶切位点，命名为XMVA15(见图11)。其中抗VEGF的VH氨基酸序列和VL氨基酸序列、抗ANG-2的VH氨基酸序列和VL氨基酸序列与XMVA13相同。
通过连接、转化及克隆筛选鉴定等常规分子生物学操作构建了XMVA10、XMVA11、XMVA13、XMVA14和XMVA15载体，用无内毒素的质粒提取试剂盒(MN)获得高质量的质粒DNA备用。
按照实施例2中所述方法，将质粒分别转染HEK293T细胞，收集上清，用ELISA方法检测上清中蛋白的表达。按照实施例3中所述的方法，在细胞中表达上述载体对HRMECs增殖的影响。按照实施例4中所述的方法，在细胞中表达上述质粒对HRMECs管腔形成的影响。按照实施例5中所述的方法，制备重组AAV病毒并鉴定。
表3.XMVA10至XMVA15的序列信息






实施例6：重组AAV病毒的制备和鉴定
用BamH I/EcoR V双酶切上述XMVA09构建体和ssAAV质粒，通过连接、转化及克隆筛选鉴定等常规分子生物学操作构建ssAAV-XMVA09载体，其载体信息见图2B。用无内毒素的质粒提取试剂盒(MN)获得高质量的质粒DNA，并使用三质粒包装系统制备重组AAV病毒，辅助质粒(phelper)、AAV的Cap和Rep蛋白表达质粒、表达目的基因质粒(ssAAV-XMVA09)按照2:1:1的质量比，和PEI促转剂形成转染复合物，转染HEK293T细胞，进行AAV-XMVA09病毒包装。分别于转染后第3天和第7天，进行两次上清的收集，即获得含有目的基因的AAV病毒颗粒。采用不同梯度的碘克沙醇(15％、25％、40％和60％)进行密度梯度离心(Beckman的超速离心机)，获得纯化的AAV病毒。对获得的纯化的AAV病毒，通过透射电子显微电镜进行AAV质量的鉴定和qPCR进行AAV病毒滴度的定量。
实施例7：XMVA09对激光诱导wAMD模型小鼠CNV的抑制作用
通过高能量激光光凝视网膜是目前国内外普遍使用的CNV动物模型，也是目前大多数治疗评估实验中的标准动物模型。其选择性破坏光感受器外节盘膜、Bruch膜、视网膜色素上皮以及部分前脉络膜毛细血管网，随后发生损伤修复反应，包括成纤维细胞、视网膜色素上皮细胞和血管内皮细胞侵入生长，最终在光凝区内形成新生血管。
SPF级6～8周雄性C57BL/6J小鼠30只(购自北京维通利华实验动物技术有限公司)，体重22-25g，具体给药方案见下表4：
表4.给药方案
由于AAV注射后需要一定时间目的基因的表达才能达到稳定水平，于第1天进行双眼玻璃体腔注射，在第22天对注射成功动物进行双眼眼 底激光光凝以诱导CNV模型。
小鼠CNV模型的建立：小鼠双眼各滴复方托吡卡胺滴眼液1-2滴散瞳，肌肉注射5％水合氯醛实施麻醉。麻醉后，双眼滴卡波姆滴眼液，放置眼底激光镜，在距离视盘约1.5-2PD处围绕视乳头避开血管进行光凝。激光参数为：波长532nm，功率80mW，光斑直径50μm，曝光时间100ms，光凝后动物双眼涂红霉素眼膏。
于第29天对小鼠进行荧光素眼底血管造影(FFA)检测：腹腔注射荧光素钠注射液(15mg/mL，10mL/kg)，注入荧光素钠后早期(1.5分钟内)、晚期(3分钟后)双眼采集清晰图片若干张，对有效光斑的荧光渗漏程度进行评级，计算3级渗漏光斑百分比及光斑渗漏平均分。结果见图12、图13，在激光诱导wAMD模型小鼠上，单次玻璃体腔注射AAV-XMVA09后对CNV形成有显著抑制作用。
有效光斑是指其附近无严重视网膜出血、在FFA中能完整显示的光斑。光斑荧光渗漏评级标准为：0级(无荧光渗漏)，1级(轻度荧光渗漏，渗漏面积为激光光斑大小的1～50％)，2级(中度荧光渗漏，渗漏面积为激光光斑大小的50～100％)，3级(重度荧光渗漏，渗漏面积大于激光光斑大小)。
