Anti-csf3r antibodies and uses

Abstract

The application belongs to the technical field of biology, and discloses an anti-CSF3R antibody and application thereof, wherein the anti-CSF3R antibody comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises VHCDR1, VHCDR2 and VHCDR3, and the light chain variable region VL comprises VLCDR1, VLCDR2 and VLCDR3. The anti-CSF3R antibody can block the interaction between CSF3R and a ligand G-CSF, and then inhibit G-CSF signal transmission, neutrophil survival and migration, and the effect is equivalent to that of a reference antibody CSL324, and the anti-CSF3R antibody can be used for treating chronic sinusitis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an anti-CSF3R antibody and application. BACKGROUND

[0002] Under normal circumstances, human neutrophils mature in the bone marrow and then enter the blood, and then migrate to the inflammation site. Neutrophils express CSF3R receptors, which activate downstream signaling pathways after binding with G-CSF, promoting and maintaining the development, survival and function of neutrophils.

[0003] Clinically, sinusitis is usually divided into polyp type (CRSwNP) and non-polyp type (CRSsNP), and is divided into neutrophil infiltration type, eosinophil infiltration type, lymphocyte / plasma cell infiltration type and mixed type according to the type of infiltrating inflammatory cells. In the polyp type of neutrophil infiltration sinusitis, neutrophils migrate to the polyp tissue and exacerbate local inflammation through various mechanisms.

[0004] The increase in the number of neutrophils in the peripheral blood is related to sinusitis. The clinical data of Zagolski et al. showed that the number of neutrophils in the blood of CRSwNP patients was significantly higher than that of healthy people (5.2×10 3 / μl vs. 3.6×10 3 / μl), and the NLR value (neutrophil to lymphocyte ratio) of recurrent CRSwNP patients was also significantly higher than that of non-recurrent (2.06 vs. 3.67). In addition, a number of studies have shown that high levels of neutrophil infiltration in tissues are also a high-risk factor for recurrent and refractory CRSwNP, and gradually increase with the number of operations.

[0005] As one of the key inflammatory cells in chronic sinusitis, neutrophils have been treated by blocking their activation or migration to the inflammation site. There is no report on controlling the condition of sinusitis by regulating the number of neutrophils in the peripheral blood, but there are studies on treating palmoplantar pustulosis and hidradenitis suppurativa using a similar approach. In palmoplantar pustulosis, neutrophils are involved in the inflammatory condition and have a significant correlation with the development of the disease. Clinical studies have shown that by giving CSF3R blocking antibody CSL324 to reduce the number of neutrophils in the peripheral blood to a safe low value, the severity of psoriasis areas has been significantly improved. In patients with hidradenitis suppurativa, the number of neutrophils in the peripheral blood is significantly increased, and there have been related clinical studies on treating the disease by inhibiting the development, migration and function of neutrophils using CSL324.

[0006] The CSF3R gene is located at chromosome 1p34.3, encoding granulocyte colony-stimulating factor receptor (G-CSFR), belonging to the type I cytokine receptor family. The receptor is composed of an extracellular ligand-binding domain, a transmembrane domain and an intracellular signaling domain, and plays a core regulatory role in granulocyte proliferation, differentiation and anti-apoptosis by activating downstream JAK / STAT, SRC family kinases and other signaling pathways.

[0007] Current treatment options for many inflammatory diseases include the use of broad-spectrum therapies such as corticosteroids, non-steroidal anti-inflammatory drugs and general immunosuppressants, and more specific biological therapies such as TNF-alpha inhibitors. Although these therapies do not directly target neutrophils, they indirectly inhibit the function of neutrophils, including reducing the release of cytokines and cytotoxic granules, inhibiting neutrophil oxidative burst, thereby reducing inflammation. A new approach to targeting the destructive effects caused by neutrophils under inflammatory conditions can be to target the neutrophils themselves through G-CSF, the main regulator of neutrophils. We have developed a monoclonal antibody antagonist against human G-CSFR, which can prevent the binding of G-CSF and subsequently inhibit G-CSF signaling, neutrophil survival and migration. This suggests that G-CSFR blockade can be a viable therapeutic strategy to reduce the number of neutrophils and the pro-inflammatory effects of neutrophils in chronic inflammatory diseases. SUMMARY

[0008] The purpose of the present application is to provide an anti-CSF3R antibody and application, which can block the interaction of CSF3R and ligand G-CSF, and subsequently inhibit G-CSF signaling, neutrophil survival and migration, and the effect is equivalent to the reference antibody CSL324, which can be used for the treatment of chronic rhinosinusitis.

