Composition and application of nanobody and hr2-fc fusion protein synergistic treatment of feline infectious peritonitis

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a composition and application for synergistically treating feline infectious peritonitis based on a nano-antibody and an HR2-Fc fusion protein. The composition comprises a nano-antibody 2F1 for neutralizing FIPV and a recombinant fusion protein HR2-Fc, wherein the nano-antibody 2F1 blocks the binding of the virus to the host cell by specific binding to FIPV S protein; the recombinant fusion protein HR2-Fc plays a role by competitively inhibiting viral membrane fusion. Further, the composition can be combined with a TNF-alpha inhibitory antibody, IFN-omega or a nucleoside analogue GS-441524, and is administered by subcutaneous injection to significantly improve the symptom relief rate. The present application synergistically intervenes from the double targets of viral invasion and membrane fusion, and provides an efficient and precise new scheme for FIP treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition and its application for the synergistic treatment of feline infectious peritonitis based on nanobodies and HR2-Fc fusion protein. Background Technology

[0002] Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV), seriously threatening the health and lives of cats. FIP has a complex pathogenesis, diverse clinical manifestations, rapid progression, and extremely high mortality rate, causing great distress to pet owners and veterinarians. Currently, treatment options for FIP remain extremely limited. Although some antiviral drugs, such as GS-441524, have shown some efficacy, they cannot completely cure the disease. Existing treatments mostly focus on symptom relief rather than directly targeting the virus itself. Therefore, a new and effective treatment method is urgently needed to improve the cure rate of FIP and the prognosis of affected cats.

[0003] In recent years, research on treatments for viral infections has gradually shifted towards immunotherapy and combination therapies. Nanobodies, due to their small molecular weight, superior stability, and specificity, have emerged as a novel antiviral therapeutic tool. In research on combating the novel coronavirus, nanobodies have demonstrated unique advantages. Several nanobodies have been identified that can efficiently neutralize SARS-CoV-2 by binding to the S1 receptor-binding domain (RBD). Related research findings have been published in authoritative journals such as Science, Nature, Nature Communications, and PNAS.

[0004] In addition to neutralizing antibodies, researchers have also discovered that the HR2 peptide can competitively bind to inhibit the interaction between HR1 and HR2, blocking the fusion of the virus with the cell membrane. This intervention can effectively prevent the virus from invading host cells, thereby reducing the risk of infection. The HR2 peptide has received widespread attention as a potential antiviral target, particularly in SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), MERS-CoV (Middle East Respiratory Syndrome Coronavirus), and more recently, SARS-CoV-2 (Novel Coronavirus). These research advances provide new insights into developing effective treatments for FIP.

[0005] Combination therapy is considered a potential approach to improve the cure rate of complex diseases, and there are many successful cases in human medicine. However, its application in the treatment of febrile diseases (FIP) is still in the exploratory stage. Although researchers are aware of the limitations of single treatment methods and have tried to combine different drugs or treatment technologies, due to a lack of in-depth understanding of the synergistic mechanisms of each component, they often simply piece together existing treatments and fail to fully realize the advantages of combination therapy.

[0006] In conclusion, there is an urgent need in the market for an innovative treatment plan that targets key aspects of feline infectious peritonitis virus (FIP) infection, integrates multiple effective treatment methods, and precisely regulates the body's immune response. This plan should not only directly attack the virus but also consider the regulation and maintenance of the host's immune response to improve the cure rate of infected cats. Summary of the Invention

[0007] The purpose of this invention is to provide a composition and application for the synergistic treatment of feline infectious peritonitis based on nanobodies and HR2-Fc fusion protein, thereby overcoming the shortcomings of the prior art, proposing a unique virus blocking scheme and combination drug strategy, and hoping to bring a revolutionary breakthrough to the treatment of feline infectious peritonitis.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a composition comprising at least one of nanobody 2F1 and recombinant fusion protein HR2-Fc; The variable region of the nanobody 2F1 has a CDR1 region as shown in SEQ ID NO.1, a CDR2 region as shown in SEQ ID NO.2, and a CDR3 region as shown in SEQ ID NO.3; The amino acid sequence of the recombinant fusion protein HR2-Fc is shown in SEQ ID NO.11.

[0009] In some other embodiments, the nanobody 2F1 further includes a backbone region having FR1 as shown in SEQ ID NO.4, FR2 as shown in SEQ ID NO.5, FR3 as shown in SEQ ID NO.6, and FR4 as shown in SEQ ID NO.7.

