C-type single domain antibodies neutralizing respiratory syncytial virus and applications

Abstract

C-type single domain antibodies neutralizing respiratory syncytial virus and applications [Solution] In the present invention, a phage surface display library based on CH2-m01sm2, a modified CH2 domain of human IgG1, was used as the backbone to screen and subsequently modify respiratory syncytial virus envelope protein F (F protein) as an antigen to obtain candidate clones. The cloned proteins were then prokaryotically expressed in E. coli and purified to identify their biological specificity and neutralizing activity. Finally, the C-type single domain antibody BBT-VC001-1 and its variants, which can neutralize respiratory syncytial virus, were obtained and can be used in the prevention and treatment of respiratory syncytial virus.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical engineering, particularly to a C-type single domain antibody that neutralizes respiratory syncytial virus and its application. [Background technology]

[0002] Respiratory syncytial virus (RSV) is a seasonal virus transmitted through the respiratory system or close contact, primarily affecting infants, young children, the elderly, and immunocompromised individuals, causing acute respiratory infections, pneumonia, and even death. However, there are no effective vaccines or specific therapeutic agents. It is difficult to achieve antiviral goals through the clinical use of ribavirin (which is nonspecific and has significant side effects) or palivizumab monoclonal antibody vaccination (which is expensive and has weak neutralizing activity), and there is an urgent need for the development of new generation drugs. Summary of the Invention [Problem to be solved by the invention]

[0003] In view of the problems of the prior art, the present application provides a C-type single domain antibody for neutralizing respiratory syncytial virus and its application. [Means for solving the problem]

[0004] This invention uses a phage surface display library based on the CH2-m01sm2 framework, a modified CH2 domain of human IgG1, and uses the respiratory syncytial virus envelope protein F (F protein) as an antigen to screen and subsequently modify candidate clones. The cloned proteins were then prokaryotically expressed in E. coli and purified to identify their biological specificity and neutralizing activity. Finally, a C-type single-domain antibody, BBT-VC001-1, capable of neutralizing respiratory syncytial virus (RSV) was isolated (also known as C-type nanobody BBT-VC001-1), which can be used for the prevention and treatment of RSV.

[0005] BBT-VC001-1 has three loop regions, and the amino acid sequences of loop 1, loop 2, and loop 3 are shown in SEQ ID No. 1, SEQ ID No. 3, and SEQ ID No. 6, respectively.

[0006] In BBT-VC001-1, the nucleotide sequences encoding loop 1, loop 2, and loop 3 in the three loop regions are shown as SEQ ID No. 39, SEQ ID No. 40, and SEQ ID No. 41, respectively.

[0007] The amino acid sequence of BBT-VC001-1 is shown in SEQ ID No. 10, and the nucleotide sequence is shown in SEQ ID No. 42.

[0008] The C-type single domain antibody BBT-VC001-1 against the RSV F protein provided by the present invention was obtained as follows: First, a phage display library was constructed using CH2 (Figure 1) as a backbone, and then the RSV F protein was used as an antigen. After screening and optimization, clone BBT-VC001-1 was obtained. The clone was expressed, purified (Figure 2), and identified. The binding activity of BBT-VC001-1 was identified by ELISA, and the experimental results showed that BBT-VC001-1 could specifically bind to the RSV F protein (Figure 3). Indirect immunofluorescence experiments showed that BBT-VC001-1 could also bind to the native F protein on the surface of the cell membrane (Figure 4). Furthermore, cell-level antiviral experiments showed that BBT-VC001-1 had a highly effective antiviral effect against respiratory syncytial virus (Figure 5). Animal experiments demonstrated that pulmonary aerosol administration of the BBT-VC001-1 antibody was effective in preventing and treating RSV-infected Balb / c mice (Figure 6).