各级光斑百分比(％)＝对应级别光斑总数÷4种光斑总数×100％。
光斑渗漏平均分＝[(0级光斑数×0)+(1级光斑数×1)+(2级光斑
数×2)+(3级光斑数×3)]÷4种光斑总数。
实例8：XMVA09对激光诱导wAMD模型恒河猴CNV的抑制作用
普通级(试验前检疫合格)5-6岁雄性恒河猴7只(购自雅安普莱美生物科技有限公司)，具体给药方案见下表5：
表5.给药方案
XMVA09组：于激光造模前21天双眼玻璃体腔注射给予AAV-XMVA09(50μL/眼)，并于第0天进行双眼眼底激光诱导CNV。
对照组：于第0天进行双眼眼底激光诱导CNV，于第21天双眼玻璃体腔注射给予PBS(50μL/眼)。
所有动物激光造模前进行光学相干断层扫描(OCT)检查，激光造模第19天及第77天进行眼底彩照(FP)、FFA、OCT检查；将IV级光斑的数量、渗漏面积及视网膜厚度作为主要药效评价指标。恒河猴CNV模型的建立：动物经盐酸氯胺酮注射液(20mg/kg)和盐酸右美托咪定注射液(0.03mg/kg)肌肉注射麻醉，双眼滴加美多丽(复方托吡卡胺滴眼液)散瞳。双眼瞳孔大于6mm后，将动物头部固定于眼科激光光凝仪前，通过眼底镜窥清视网膜结构后，围绕黄斑中心一个视盘距离进行激光光凝，每眼9个点。激光参数为：波长532nm，功率650mW-700mW，光斑直径50μm，曝光时间0.1秒。FP和FFA测量荧光素渗漏面积，具体检查方法如下：(1)动物经盐酸氯胺酮注射液(20mg/kg)和盐酸右美托咪定注射液(0.03mg/kg)肌肉注射麻醉。(2)双眼各滴加1-2滴复方托吡卡胺滴眼液散瞳。(3)散瞳后观察动物瞳孔，若瞳孔在光下大于6mm则通过眼底照相机拍摄以黄斑为中心的眼底彩照。(4)之后自下肢隐静脉快速推注0.075mL/kg剂量的10％荧光素钠注射液(历设得，Alcon)。在荧光素注射后的早期(1分钟内)，中期(5分钟)和晚期(10分钟)，通过眼底照相机拍摄以黄斑为中心的FFA照片。(6)通过Image J软件(版本1.53e,NIH,Wayne,Rasband,USA)，自动测量FFA晚期(10min分钟)照片中IV级光斑的荧光素渗漏面积，作为FFA的药效评价指标。激光光斑的荧光素渗漏分级标准为：I级(光斑无高荧光)，II级(光斑高荧光但无渗漏)，III级(光斑高荧光，轻度荧光素渗漏，但渗漏不超过光斑边缘)，IV级(光斑高荧光，显著荧光素渗漏，且渗漏超过光斑边缘)。
OCT测量视网膜厚度变化，具体检查方法如下：(1)动物完成FP和FFA检查后，置于OCT仪器前，调整动物双眼平视扫描镜头。(2)以黄斑为中心采用随访模式进行多层线状扫描，扫描区域覆盖所有激光点。(3)选取造模后视网膜厚度变化最明显且视网膜边界清晰的层面，使用仪器自带的测量软件测定该区域视网膜厚度最大值作为OCT的药效评价指标。
结果见图14、图15、图16、图17。各组动物激光造模第19天及第77天时，FFA检查可见眼底激光光斑处有不同的荧光渗漏表现。与对照组比较，单次玻璃体腔注射AAV-XMVA09对激光诱导wAMD模型恒河猴CNV的IV级光斑的数量、渗漏面积及视网膜厚度有明显的改善作用。
实例9：高糖诱导HRMECs通透性增加模型
用0.1％的明胶对24孔板进行包板预处理，37℃，1-2个小时后，干燥数分钟；再用含10％FBS+1％PS+1％ECGS的ECM(Sciencell)的完全培养基，将HRMECs重悬后，按照3×105/孔的标准，均匀平铺在孔板中；次日，用含0.5％FBS的低血清培养基开始进行饥饿处理，共7小时；再将HRMECs暴露于30mM的高糖环境中，过夜；对照组未经高糖处理；次日，开始进行细胞的膜蛋白VE-cadherin和细胞核DAPI的免疫荧光共染色，荧光显微镜观察及拍照。结果见图18，高糖处理后的HRMECs的细胞膜连续性被破坏，细胞间隙增大，通透性增加。