[0010] In some other embodiments, the recombinant fusion protein HR2-Fc comprises the HR2 peptide and Fc fragment of the FIPV S protein; The amino acid sequence of the HR2 peptide in the FIPV S protein is shown in SEQ ID NO.8; The amino acid sequence of the Fc fragment is shown in SEQ ID NO.9; The HR2 peptide and Fc fragment in the FIPV S protein are linked by a linker fragment. The connecting segment is (GGGGS)n, where n is a natural number from 1 to 6; Add a secretion signal peptide as shown in SEQ ID NO.10 to the N-terminus.

[0011] In some other embodiments, at least one of the following components is also included: (1) Tumor necrosis factor α inhibitory antibody; (2) Interferon ω; (3) Nucleoside analogue GS-441524.

[0012] In some other embodiments, the tumor necrosis factor α inhibitory antibody is ozolazumab.

[0013] In some other embodiments, a composition includes at least one of nanobody 2F1, recombinant fusion protein HR2-Fc, and GS-441524; it can be used as... Specifically, it includes any one, two, or three of the following: nanobody 2F1, recombinant fusion protein HR2-Fc, and GS-441524; Alternatively, any three, four, or five of the following: nanobody 2F1, recombinant fusion protein HR2-Fc, GS-441524, interferon ω, and ozolazumab.

[0014] In some other embodiments, the mixing mass ratio of the nanobody 2F1 and HR2-Fc is (0.5-2):(0.5-2). Alternatively, the mass ratio of the nanobody 2F1 and GS-441524 is (0.5-2):(4.5-5.5). Alternatively, the mixing mass ratio of HR2-Fc and GS-441524 is (0.5-2):(4.5-5.5). Alternatively, the mass ratio of the nanobody 2F1, HR2-Fc, and GS-441524 is (0.5-2):(0.5-2):(4.5-5.5). Alternatively, the mixed mass ratio of GS-441524, interferon ω, and ozolazumab is (4.5-5.5):(0.05-0.15):(0.15-0.25). Alternatively, the mass ratio of nanobody 2F1, recombinant fusion protein HR2-Fc, GS-441524, and ozolazumab may be (0.5-2):(0.5-2):(4.5-5.5):(0.15-0.25). Alternatively, the mass ratio of nanobody 2F1, recombinant fusion protein HR2-Fc, GS-441524, and interferon ω may be (0.5-2):(0.5-2):(4.5-5.5):(0.05-0.15): Alternatively, the mixed mass ratio of recombinant fusion protein HR2-Fc, GS-441524, interferon ω, and ozolazumab is (0.5-2):(4.5-5.5):(0.05-0.15):(0.15-0.25). Alternatively, the mass ratio of nanobody 2F1, recombinant fusion protein HR2-Fc, GS-441524, interferon ω, and ozolazumab is (0.5-2):(0.5-2):(4.5-5.5):(0.05-0.15):(0.15-0.25).

[0015] In a second aspect, the present invention provides the use of the composition described in the first aspect in the preparation of a product for treating feline infectious peritonitis.

[0016] In some other embodiments, the feline infectious peritonitis has the following symptoms: (1) The FIPV nucleic acid test was positive; (2) White ball ratio ≤ 0.5 (3) Depression and loss of appetite; (4) Fluid accumulation in the chest or abdomen.

[0017] In some other embodiments, the product is a drug, and the dosage form of the drug is an injection.

[0018] In some other embodiments, the dosage range of the nanobody 2F1 in the drug is 0.5-2 mg / kg; And / or, the dose range of the recombinant fusion protein HR2-Fc is 0.5-2 mg / kg.

[0019] In some other embodiments, the drug may further include pharmaceutically acceptable excipients and / or carriers.

[0020] The beneficial effects of this invention are: This invention combines FIPV neutralizing nanobodies with HR2 fusion protein to inhibit viral invasion and membrane fusion, two critical critical points, resulting in a significant improvement in antiviral efficacy compared to traditional single-treatment modalities. Through ingenious combination and flexible use with multiple components such as TNF-α inhibitory antibodies, IFN-ω, and GS-441524, it comprehensively regulates the body's antiviral immune response mechanism, effectively inhibiting viral replication and significantly reducing inflammatory damage. This allows for personalized and precise treatment plans tailored to cats at different stages of the disease and with varying individual differences, potentially significantly improving the cure rate of feline infectious peritonitis (FIP), reducing mortality, comprehensively improving the quality of life of affected cats, and optimizing prognosis. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is an SDS-PAGE image of purified FCoV S1; Figure 2 This is an SDS-PAGE image of the purified nanobody; Figure 3 This demonstrates the neutralizing effect of nanobodies on FCoV. Figure 4 This is an SDS-PAGE image of purified HR2-Fc. Figure 5 This is to demonstrate the neutralizing effect of HR2-Fc on FCoV; Figure 6 This is an SDS-PAGE image of purified FeIFN-ω. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the embodiments employ conventional techniques in the art. Unless otherwise specified, all chemical reagents are commercially available analytical grade reagents.