[0009] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] In a first aspect, the present application provides an anti-CSF3R antibody, which comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises VHCDR1, VHCDR2 and VHCDR3, and the light chain variable region VL comprises VLCDR1, VLCDR2 and VLCDR3.

[0011] The sequence of VHCDR1 is shown as SEQ ID NO: 3, the sequence of VHCDR2 is shown as SEQ ID NO: 4, the sequence of VHCDR3 is shown as SEQ ID NO: 5, the sequence of VLCDR1 is shown as SEQ ID NO: 6, the sequence of VLCDR2 is shown as SEQ ID NO: 7, and the sequence of VLCDR3 is shown as SEQ ID NO: 8.

[0012] the sequence of the VH CDR1 is shown as SEQ ID NO: 11, the sequence of the VH CDR2 is shown as SEQ ID NO: 12, the sequence of the VH CDR3 is shown as SEQ ID NO: 13, the sequence of the VL CDR1 is shown as SEQ ID NO: 14, the sequence of the VL CDR2 is shown as SEQ ID NO: 15, and the sequence of the VL CDR3 is shown as SEQ ID NO: 16.

[0013] In the above technical solution, the sequence of the heavy chain variable region VH is shown as SEQ ID NO: 1, and the sequence of the light chain variable region VL is shown as SEQ ID NO: 2; or

[0014] the sequence of the heavy chain variable region VH is shown as SEQ ID NO: 9, and the sequence of the light chain variable region VL is shown as SEQ ID NO: 10.

[0015] In the above technical solution, the anti-CSF3R antibody is a murine antibody.

[0016] In a second aspect, the present application provides a nucleotide molecule encoding the above-mentioned anti-CSF3R antibody.

[0017] In a third aspect, the present application provides an expression vector containing the above-mentioned nucleotide molecule.

[0018] In a fourth aspect, the present application provides a host cell containing the above-mentioned expression vector or having the above-mentioned nucleotide molecule integrated into the genome.

[0019] In a fifth aspect, the present application provides use of the above-mentioned anti-CSF3R antibody in the preparation of a medicament for treating chronic rhinosinusitis.

[0020] In the above technical solution, the anti-CSF3R antibody achieves the therapeutic effect of chronic rhinosinusitis by blocking the interaction between CSF3R and the ligand G-CSF, thereby inhibiting G-CSF signaling, neutrophil survival and migration.

[0021] In a sixth aspect, the present application provides a medicament for treating chronic rhinosinusitis, the medicament comprising the above-mentioned anti-CSF3R antibody.

[0022] In the above technical solution, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0023] The anti-CSF3R antibodies 2A6 and 56D9E10 of the present application can block the interaction of CSF3R with ligand G-CSF, and then inhibit G-CSF signaling, neutrophil survival and migration, and the effect is equivalent to that of the reference antibody CSL324, and can be used for treating chronic rhinosinusitis. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a graph of the binding activity experiment of the antibody to the CSF3R protein, wherein, Figure 1 A is a human CSF3R protein, Figure 1 B is a monkey CSF3R protein.

[0025] Figure 2 is a graph of the binding activity experiment of the antibody to the CSF3R expressing cell, wherein, Figure 2 A is CHOS-CSF3R cell, Figure 2 B is 293T-CSF3R cell, Figure 2 C is Baf3-CSF3R cell.

[0026] Figure 3 is a graph of the blocking activity experiment of the antibody to the binding of CSF3R protein to ligand G-CSF.

[0027] Figure 4 is a graph of the blocking activity experiment of the antibody to the binding of CSF3R expressing cell to ligand G-CSF, wherein, Figure 4 A is CHOS-CSF3R cell, Figure 4 B is 293T-CSF3R cell, Figure 4 C is Baf3-CSF3R cell.

[0028] Figure 5 is a graph of the cell proliferation inhibition ability experiment of the antibody.

[0029] Figure 6 is a graph of the neutrophil survival inhibition ability experiment of the antibody.

[0030] Figure 7 is a graph of the pSTAT3 signaling blocking ability experiment of the antibody.

[0031] Figure 8 is a graph of the neutrophil migration blocking ability experiment of the antibody, wherein, Figure 8 A is MIP-2, Figure 8 B is CXCL8.