免疫荧光染色的步骤：弃培养基上清，余200μL的量，再补加200μL的4％的PFA开始进行梯度固定，室温固定20分钟；弃上清后，直接加200μL的PFA继续室温固定10分钟；弃固定液后，用PBS进行洗涤，洗涤3次，每次5分钟；用PBS对VE-cadherin(Santa Cruz Biotechnology Inc)按照1:150进行稀释后，300μL/孔，室温孵育2小时；弃上清，用PBS进行洗涤，洗涤3次，每次5分钟；用PBS对Donkey anti-Mouse IgG(H+L)Highly Cross-Adsorbed Secondary Antibody,Alexa FluorTM 488(Invitrogen)按照1:200稀释后，每孔200μL，37℃共孵育1小时；弃上清，用PBS进行洗涤，洗涤3次，每次5分钟；最后加入含DAPI的封片剂(碧云天)100μL/孔，荧光显微镜观察及拍照。
实例10：基于VE-cadherin指标判断XMVA09蛋白对HRMECs通透性的调控效应
用0.1％的明胶对24孔细胞培养板进行预包被，4度过夜或37℃2小时；弃去包被液，开始接种HRMECs细胞，3×105/孔；次日，待单层细胞达到一定的汇合度，开始用含0.5％FBS的低血清培养基饥饿处理7小时； 用未转染的HEK293T细胞上清、ssAAV-XMVA09质粒转染HEK293T表达的细胞上清分别与高糖(30mM的葡萄糖)共孵育，对HRMECs过夜处理，对照组为无高糖处理的非造模组；进行VE-cadherin的免疫荧光染色，详细步骤如实施例9，在荧光显微镜下进行拍照分析。结果见图19，ssAAV-XMVA09所表达蛋白对高糖处理后的HRMECs的通透性的增加具有显著抑制效应。
实例11：基于生物素-亲和素系统指标判断XMVA09蛋白对HRMECs通透性的调控效应
用EZ-LinkTM NHS-LC-LC-生物素对明胶(gelatin)处理，获得生物素化的明胶；用该生物素化的明胶包被细胞培养板，4℃过夜或37℃2小时；直接在培养板中接种HRMECs，细胞的接种量为3×105/孔；次日，先用含0.5％FBS的低血清培养基开始进行饥饿处理7小时；再用未转染的HEK293T细胞上清、ssAAV-XMVA09质粒转染HEK293T表达的细胞上清分别与高糖(30mM的葡萄糖)共孵育，对HRMECs处理17小时，对照组为无高糖处理的非造模组；用FITC-avidin进行染色，再用4％PFA固定后，开始AF594-phalloidin(Invitrogen A12381)的染色，再加入含DAPI的抗荧光淬灭剂，在荧光显微镜下进行拍照分析。结果见图20，ssAAV-XMVA09所表达蛋白对高糖处理后的HRMECs的通透性的增加具有显著抑制效应。
实例12：糖尿病小鼠模型构建
药物诱导是目前建立糖尿病动物模型的方法之一，能够较好模拟糖尿病临床病理表现及临床特征。链脲佐菌素(STZ)是诱发糖尿病最常用的药物，其主要通过破坏胰岛β细胞导致高血糖，继而引起毛细血管周细胞丢失、血管层变薄及血-视网膜屏障破坏，进一步导致血管渗漏形成，是研究DME的良好模型。
糖尿病小鼠模型的建立：
SPF级6～8周雄性C57BL/6J小鼠80只(购自浙江维通利华实验动物技术有限公司)，体重20-25g，将小鼠随机分为空白组、DME造模对照组、DME造模组。造模前测量三组小鼠空腹血糖及体重：禁食6小时(自 由饮水)后，尾尖采血进行血糖测量并称重，此时为造模第0天。于第1天开始造模，小鼠禁食(自由饮水)6小时，空白组无处理，DME造模对照组连续5天单次腹腔注射50mM柠檬酸钠溶液(Sigama)60mg/kg，DME造模组连续5天单次腹腔注射STZ(Sigama)60mg/kg，注射后自由摄食和饮水，定期测量血糖及体重并记录，造模持续135天。