[0024] This invention provides a composition for treating feline infectious peritonitis, its preparation method, and its application, wherein... The FIPV neutralizing nanobody 2F1 can specifically bind to the FIPV S protein, thereby effectively blocking the binding of the virus to the host cell receptor and inhibiting FIPV infection.

[0025] The nanobody 2F1 includes three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein: CDR1 has the amino acid sequence shown in SEQ ID NO.1, CDR2 has the amino acid sequence shown in SEQ ID NO.2, and CDR3 has the amino acid sequence shown in SEQ ID NO.3; the FIPV neutralizing nanobody 2F1 also includes four frame regions (FR1, FR2, FR3, and FR4), wherein: the amino acid sequence of frame region FR1 is shown in SEQ ID NO.4; the amino acid sequence of frame region FR2 is shown in SEQ ID NO.5; the amino acid sequence of frame region FR3 is shown in SEQ ID NO.6; and the amino acid sequence of frame region FR4 is shown in SEQ ID NO.7.

[0026] The recombinant protein HR2-Fc, formed by fusing the HR2 peptide and Fc fragment of the FIPV S protein, competitively blocks the fusion process between FIPV and the host cell membrane, thereby inhibiting viral entry. The amino acid sequence of the HR2 peptide is shown in SEQ ID NO. 8, the amino acid sequence of the feline Fc is shown in SEQ ID NO. 9, the HR2 and Fc fragments are linked by a (GGGGS)3 linker, and a secretion signal peptide is added to the N-terminus, as shown in SEQ ID NO. 10. The complete amino acid sequence of HR2-Fc is shown in SEQ ID NO. 11.

[0027] The specific amino acid sequences involved are as follows: The amino acid sequence of CDR1 in nanobody 2F1: GDISGAYA (SEQ ID NO.1) The amino acid sequence of CDR2 of nanobody 2F1: MDPEGRK (SEQ ID NO.2) The amino acid sequence of CDR3 of nanobody 2F1: RLITGTGTDY (SEQ ID NO.3) The amino acid sequence of FR1 in nanobody 2F1: QVQLQESGGDLVQPGGSLTLTCVIS (SEQ ID NO.4) The amino acid sequence of FR2 of nanobody 2F1: WTWYRRAPEQEREMVAV(SEQ ID NO.5) The amino acid sequence of FR3 of nanobody 2F1: NYGDSVKGRFTITRDKTKRLMTLRMNSLKPEDTAAYYW (SEQ ID NO.6) The amino acid sequence of FR4 of nanobody 2F1: WGQGTQVTVSS (SEQ ID NO.7) A recombinant protein, HR2-Fc, is provided, which is a fusion of the HR2 peptide and the Fc fragment of the FIPV S protein. This fusion protein can competitively block the fusion process between FIPV and the host cell membrane, thereby inhibiting viral entry. The amino acid sequence of the HR2 peptide is shown in SEQ ID NO.8, the amino acid sequence of the cat Fc is shown in SEQ ID NO.9, the HR2 and Fc fragments are linked by a (GGGGS)3 linker, and a secretion signal peptide is added to the N-terminus, as shown in SEQ ID NO.10. The complete amino acid sequence of HR2-Fc is shown in SEQ ID NO.11.

[0028] The amino acid sequence of the HR2 peptide: FNATYLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTLVNLEWLNRIE (SEQ ID NO.8) The amino acid sequence of cat Fc: PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKG QPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO.9) The amino acid sequence with the secretion signal peptide added to the N-terminus is as follows: MESPAQLLFLLLLLWLPDTQA (SEQ ID NO.10) The complete amino acid sequence of HR2-Fc: MESPAQLLFLLLLLWLPDTQAFNATYLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTLVNLEWLNRIEGGGGSGGGGSGGGGSPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNST YRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO.11) Example 1 A method for preparing FIPV neutralizing nanobodies 1.1 Recombinant expression of FIPV S1 protein Based on the amino acid sequence of the FCoV S protein from NCBI (GenBank accession no. ACS44218.1), the Tyr250-Ser674 sequence was synthesized and ligated into the pET30a (+) vector via NdeI and XhoI restriction sites to obtain the recombinant expression plasmid pET30a-FCoV S1. pET30a-FCoV S1 was transformed into BL21(DE3) competent cells, and single colonies were picked and cultured in LB-K medium at 37°C with shaking overnight. The next day, the bacterial culture was diluted 1:100 with 100 mL of fresh LB-K medium and cultured at 37°C with shaking for 3 hours until the bacterial culture reached OD. 600 =Approximately 0.8, add 1 mM IPTG to a final concentration, and induce overnight at 30℃ and 200 rpm. The next day, collect bacterial cells by centrifugation at 8000 rpm and 4℃ for 10 minutes. Add 2 mL of bacterial lysis buffer to every 100 mg of bacterial cells (wet weight) and perform ultrasonic lysis. Collect the precipitate by centrifugation at 12000 rpm and 4℃ for 10 minutes. Dissolve the precipitate in Binding Buffer containing 8 M urea and perform nickel column affinity purification. The eluted protein is refolded by urea gradient dialysis to obtain FCoV S1 protein. Results are shown in […]. Figure 1 .