[0009] The amino acid sequence point mutants of the BBT-VC001-1 antibody in this study, designated 2C, M17, Zh1, B5, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, KS, 2H, 2H-KS, F11, F11-KS, 2H-F11, and 2H-F11-KS, have similar binding and antiviral activities to the BBT-VC001-1 antibody (Figures 7-10). At the same time, the corresponding sites of other antibody fragments were replaced with the three loop regions of BBT-VC001-1 (resulting antibodies: hIgG1 CH2-com, hIgG2 CH2-com, hIgG3 CH2-com, and hIgG4 CH2-com). The resulting combinations still maintained binding and antiviral activity (Figure 11). BBT-VC001-1 was bound to peptides (ABD and 16L) or proteins (VH) to form fusion peptides or fusion proteins, and its antigen-binding activity was unchanged (Figure 12), and the sequence numbers of all the above mutants and antibodies are listed in Table 1 below.

[0010] The present invention also clarifies the application of the above-mentioned C-type single domain antibody that neutralizes respiratory syncytial virus to the preparation of prophylactic and therapeutic drugs, detection probes, fusion peptides, fusion proteins, and labeled antibodies against respiratory syncytial virus. The C-type single domain antibody that neutralizes respiratory syncytial virus of the present invention can be used to prepare nasal sprays, nasal drops, nebulizers, intramuscular injection preparations, and intravenous preparations related to the prevention and treatment of respiratory syncytial virus, and the concentration of each preparation is preferably in the range of 0.01 ng / mL to 1 g / mL.

[0011] The term "C-type single domain antibody" as used herein refers to a class of antibodies whose backbone is the CH2 domain of the antibody constant region. Compared to full-length monoclonal antibodies, single domain antibodies have superior tissue penetration and can preferentially bind to spatially hindered epitopes. [Effects of the Invention]

[0012] The beneficial effects of the present invention are as follows: The C-type single domain antibody of the present invention is an antibody against the RSV F protein and can inhibit viral entry into cells. It can be used for the prevention, treatment, and diagnosis of RSV, as well as for further research into the mechanism of RSV infection. Compared to full-length monoclonal antibodies (molecular weight approximately 150 kD), the C-type single domain antibody of the present invention has a smaller molecular weight (12-15 kD), excellent tissue penetration, and the ability to bind to antigen epitopes with steric hindrance effects. It can be expressed in multiple expression systems, including prokaryotes, yeast, and mammalian cells, and can be produced at low cost and with a short cycle. C-type single domain antibodies can be administered via a wide range of routes, including nasal drops, nasal sprays, sprays, atomized inhalation, intramuscular injection, subcutaneous injection, and intravenous injection. [Brief explanation of the drawings]