[0032] Figure 9 is a graph of the expression reduction ability experiment of the antibody to G-CSF induced CXCR1 and CXCR2, wherein, Figure 9 A is the expression change of CXCR1, Figure 9B is the change in expression of CXCR2. DETAILED DESCRIPTION

[0033] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in combination with specific examples. The present application can be implemented in many different forms, and should not be understood as being limited to the examples set forth herein. On the contrary, these examples are provided so that the present disclosure will be thorough and complete, and will fully convey the idea of the present application to those skilled in the art, which will be limited only by the claims.

[0034] The present application immunizes mice with the extracellular segment of human CSF3R protein (uniprot: Q99062, aa25-332), and investigates the binding of the antibody to CSF3R and its blocking of the binding to the ligand in the preliminary screening stage, screens out the antibody that specifically binds to CSF3R and has the blocking function, and in the subsequent screening verification, uses the anti-CSF3R antibody CSL324 as a benchmark antibody, and the in vitro activity detection shows that the antibody of the present application can simultaneously bind to human and monkey CSF3R, and can block the interaction between CSF3R and the ligand G-CSF, and then inhibit G-CSF signaling, neutrophil survival and migration, and the effect is equivalent to that of the benchmark antibody CSL324, and can be used for treating chronic sinusitis.

[0035] Example 1 - Generation of murine anti-human CSF3R antibodies

[0036] Human CSF3R was overexpressed on CHOS cells (Invitrogen), 293T cells (ATCC) and Baf3 cells (ATCC) by lentivirus infection method (MOI = 3-10, 5 μg / ml polybrene). Lentivirus was provided by Shanghai Jikai Gene Medical Technology Co., Ltd., and after 72 hours of cell infection, the corresponding antibiotics were added for continued culture for 2-4 weeks, expansion and freezing, to obtain three CSF3R overexpression cell lines CHOS-CSF3R, 293T-CSF3R and Baf3-CSF3R for subsequent experiments.

[0037] In order to obtain anti-human CSF3R antibodies, Balb / c mice (Beijing Vantoll Life-Science Experimental Animals Technology Co., Ltd., strain code 216) were immunized with the extracellular segment of CSF3R protein; complete Freund's adjuvant CFA (InvivoGen, Inc., item number vac-cfa-60) was used as the primary adjuvant, and then IFA (InvivoGen, Inc., item number vac-ifa-60) was used as the adjuvant; the immunization route was subcutaneous multiple points. After the completion of immunization, the hybridoma and phage display methods were used to screen the target antibodies.

[0038] Hybridoma screening: After multiple immunization, the spleen cells of the immunized mice were fused with mouse myeloma cells SP2 / 0 using electrofusion method to obtain B cells that can express antibodies and can proliferate in vitro, and were cultured in HAT selection medium. The fused hybridoma cells were plated in 96-well cell culture plates, and the supernatant was screened for its ability to bind to human CSF3R protein, its ability to bind to monkey CSF3R protein, and its ability to block the binding of human CSF3R protein and ligand G-CSF. The positive clones were subcloned for 2-3 rounds.

[0039] Mouse anti-CSF3R binding screening: In the screening, human or monkey CSF3R protein was used for plating, incubated at 4°C overnight, and the supernatant was removed the next day. Each well was added with 200 μL of washing solution to wash the plate 3 times. Each well was added with 200 μL of BSA solution, and the plate was blocked at room temperature for 2 hours. The supernatant was removed, and each well was added with 200 μL of washing solution to wash the plate 3 times, and the supernatant was removed. Each well was added with 100 μL of hybridoma supernatant, and incubated at room temperature for 1 h, and the supernatant was removed. Each well was added with 200 μL of washing solution to wash the plate 3 times, and the supernatant was removed. Each well was added with 100 μL of secondary antibody solution (HRP goat anti-mouse IgG, abcam catalog number ab205719), and incubated at room temperature for 30 min, and the supernatant was removed. Each well was added with 200 μL of washing solution to wash the plate 3 times, and the supernatant was removed. Each well was added with 100 μl of TMB solution, and after color development, 100 μl of sulfuric acid stop solution was added to each well, and the machine (TECAN, model Spark 10M) was used to detect OD450nm.