[0029] 1.2 Screening for anti-FCoV S1 nanobodies 1.2.1 Alpaca Immunization Adult alpacas of moderate size, good health, robustness, no signs of injury or discomfort, and in good spirits were selected. FCoV S1 protein and Gerbu adjuvant were mixed at a 1:1 ratio and injected into the alpaca's neck lymph nodes on both sides, with four injection points on each side, 0.5 mL per point. A total of four immunizations were administered, with each injection two weeks apart. Peripheral blood was then collected from the alpacas for constructing a phage display library.

[0030] 1.2.2 Isolation of alpaca lymphocytes Peripheral blood collected from alpacas was used to isolate lymphocytes using a camel peripheral blood lymphocyte separation kit (Tianjin Haoyang Company, catalog number LTS1076) according to the instructions. The separation was performed at a rate of 2.5 × 10⁶ cells per cell. 7 Add 1 mL of RNA separation reagent to each live cell, take 1 mL for RNA extraction, and place... Store at 80℃.

[0031] 1.2.3 RNA Extraction Repeatedly pipet 1 mL of Tipure Isolation Reagent containing lymphocytes and let stand for 5 minutes; add 200 μL of chloroform, shake vigorously vertically for 30 seconds, and let stand for another 5 minutes; centrifuge at 12000 g for 15 minutes at 4°C, and transfer the aqueous phase to a new EP tube; add an equal volume of isopropanol and let stand for 10 minutes; centrifuge at 12000 g for 10 minutes at 4°C and discard the supernatant; wash with 1 mL of pre-cooled 70% ethanol, centrifuge at 7500 g for 5 minutes at 4°C, discard the supernatant and dry for 5 minutes; add 30 μL of RNase Free water dissolves the precipitate.

[0032] 1.2.4 Reverse transcription to synthesize cDNA According to the reverse transcription kit instructions (ABM Bio All-In-One 5×RT Mastermix), the RNA obtained in step 1.2.3 was used as a template for reverse transcription of cDNA.

[0033] 1.2.5 Amplification of antibody variable region genes The cDNA obtained from reverse transcription was used as a template for the first round of PCR. The primer sequences for the first round of PCR are as follows: CALL001:GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO.12) CALL002:GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO.13) The conditions and procedure for the first round of PCR reaction are as follows: 95℃ for 5 minutes; 95℃ 30 s, 57℃ 30 s, 72℃ 30 s, 25cycles; 72℃ for 7 minutes.

[0034] The band of approximately 700 bp was recovered using an agarose gel extraction kit, and the nucleic acid concentration was adjusted to 5 ng / μL with water.

[0035] The first-round PCR product was used as a template for the second-round PCR. The primer sequences for the second-round PCR reaction are as follows: VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO.14) VHH-For: CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT (SEQ ID NO.15) The conditions and procedures for the second round of PCR reaction are as follows: 95℃ for 5 minutes; 95℃ 30 s, 55℃ 30 s, 72℃ 30 s, 25cycles; 72℃ for 5 minutes.

[0036] The second-round PCR products were purified using a PCR product recovery kit.

[0037] 1.2.6 Carrier Construction The pMES4 vector (purchased from Biovector) and the second-round PCR product were double-digested with PstI and BstEII, respectively. 2 μg of the digested vector and 2 μg of the digested second-round PCR product were added to 40 μL of T4 DNA ligase, and buffer and water were added to a total volume of 150 μL. The mixture was incubated overnight at 16°C, and the ligation product was recovered. The ligation was then completed using a PCR product recovery kit, followed by elution with 20 μL of water.