[0013] [Figure 1] Schematic diagram of the structure of a C-type single domain antibody. The backbone of a C-type single domain antibody is derived from the CH2 domain of the constant region of an IgG1 antibody and contains three flexible loop regions (loop 1, loop 2, and loop 3), which correspond to the three complementarity-determining regions (CDRs) in the variable region of an antibody. [Figure 2] Expression and purification of the C-type single domain antibody BBT-VC001-1. The target protein was detected by polyacrylamide gel electrophoresis (SDS-PAGE). Lanes from left to right show protein molecular weight standards (markers) and purified BBT-VC001-1. [Figure 3] The binding of BBT-VC001-1 to the human respiratory syncytial virus envelope protein F was measured by ELISA. The EC50 binding activity of BBT-VC001-1 to the F protein was 28 nM, and bovine serum albumin (BSA) was used as a negative control. [Figure 4]Indirect immunofluorescence analysis confirmed that BBT-VC001-1 binds to the native F protein on the surface of human respiratory syncytial virus. BBT-VC001-1 binds to the envelope protein F on the surface of the virus particle and fluoresces when stained with a fluorescent secondary antibody. [Figure 5] BBT-VC001-1 was tested for neutralization of human respiratory syncytial virus (RSV). The IC50 for BBT-VC001-1 to inhibit respiratory syncytial virus type A (RSV A) was 8.326 ng / mL (Figure 5A), and the IC50 for inhibiting respiratory syncytial virus type B (RSV B) was 8.419 ng / mL (Figure 5B). [Figure 6] Preventive and therapeutic effects of BBT-VC001-1 on human respiratory syncytial virus infection in Balb / c mice. Prophylactic pulmonary aerosol administration of BBT-VC001-1 prevented weight loss in mice (Figure 6A) and reduced the viral load (RNA copy number) in the lungs (Figure 6B). Furthermore, therapeutic pulmonary aerosol administration of BBT-VC001-1 significantly reduced the viral load (viral titer) in the lungs (Figure 6C). [Figure 7] The binding activities of the mutants obtained by point mutation in the loop and / or framework regions of antibody BBT-VC001-1 were equivalent to that of BBT-VC001-1 in terms of EC50 values. [Figure 8] Among the variants of antibody BBT-VC001-1 shown in Figure 7, the variants numbered 2C, M17, Zh1, B5, M2, M3, and M4 have IC50 values ​​comparable to those of BBT-VC001-1 in antiviral activity. [Figure 9] Among the variants of antibody BBT-VC001-1 shown in Figure 7, the variant numbered M517-M11 has an IC50 value comparable to that of BBT-VC001-1 in antiviral activity. [Figure 10]Among the variants of antibody BBT-VC001-1 shown in Figure 7, the variants numbered KS, 2H, 2H-KS, F11, F11-KS, 2H-F11, and 2H-F11-KS have IC50 values ​​comparable to those of BBT-VC001-1 in antiviral activity. [Figure 11] The three loop region sequences of BBT-VC001-1 were used to replace the loop regions of the CH2 backbone of different types of antibodies, and the resulting combinations exhibited binding activity (Figure 11A) and antiviral activity (Figure 11B). [Figure 12] Binding activity of BBT-VC001-1 after binding to peptides or proteins. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is described in detail below in relation to the embodiments and accompanying drawings, and the following embodiments are implemented under the premise of the technical scheme of the present invention, and detailed embodiments and specific operation processes are shown, but the protection scope of the present invention is not limited to the following embodiments.

[0015] Example 1 Expression and purification of human respiratory syncytial virus envelope protein recF According to the respiratory syncytial virus gene sequence (GenBank No. M11486.1), the gene encoding the respiratory syncytial virus envelope protein F was fused with a gene encoding the Fc fragment of antibody IgG1 to prepare the recombinant F protein (recF) gene. This gene was ligated and introduced into the vector pSecTag2A, and the eukaryotic expression plasmid pSecTag2A-recF was cloned. One day before transfection, 40 mL of 293F cells (cell density 5–10 × 10) were transfected. 5Cells (control, 1 / mL) were seeded into a 125 mL suspension cell culture flask. 40 μg of plasmid (pSecTag2A-recF) was diluted in 4 mL of culture medium and gently mixed. Next, 120 μL of PEI (polyethyleneimine) was diluted in the culture medium and gently mixed. After 20 minutes of incubation at room temperature, the mixture was added dropwise to the cells. The cells were placed in a suspension incubator and cultured at 250 rpm / min at 37°C. After 144 hours, the culture supernatant was collected and protein expression was detected by Western blot using HRP-anti-human Fc.

[0016] After detecting target protein expression, we scaled up the cell culture and transfection to express large amounts of the recF protein. The culture supernatant was collected, and the target protein was purified using Protein A purification medium. The buffer was replaced by ultrafiltration using ultrafiltration tubes with a 10 kD molecular weight cutoff. After final concentration, purity was confirmed by SDS-PAGE.

[0017] <Example 2> Phage display library construction and screening Using human CH2 as a backbone, a phage display library was constructed according to existing literature (Gong R., et al., PLoS One, 2012, 7:e42288) and screened using the eukaryotically expressed antigen recF. Purified antigen was bound to magnetic beads and then screened using the phage display library. recF-specific phage were captured by the antigen, yielding candidate clones. These candidate clones were further affinity matured (Wang R., et al., Virol Sin., 2021, 11:1-11), yielding the C-type single domain antibody BBT-VC001-1.