结果见图21，造模第19天，小鼠空腹血糖值持续约三周高于16.7mmol/L，糖尿病小鼠模型构建成功。
实施例13：糖尿病小鼠模型DME表型验证
于造模第81天，对实施例12中的三组小鼠各取部分小鼠采用伊文思蓝尾静脉注射方法，解剖分离视网膜，荧光显微镜下观察血管渗漏。具体操作如下：伊文思蓝(Sigama)50mg/mL尾静脉注射50μL/只，待血液循环1.5小时后，将小鼠安乐死，摘取双眼于4％多聚甲醛(Biosharp)中固定45分钟，剥离眼球视网膜进行铺片，于荧光显微镜下观察并拍照。
结果见图22，DME造模组视网膜血管渗漏较DME造模对照组及空白组都有明显增多，DME造模对照组与空白组视网膜之间无明显差异，糖尿病小鼠模型具有DME表型。
实施例14：XMVA09对糖尿病小鼠模型视网膜血管渗漏的抑制作用
于造模后第95天对实施例12中的DME造模组小鼠注射给药，具体给药方案见下表6：
表6.给药方案
于注射后第29天，对上述XMVA09组、对照组、及实施例12中的DME造模对照组采用伊文思蓝尾静脉注射方法，解剖分离视网膜，荧光显微镜下观察血管渗漏。
结果见图23，DME造模对照组视网膜血管渗漏百分比显著低于对照组，XMVA09组视网膜血管渗漏百分比显著低于对照组，表明单次玻璃体腔注射AAV-XMVA09后对糖尿病小鼠模型视网膜血管渗漏有显著抑制作用。
统计分析
采用GraphPad Prism 8.0软件进行数据处理和统计分析。统计学水平设在5％或p≤0.05，计算各项分析指标的平均数和标准误(Mean±SEM)，p≤0.05即为差异具有统计学意义。

Claims (16)

  1. 一种双特异性抗体，其包括抗VEGF-A和ANG-2的结合结构域，所述双特异性抗体重链可变区(VH)和轻链可变区(VL)从N-末端到C-末端按照如下顺序排列：
    1)VH抗VEGF-A-VL抗VEGF-A-VH抗ANG-2-VL抗ANG-2；
    2)VL抗ANG-2-VH抗VEGF-A-VL抗VEGF-A-VH抗ANG-2；或
    3)VL抗VEGF-A-VH抗ANG-2-VL抗ANG-2-VH抗VEGF-A；
    其中，所述VH抗VEGF-A包括SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2；SEQ ID NO:3所示的CDR3；
    所述VL抗VEGF-A包括SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2；SEQ ID NO:6或SEQ ID NO:47所示的CDR3；
    所述VH抗ANG-2包括SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2；SEQ ID NO:9所示的CDR3；
    所述VL抗ANG-2包括SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2；SEQ ID NO:12所示的CDR3。
  2. 如权利要求1所述的双特异性抗体，其中
    所述VH抗VEGF-A包括SEQ ID NO:13所示的序列或与SEQ ID NO:13序列同一性为70％、80％、90％、95％、或99％的序列；
    所述VL抗VEGF-A包括SEQ ID NO:14所示的序列或与SEQ ID NO:14或SEQ ID NO:48序列同一性为70％、80％、90％、95％、或99％的序列；
    所述VH抗ANG-2包括SEQ ID NO:15所示的序列或与SEQ ID NO:15序列同一性为70％、80％、90％、95％、或99％的序列；