[0038] 1.2.7 Electroconversion and Storage Capacity Measurement Take 20 μL of the purified ligation product and add it to the bottom of a pre-cooled 2 mm electroporation cuvette containing 200 μL of *E. coli* TG1 competent cells. Mix and incubate on ice for 30 min. Wipe the cuvette clean and place it in an electroporator (Gene Pulser Xcell™, BIO-RAD) for electroporation. The electroporation parameters are set to 2.5 kV, 25 μF, and 200 Ω. Immediately after electroporation, add 800 μL of SOC medium, mix well, and transfer to a sterile EP tube. Incubate at 37°C and 200 r / min for 2 h. After incubation, aspirate 100 μL of bacterial culture and follow the 10... -1 10 -2 10-3 10 -4 10 -5 Up to 10 -6 Gradient dilution. Dilute 10... -4 10 -5 10 -6 100 μL of bacterial culture from each dilution was aspirated and plated onto 2YT-A plates, and incubated overnight at 37°C. Single colonies were counted on each dilution plate, and plates with an appropriate number of single colonies were selected. The library volume was calculated based on the dilution. Twenty single clones were randomly picked using a sterile pipette tip for colony PCR identification. The primer sequences for PCR identification are as follows: pMES-F: GCCGCTGGATTGTTATTACTC (SEQ ID NO. 16) pMES-R: CTTTCAACAGTGGAACCGTAG (SEQ ID NO. 17) The conditions and procedures for PCR identification reactions are as follows: 95℃ for 5 minutes; 95℃ 30 s, 55℃ 30 s, 72℃ 30 s, 30cycles; 72℃ for 5 minutes.

[0039] The PCR positivity rate was calculated based on the electrophoresis results, and the storage capacity was estimated.

[0040] Library capacity = number of clones × dilution factor × positive rate × 10 × library volume.

[0041] Storage capacity = 126 × 10 6 ×95%×10×2=2.39*10 9 CFU.

[0042] The calculated storage capacity is 2.39 * 10 9 CFU.

[0043] 1.2.8 Phage Display of Nanobodies The revived bacterial culture was inoculated into four 10 mL 2YT-AG culture media and cultured at 37°C and 200 rpm until the culture OD reached its maximum. 600 =0.5. Add 4×10 to every 10 mL of bacterial culture. 10PFU VCSM13 was incubated statically at 37°C for 30 minutes. The cells were centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was discarded. The cells were resuspended in 100 mL of 2×YT-AK medium (containing ampicillin and kanamycin) and incubated overnight at 37°C and 200 rpm. The overnight culture was centrifuged at 10800 g at 4°C for 15 min to collect the supernatant. 10 mL of PEG / NaCl (20% / 2.5M) solution was added to every 40 mL of supernatant and mixed thoroughly. The mixture was incubated on ice for 2 h, then centrifuged at 10800 g at 4°C for 30 min. The supernatant was discarded, and the precipitate was resuspended in 8 mL of ice-cold PBS. 2 mL of pre-chilled PEG / NaCl was added and mixed thoroughly. The mixture was incubated on ice for 1 h, then centrifuged at 3300 g at 4°C for 30 min. The precipitate was resuspended in 1 mL of PBS.

[0044] Phage titer determination: TG1 culture was cultured to OD600=0.4, and the phage was serially diluted with sterile PBS. The serially diluted TG1 phage culture (1:20) was mixed and incubated at 30℃ for 30 min. 100 μL was then spread on 2YT-AG solid plates. The plaque formation in the culture plates was observed the next day. Plaques in the dilution gradient plates with a number of plaques between 30 and 300 were counted and the phage titer (cfu) was calculated and displayed according to the following formula.

[0045] Phage titer (cfu / mL) = dilution factor × 2 × number of plaques × 100 1.2.9 Solid-phase panning of phage display libraries Dilute FCoV S1 antigen to 10 μg / mL with CBS, coat each well with 100 μL, incubate overnight at 4°C, and wash 5 times with PBST; add 250 μL of 1% BSA to each well, block at 37°C for 2 h, and wash 5 times with PBST; add 100 μL of diluted CBS to each well. 11 Display phages at cfu / mL were incubated at 37°C for 2 h, followed by 15-25 washes with PBST. After the final wash, 100 μL of glycine solution (0.2 M, pH 2.2) was added to each well, and the plates were incubated on a horizontal shaker for 15 min. The eluent from each well was then added to an EP tube containing 15 μL of Tris solution, and the titers were measured after pooling. The panning stringency was increased as needed based on the results of each round, for a total of 3-4 pannings.

[0046] 1.2.10 Phage ELISA screening for positive clones Dilute FCoV S1 antigen to 5 μg / mL with CBS, coat each well with 100 μL, incubate overnight at 4°C, and wash 5 times with PBST (0.05%). Add 250 μL of 5% BSA to each well, block at 37°C for 1 h, and wash 5 times with PBST (0.05%). Add 100 μL of overnight cultured monoclonal phage supernatant to each well, incubate at 37°C for 1 h, and wash 5 times with PBST (0.05%). Add 100 μL of HRP-labeled mouse anti-M13 secondary antibody to each well, incubate at 37°C for 1 h, and wash 5 times with PBST (0.05%). Add 100 μL of TMB chromogenic solution to each well, incubate at room temperature in the dark for 15-30 min, and add 100 μL of 2 M sulfuric acid stop solution to each well. Read the values ​​at 450 nm using a microplate reader. Fifty phage ELISA-positive clones were selected and sent for sequencing. Analysis revealed 11 different nanobody sequences.