[0018] Sequence analysis revealed that BBT-VC001-1 encodes a gene sequence. The three loop regions of BBT-VC001-1 are loop 1, loop 2, and loop 3, and their respective coding gene sequences and amino acid sequences are shown in Table 1 below.

[0019] Example 3 Expression and purification of BBT-VC001-1 BBT-VC001-1 was expressed and purified according to previous literature (Gong R., et al., Methods Mol Biol., 2012, 899:85-102). The prokaryotic expression vector for BBT-VC001-1 was transferred to E. coli HB2151. One clonal colony was selected and inoculated into SB medium (1 L of medium contains 30 g of tryptone, 20 g of yeast extract, and 10 g of MOPS, pH adjusted to 7.0 with NaOH) containing 100 μg / mL ampicillin. When the OD600 reached 0.7-1.0, IPTG was added to a final concentration of 200 μg / mL and expression was induced at 37°C and 220 rpm for 14-16 hours. The E. coli cells were collected by centrifugation at 6000 rpm at 4°C for 15 minutes, the medium discarded, and the pellet resuspended in PBS (pH 7.0). It was treated with polymyxin B for 1 hour and then centrifuged to collect the supernatant. The protein was purified using Ni-NTA filter media, and its purity was verified by SDS-PAGE. The results are shown in Figure 2. The product was then concentrated by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa. The resulting BBT-VC001-1 contained a 6xHis tag and a FLAG tag at the C-terminus.

[0020] Example 4 Determination of binding of BBT-VC001-1 to recombinant envelope protein recF using Elisa recF (4 μg / mL) was applied to an ELISA plate, incubated overnight at 4°C, and then blocked with PBS + 3% milk for 1 hour at 37°C. Serial dilutions of BBT-VC001-1 were added and incubated for 2 hours at 37°C. Plates were then washed four times with PBST (PBS + 0.05% Tween 20), incubated with HRP-Anti-FLAG monoclonal antibody for 1 hour at 37°C, washed four times with PBST, and ABTS was added for color development. Bovine serum albumin (BSA) was used as a negative control. EC values ​​for BBT-VC001-1 binding to recF were calculated. 50 The binding activity of the antibody was 28 nM and it did not bind to BSA. The results are shown in Figure 3.

[0021] <Example 5> Indirect immunofluorescence assay for the binding of BBT-VC001-1 to the native F protein on the surface of human respiratory syncytial virus particles 100 μL of Hep-2 (2 × 10 5 Cells (100 pfu / mL) were seeded into 96-well plates and cultured for 14–16 hours. The cells were washed with PBS and infected with respiratory syncytial virus (RSV) (100 pfu / well) for 1 hour. The virus was discarded, and 100 μL of DMEM containing 2% fetal bovine serum was added. Cell culture was continued at 37°C. After approximately 48 hours, the cells were fixed with 4% formaldehyde solution for 30 minutes and blocked with 5% BSA for 1 hour. BBT-VC001-1 was used as the primary antibody, and FITC-Anti-His was used as the secondary antibody. The staining results were observed using a fluorescence microscope (Figure 4).

[0022] Example 6 BBT-VC001-1 antibody inhibits human respiratory syncytial virus replication in Hep-2 cells 100 μL of Hep-2 (2 × 10 5Cells (1000µL / mL) were seeded into a 96-well plate and cultured for 14–16 hours. Respiratory syncytial virus (types A and B) was mixed with different concentrations of BBT-VC001-1 antibody and incubated for 1 hour. After incubation, the antibody was added to the cells, which had been washed with PBS, and cultured at 37°C. After 24–48 hours, the cells were fixed with 4% formaldehyde solution for 30 minutes and blocked with 5% BSA for 1 hour. IgG-mpe8 was used as the primary antibody, and FITC-Anti-human was used as the secondary antibody. The cells were then photographed and analyzed using a high-content cell analyzer. Based on the test results, the viral infection inhibition rate for each well was calculated relative to the positive and negative control wells. The IC value for BBT-VC001-1's inhibition of respiratory syncytial virus type A was calculated. 50 The IC for type B inhibition was 8.326 ng / mL. 50 was 8.419 ng / mL (Figure 5).