    所述VL抗ANG-2包括SEQ ID NO:16所示的序列或与SEQ ID NO:16序列同一性为70％、80％、90％、95％、或99％的序列。
  3. 如权利要求1所述的双特异性抗体，其中所述抗体包括SEQ ID NO:17、18、19、49、51或53所示的序列或与SEQ ID NO:17、18、19、49、51或53序列同一性为70％、80％、90％、95％、或99％的序列。
  4. 如权利要求1所述的双特异性抗体，所述双特异性抗体的N-末端包含信号肽序列或标签序列，优选所述信号肽序列为如SEQ ID NO:23所示的CD5-sp信号肽。
  5. 如权利要求4所述的双特异性抗体，其中所述抗体包括SEQ ID NO:20、21、22、50、52或54所示的序列或与SEQ ID NO:20、21、22、50、52或54序列同一性为70％、80％、90％、95％、或99％的序列。
  6. 如权利要求1所述的双特异性抗体，其中所述重链可变区(VH)和轻链可变区(VL)之间由(G4S)n链接，其中n＝1至4间任意整数。
  7. 如权利要求1所述的双特异性抗体，其中所述双特异性抗体的结构为：
    VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2；
    VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2；
    VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S-VH抗VEGF-A；
    CD5-sp-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗 ANG-2；
    CD5-sp-VL抗ANG-2-G4S-VH抗VEGF-A-(G4S)3-VL抗VEGF-A-G4S-VH抗ANG-2；或
    CD5-sp-VL抗VEGF-A-G4S-VH抗ANG-2-(G4S)3-VL抗ANG-2-G4S-VH抗VEGF-A。
  8. 一种核酸序列，其编码如权利要求1-7所述的双特异性抗体。
  9. 一种载体，其包括编码如权利要求1-7所述的双特异性抗体的核酸序列，所述载体优选为AAV病毒载体。
  10. 如权利要求9所述的AAV病毒载体，进一步包括：5’ITR和3’ITR、启动子、polyA序列。
  11. 一种AAV病毒颗粒，包括如权利要求9或10所述的AAV病毒载体和衣壳蛋白。
  12. 如权利要求11所述的AAV病毒颗粒，其中所述衣壳蛋白的血清型为AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、或AAV9。
  13. 一种药物组合物，包括如权利要求1-7所述的双特异性抗体、如权利要求8所述的核酸序列、如权利要求9或10所述的载体、如权利要求11或12所述的AAV病毒颗粒中的至少一种，以及药学上可接受的载体。
  14. 如权利要求1-7所述的双特异性抗体、如权利要求8所述的核酸序列、如权利要求9或10所述的载体、如权利要求11或12所述的AAV病毒颗粒或如权利要求13所述的药物组合物在制备用于治疗或预防癌症、眼内新生血管综合征、类风湿性关节炎、银屑病、增殖性视网膜病变、年龄相关性黄斑变性或糖尿病性黄斑水肿的药物中的用途。
  15. 如权利要求14所述的用途，所述年龄相关性黄斑变性为湿性年龄相关性黄斑变性。
  16. 如权利要求14或15所述的用途，其中所述药物通过玻璃体腔、视网膜下腔或者脉络膜上腔注射施用。
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