[0047] 1.2.11 Induction, Expression, and Purification of Nanobodies The original strain TG1 glycerol bacteria containing the above 11 nanobody nucleic acids was inoculated into 5 mL of fresh LB-A medium at a ratio of 1:1000 and cultured overnight at 37°C and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the manufacturer's instructions. After verification, 1 μL of the plasmid was transformed into 100 μL of competent cells, gently mixed, placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, and cooled on ice for 2 minutes. 600 μL of LB medium was added to the centrifuge tube, and the mixture was cultured at 37°C with shaking for 60 minutes. 100 μL of the supernatant was taken and spread on an LB-A plate, and incubated upside down at 37°C overnight.

[0048] Pick the above single colonies and place them in LB-A medium, incubate overnight at 37°C with shaking. The next day, add the bacterial suspension to 100 mL of fresh LB-A medium at a ratio of 1:100, and incubate at 37°C with shaking for 3 hours until the bacterial growth rate reaches OD. 600 =Approximately 0.8, add 1 mM IPTG to a final concentration, and induce overnight at 30°C and 200 rpm. The next day, collect the bacterial cells by centrifugation at 8000 rpm and 4°C for 10 minutes, and resuspend the precipitate in 1.5 mL of pre-chilled TES buffer. After incubating on ice for 2 minutes, gently vortex for 30 seconds, repeating this cycle 6 times. Add 3.0 mLTES / 4 (TES diluted 4-fold with water), gently vortex for 30 seconds, and incubate on ice for 2 minutes, repeating the vortexing and ice-incubation steps 6 times. Centrifuge at 9000 rpm and 4°C for 10 minutes, and collect approximately 4.5 mL of supernatant (periplasmic extract). Obtain the nanobody protein using nickel column affinity purification; results are shown in [Figure missing]. Figure 2 Except for 2F1, all 10 other nanobodies were expressed.

[0049] 1.2.12 Determination of the affinity between nanobodies and antigens The FCoV S1 antigen was conjugated to the chip, and nanobody solutions at concentrations of 200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, and 2 μg / mL were used. The affinity between the nanobody and the antigen was tested using the instrument's built-in template method (with injection conditions of 60 s, 30 μL / min; dissociation time of 600 s; and regeneration conditions of 30 s, 30 μL / min). Binding and dissociation curves at several suitable concentration gradients were fitted using a 1:1 binding mode to obtain the affinity values, binding constants, and dissociation constants, among other important parameters. The results are shown in Table 1. Seven nanobody strains specifically bound to the FCoV S1 protein conjugated on the chip.

[0050] Table 1. Affinity values, binding constants, and dissociation constants of nanobodies

[0051] Table 1 shows that the seven nanobodies can specifically bind to the FCoV S1 protein, with 2D2 exhibiting the highest affinity (0.1 nM); 1A1 / 2F1 / 2G1 showing affinity between 1 and 10 nM; and 1A8 / 1B3 / 2A9 showing slightly lower affinity. Although the seven nanobodies demonstrate binding performance, their binding interface must partially overlap with the RBD (Restricted Baseline Diode) to exert their blocking function through steric hindrance or conformational regulation. To clarify the correlation between binding properties and functional activity, functional verification will be performed through the following cell experiments.

[0052] 1.3 Screening for FCoV neutralizing nanobodies Fcwf-4 was prepared to a concentration of 5 × 10⁻⁶. 4 Cell suspensions of 10 cells / mL were seeded into 48-well plates and incubated at 37°C for 24 hours. 20 μM nanobody was incubated with NTU156 virus (MOI 0.1) for 1 hour, then the mixture was transferred to Fcwf-4 cell culture plates that had been cultured for 24 hours and incubated for another hour. After 1 hour, the supernatant was carefully removed from each well, and DMEM medium containing 2% fetal bovine serum was added to each well. 48 hours after infection, the supernatant was collected from each well and co-incubated with fresh Fcwf-4 cells for 1 hour. The supernatant was removed again, and DMEM medium containing 2% fetal bovine serum was added to each well. 72 hours after infection, the cells were fixed, stained, and the inhibitory effect of the nanobody on FCoV was analyzed. The results are shown below. Figure 3 2F1 has a significant ability to neutralize FCoV.