[0023] Example 7 Protective effect of BBT-VC001-1 against human respiratory syncytial virus infection in a mouse model Viral prophylaxis: Before viral infection, Balb / c mice were anesthetized and administered 50 μL of BBT-VC001-1 at a dose of 2.5 mg / kg by pulmonary nebulization (prophylaxis group). Control mice were administered 50 μL of PBS by pulmonary nebulization (blank and PBS groups). Six hours later, each mouse in the prophylaxis and PBS groups was administered 1 × 10 7 Mice were infected by intranasal instillation of FFU RSV A2 virus. Subsequently, the mice were weighed daily. Four days after infection, some mice were anesthetized and sacrificed, and lung tissue was collected to measure viral load. The results are shown in Figure 6A. Compared with the PBS group, the prophylactic group had significantly increased body weight and reduced viral load in the lung tissue.

[0024] Virus treatment: First, mice were anesthetized and each mouse was inoculated with 1 × 10 7Mice were infected with FFU RSV A2 by intranasal instillation. Three hours later, different doses of BBT-VC001-1 (5 mg / kg and 20 mg / kg) were administered by aerosol in a volume of 50 μL per mouse. The control group received 50 μL of PBS by aerosol into the lungs. Subsequently, the antibody was administered once on days 1 and 2. On day 4 post-infection, the mice were anesthetized and sacrificed, and lung tissue was collected for viral titer determination. The results are shown in Figure 6C, which demonstrates a significant reduction in viral titer in the lung tissue of mice administered the antibody.

[0025] Example 8 Construction and activity of point mutants, combinations, and fusions of BBT-VC001-1 antibody Construction and activity of point mutants: Single or multiple point mutations were performed in the loop and / or backbone regions of BBT-VC001-1 to obtain different sequences: 2C (L4S, L14F), M17 (V67I), Zh1 (L4S, L14F, V67I), B5 (V15M, I37V, D49H, V53T, S65R), M2 (S2T, V15M, D49H, V53T, S65R, K81R, K114N), M3 (V15M, D49H, A51G, V53T, S65R), M4 (V15M, D49H, V53T, S65R), M5 (V15M, I37V, D49H, V53T, S65R, E91V, S111A), M6 (V3I, V15M, S27F, I37V, V48M, D49H, V53T, S65R, E91V), M7(K11N, V15M, I37V, V46E, D49H, V53T, D57E, S65R, L73P, E91V), M8(V15M, I37V, D49H, V53T, K54N, S65R, N79S, D101N), M9(V15M, I37V, K38N, D49H, V53T, S65R, E97D), M10(K9E, V15M, I37V, D49H, V53T, S65R, T71I, K112I), M11(E91V), KS(K112I, A113K, K114S), 2H(L4H, L6H, L73K), 2H-KS(L4H, L6H, L73K, K112I, A113K, K114S), F11(P10S, K11N, T13A,V72I), F11-KS(P10S, K11N, 2H-F11-KS(L4H, L6H, P10S, K11N, T13A,V72I, L73K, K112I, A113K, The mutant (K114S) was obtained and synthesized by a gene synthesis company, and constructed into an expression vector for protein expression. The protein expression method was as described in Example 3. The obtained point mutant proteins were tested by ELISA and antiviral experiments, and the detection methods were as described in Examples 4 and 6, respectively. The test results are shown in Figures 7 to 10.The binding activity of these point mutant proteins to the recombinant RSV envelope protein recF ranged from 20 nM to 150 nM, and their antiviral activity against the RSV A2 strain ranged from 3 ng / mL to 55 ng / mL. The results showed that mutants obtained by making several point mutations in the loop and / or backbone regions of the BBT-VC001-1 protein still retained activity.