[0053] Example 2 A method for preparing a recombinant protein fused with the HR2 peptide and Fc fragment of FIPV S1 protein and its neutralizing effect on FCoV. 2.1 Recombinant expression of HR2-Fc protein 2.1.1 Construction of recombinant plasmids Combine the FIPV S1 protein (GenBank ACS44218.1) Phe1339-Glu1388 peptide FNATYLNLTGEIDDLEFRSEKLHNTTVELAILIDNINNTLVNLEWLNRIE (SEQ ID NO.8) and the IgG1 Fc fragment (GenBankBAA32229.1) Pro117-Lys335 PPEMLGGPSIFIFPPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKG QPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGK (SEQ ID NO.9) The signal peptide sequence MESPAQLLFLLLLWLPDTQA (SEQ ID NO.10) was added to the N-terminus via (GGGGS)3linker to obtain the full-length HR2-Fc recombinant protein sequence, as shown in SEQ ID NO.11. After codon optimization, the entire gene was synthesized and cloned into the pcDNA3.1 plasmid using HindIII and EcoRI to obtain the pcDNA3.1-HR2-Fc recombinant plasmid.

[0054] 2.1.2 Expression and purification of HR2-Fc protein The pcDNA3.1-HR2-Fc plasmid was extracted using an endotoxin-free plasmid extraction kit and transfected into 293 cells in logarithmic growth. After 72 hours of cell culture, the cell culture medium was transferred to 50 mL centrifuge tubes and centrifuged at 12000 g for 5 minutes. The supernatant was collected, filtered through a 0.22 μm filter membrane, and purified using Protein A affinity chromatography. Protein expression was detected by SDS-PAGE. The results are shown in the figure. Figure 4 .from Figure 4 It can be seen that a single main band appears at about 55 kD, which is larger than the theoretical value (33 kD). Glycosylation modification is the main reason for the difference in molecular weight.

[0055] 2.2 The blocking effect of HR2-Fc protein on FCoV Fcwf-4 was prepared to a concentration of 5 × 10⁻⁶. 4 Cell suspensions of HR2-Fc at a concentration of 1,000 cells / mL were seeded into 48-well plates and incubated at 37°C for 24 hours. 1, 5, 10, 20, and 30 μM HR2-Fc were incubated with NTU156 virus (MOI 0.1) for 1 hour, then the mixture was transferred to Fcwf-4 cells cultured for 24 hours and incubated for another hour. After 1 hour, the supernatant was carefully removed from each well, and DMEM medium containing 2% fetal bovine serum was added to each well. 48 hours after infection, the supernatant was collected from each well and co-incubated with fresh Fcwf-4 cells for 1 hour. The supernatant was removed again, and DMEM medium containing 2% fetal bovine serum was added to each well. 72 hours after infection, the cells were fixed, stained, and the inhibitory effect of HR2-Fc on FCoV was analyzed. The results are shown below. Figure 5 .from Figure 5 It can be seen that HR2-Fc has a blocking effect on FCoV in a concentration-dependent manner.

[0056] Example 3 IFN-ω protein recombinant expression A His6 tag was added to the C-terminus of the feline IFN-ω protein (GenBank P35849.1) to obtain the full-length FeIFN-ω recombinant protein sequence, as shown in SEQ ID NO.18. The entire gene was synthesized after codon optimization and cloned into the pcDNA3.1 plasmid using HindIII and EcoRI to obtain the pcDNA3.1-FeIFN-ω recombinant plasmid. The pcDNA3.1-FeIFN-ω plasmid was extracted using an endotoxin-free plasmid extraction kit and transfected into logarithmically growing 293 cells. After 72 hours of cell culture, the cell culture medium was transferred to 50 mL centrifuge tubes, centrifuged at 12000 g for 5 minutes, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane and purified using nickel column affinity chromatography. Protein expression was detected by SDS-PAGE, and the results are shown below. Figure 6 .from Figure 6 It can be seen that a single main band appears at approximately 20 kD, which is consistent with the theoretical molecular weight (20 kD).

[0057] MALPSSFLVALVALGCNSVCSLGCDLPQTHGLLNRRALTLLGQMRRLPASSCQKDRNDFAFPQDVFGGDQSHKAQALSVVHVTNQKIFHFFCTEASSSAAWNTTLLEEFCTGLDRQLTRLEACVLQEVEEGEAPLTNEDIHPEDSILRNYFQRLSLYLQEKKYSPCAWEIVRAEIMRSLYYSSTALQKRLRSEKHHHHHH (SEQ ID NO.18) Example 4 Efficacy studies of FIPV neutralizing nanobodies, HR2-Fc, and their combinations in the treatment of feline infectious peritonitis. Grouping: Clinically infected cats with FIP (defined as clinically infected with FIPV if all of the following symptoms are present: ① positive FIPV nucleic acid test; ② albumin / globulin ratio ≤ 0.5; ③ lethargy and loss of appetite; ④ effusion in the chest or abdomen), 6 cats in each group.