[0026] Combination Construction and Activity: The loop regions of the CH2 backbones of different types of antibodies (human IgG1 CH2 region, IgG2 CH2 region, IgG3 CH2 region, and IgG4 CH2 region) were replaced with three loop region sequences (loop 1, loop 2, and loop 3) from BBT-VC001-1. The resulting combinations were named hIgG1 CH2-com, hIgG2 CH2-com, hIgG3 CH2-com, and hIgG4 CH2-com, respectively. These combinations were subjected to gene synthesis, protein expression, and activity detection (using the same methods as above). The test results are shown in Figure 11. hIgG1 CH2-com, hIgG2 CH2-com, hIgG3 CH2-com, and hIgG4 CH2-com all possess binding and antiviral activity at the cellular level. These results suggest that the three loop regions of BBT-VC001-1 can be arranged on different scaffolds and function.

[0027] Fusion Construction and Activity: BBT-VC001-1 was conjugated to a peptide (16L and ABD) or a protein (VH), designated BBT-VC001-1-16L, BBT-VC001-1-ABD, and BBT-VC001-1-VH, respectively. Gene synthesis, protein expression, and activity detection were then performed (using the same methods as above). The test results are shown in Figure 12. BBT-VC001-1 maintained its binding activity after fusion with a polypeptide or protein. The results indicated that BBT-VC001-1 could be used to prepare fusion polypeptides and proteins for the prevention and treatment of human respiratory syncytial virus.

[0028] Finally, a table of sequence information for all antibodies and fragments related to the above examples is attached.

[0029] Sequence information of antibodies and their loops JPEG0007784191000001.jpg143170JPEG0007784191000002.jpg197170

[0030] Those skilled in the art can better understand and grasp the present invention with the help of the embodiments. However, the protection scope and claims of the present invention are not limited to the examples provided. Based on the embodiments of the present invention, all other embodiments obtained by ordinary skilled in the art without creative work shall fall within the protection scope of the present invention.

Claims

1. A C-type single domain antibody for neutralizing respiratory syncytial virus, wherein the C-type single domain antibody is composed of only a CH2 region without any variable region or CH3 region, and the CH2 region contains three loop regions, i.e., loop 1, loop 2, and loop 3, wherein the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 6, respectively; Alternatively, the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, respectively; Alternatively, the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 6, respectively; Alternatively, the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:7, respectively; Alternatively, the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:7, respectively; Alternatively, the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:8, respectively; Alternatively, the amino acid sequences of loop 1, loop 2, and loop 3 are set forth in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 9, respectively; Alternatively, a C-type single domain antibody, characterized in that the amino acid sequences of loop 1, loop 2, and loop 3 are shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 7, respectively.

2. The C-type single domain antibody according to claim 1, characterized in that the amino acid sequence of the C-type single domain antibody is selected from any one of SEQ ID NOs: 10 to 35.

3. Use of a type C single domain antibody according to claim 1 or 2 for preparing a drug, a detection probe, a fusion polypeptide, a fusion protein or a fusion antibody for the prevention and treatment of respiratory syncytial virus.

4. 3. Use of a type C single domain antibody according to claim 1 or 2 for the preparation of a nasal drop, nasal spray, nebuliser or injection related to the prevention and treatment of respiratory syncytial virus.

5. An antibody fusion polypeptide of respiratory syncytial virus, characterized in that the amino acid sequence is set forth in SEQ ID NO:

36.

6. An antibody fusion polypeptide of respiratory syncytial virus, characterized in that the amino acid sequence is set forth in SEQ ID NO:

37.

7. An antibody fusion protein of respiratory syncytial virus, characterized in that the amino acid sequence is set forth in SEQ ID NO:

38.

8. The C-type single domain antibody according to claim 1, characterized in that the nucleotide sequences encoding the loop 1, the loop 2 and the loop 3 are shown in SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, respectively.

9. The C-type single domain antibody according to claim 2, characterized in that the nucleotide sequence encoding said C-type single domain antibody is shown in SEQ ID NO: 42.

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