[0058] Treatment: Different combinations of FIPV neutralizing nanobodies 2F1, HR2-Fc, FeIFN-ω, TNFα antibody (oszolatumab in this example), and GS-441524 were administered once daily for 28 days. Specifically, the FIPV neutralizing nanobodies 2F1 and HR2-Fc were injected subcutaneously at a dose of 1 mg / kg, FeIFN-ω at a dose of 0.1 mg / kg, TNFα antibody at a dose of 0.2 mg / kg, and GS-441524 at a dose of 5 mg / kg.

[0059] Clinical symptom score: This is the clinical symptom score for FIP cats. Examination reveals serous effusion and granulomatous lesions in the body cavity. Based on the severity of the lesions, it is divided into four levels: no obvious symptoms, mild, moderate, and severe. No obvious symptoms and mild symptoms indicate symptom relief. The proportion of cats with symptom relief to the total number of cats in this group is the symptom relief rate.

[0060] Experimental results: After each group of treatments, the symptom relief rate of FIP cats was calculated, and the results are shown in Table 2.

[0061] Table 2 Symptom relief rate in FIP cats

[0062] Table 2 shows that multiple drug combinations exhibit synergistic effects in treating FIP. For example, on Day 28, the 2F1 + HR2-Fc combination achieved a remission rate of 67%, significantly higher than either drug alone, indicating a synergistic effect. The 2F1 + HR2-Fc + GS-441524 combination achieved a remission rate of 83%, significantly higher than either drug alone, indicating a synergistic effect among the three. This synergistic effect was already evident in the early stages (Days 7 and 14), with the remission rate gradually increasing from 50% on Day 7 to 83% on Day 28, showing a time-dependent increasing trend. FeIFN-ω and ozolazumab were ineffective alone, but they improved the remission rates of 2F1, HR2-Fc, and GS-441524, indicating that FeIFN-ω and ozolazumab exert a synergistic adjuvant effect by modulating immunity or enhancing antiviral activity. The five-component combination of 2F1+HR2-Fc+GS-441524+FeIFN-ω+ozolazumab achieved a 67% remission rate on Day 7 and stabilized at 83% after Day 14, demonstrating a rapid and durable synergistic effect.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition, characterized in that, Including at least one of nanobody 2F1 and recombinant fusion protein HR2-Fc; The variable region of the nanobody 2F1 has a CDR1 region as shown in SEQ ID NO.1, a CDR2 region as shown in SEQ ID NO.2, and a CDR3 region as shown in SEQ ID NO.3; The amino acid sequence of the recombinant fusion protein HR2-Fc is shown in SEQ ID NO.

11.

2. The composition according to claim 1, characterized in that, The nanobody 2F1 further includes a backbone region having FR1 as shown in SEQ ID NO.4, FR2 as shown in SEQ ID NO.5, FR3 as shown in SEQ ID NO.6, and FR4 as shown in SEQ ID NO.

7.

3. The composition according to claim 1, characterized in that, The recombinant fusion protein HR2-Fc includes the HR2 peptide and Fc fragment of the FIPV S protein; The amino acid sequence of the HR2 peptide in the FIPV S protein is shown in SEQ ID NO.8; The amino acid sequence of the Fc fragment is shown in SEQ ID NO.9; The HR2 peptide and Fc fragment in the FIPV S protein are linked by a linker fragment. The connecting segment is (GGGGS)n, where n is a natural number from 1 to 6; Add a secretion signal peptide as shown in SEQ ID NO.10 to the N-terminus.

4. The composition according to claim 1, characterized in that, It also includes at least one of the following components: (1) Tumor necrosis factor α inhibitory antibody; (2) Interferon ω; (3) Nucleoside analogue GS-441524.

5. The composition according to claim 4, characterized in that, The tumor necrosis factor α inhibitory antibody is ozolazumab.

6. Use of the composition according to any one of claims 1-5 in the preparation of a product for treating feline infectious peritonitis.

7. The application according to claim 6, characterized in that, The symptoms of feline infectious peritonitis are as follows: (1) The FIPV nucleic acid test was positive; (2) White ball ratio ≤ 0.5; (3) Depression and loss of appetite; (4) Fluid accumulation in the chest or abdomen.

8. The application according to claim 6, characterized in that, The product is a drug, and the dosage form of the drug is an injection.

9. The application according to claim 8, characterized in that, In the drug, the dosage range of nanobody 2F1 is 0.5-2 mg / kg; And / or, the dose range of the recombinant fusion protein HR2-Fc is 0.5-2 mg / kg.

10. The application according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients and / or carriers.

Images