[0040] Mouse anti-CSF3R blocking screening: In the screening, human CSF3R protein was used for plating, incubated at 4°C overnight, and the supernatant was removed the next day. Each well was added with 200 μL of washing solution to wash the plate 3 times. Each well was added with 200 μL of BSA solution, and the plate was blocked at room temperature for 2 hours. The supernatant was removed, and each well was added with 200 μL of washing solution to wash the plate 3 times, and the supernatant was removed. Each well was added with 50 μL of hybridoma supernatant and 50 μL of biotinylated ligand G-CSF, and incubated at room temperature for 1 h, and the supernatant was removed. Each well was added with 200 μL of washing solution to wash the plate 3 times, and the supernatant was removed. Each well was added with 100 μL of secondary antibody solution (HRP Streptavidin, BioLegend catalog number 405210), and incubated at room temperature for 30 min, and the supernatant was removed. Each well was added with 200 μL of washing solution to wash the plate 3 times, and the supernatant was removed. Each well was added with 100 μl of TMB solution, and after color development, 100 μl of sulfuric acid stop solution was added to each well, and the machine (TECAN, model Spark 10M) was used to detect OD450nm.

[0041] Phage display: Spleen cells of immunized mice were isolated after multiple immunization, total RNA was extracted and immune library was constructed, the first round of panning was performed using biotin-labeled human CSF3R protein extracellular segment. Take 150ul beads (Dynabeads™ M-280 Streptavidin, ThermoFisher, 11206D) with 50ug protein and make up to 1ml, rotate at room temperature for 1-2h, after incubation, place in magnetic stand, wash with PBST, 5min each time, discard the supernatant, wash 5 times. Add 1x10 13 Phage library and make up to 1ml, rotate at room temperature for 1-2h, wash with PBST, 5min each time, wash 5 times to wash away the unbound phage. Finally add 500ul TEA and rotate for 10min, aspirate the supernatant, add 1M Tris-HCl 250ul, dissociate the phage specifically bound to human CSF3R, and infect E. coli TG1 in the logarithmic growth phase to produce and purify phage for the next round of screening. The second round of screening used monkey CSF3R extracellular protein (uniprot: A0A7N9CCR5, aa25-332), and the screening process was the same as the first round. In this way, positive clones were enriched, achieving the purpose of screening CSF3R-specific antibodies from the antibody library using phage display technology.

[0042] Phage supernatant ELISA screening of specific single positive clones: After 2 rounds of panning, the positive phage obtained was used to infect the SS320 strain (Lucigene) in the logarithmic growth phase and plated. Single colonies were picked and cultured, 50ul IPTG (final concentration 1mM) was added, and induction was performed at 30°C overnight. The plates were coated with human and monkey CSF3R at 4°C overnight, and the obtained phage sample (control group was blank phage) was added, and reacted at room temperature for 1 hour. After washing, mouse anti-HA tag antibody (Abclonal, AE025) was added, and reacted at room temperature for 1 hour. After washing, TMB color developing solution was added, and 100ul sulfuric acid was added to each well after color development. The machine (TECAN, model Spark 10M) was used to detect OD450nm. When the OD value of the sample well was more than 3 times the OD value of the control well, it was judged as a positive clone well. The bacteria in the positive clone well were transferred to LB liquid containing 100ug / ml ampicillin for culture to extract plasmid and perform sequencing.

[0043] The binding and blocking of murine antibodies 2A6 and 56D9E10 to CSF3R are shown in Table 1.

[0044] Table 1 Binding and blocking of murine antibodies to CSF3R

[0045]

[0046] Example 2 - Determination of variable region sequences of murine anti-CSF3R antibodies

[0047] Hybridoma cells were collected by centrifugation, and 5-10 x 10 6 The cells were added with 1 mL TRIzol and 0.2 mL chloroform, and shaken vigorously for 15 seconds, and left at room temperature for 3 minutes. The water phase was collected by centrifugation, and 0.5 mL isopropanol was added, and left at room temperature for 10 minutes, and then the precipitate was collected. After ethanol washing and drying, the RNA was obtained. The template RNA and primers were added into a pre-cooled centrifuge tube, and the primers were allowed to pair with the template correctly, and then the reverse transcription process was performed, and then PCR amplification was performed. In a microcentrifuge tube, 2.5 μL of a dNTP / ddNTP mixture was added, and the mixture was incubated at 37°C for 5 minutes, and was ready for use. In an empty microcentrifuge tube, 1 pmol of PCR amplification double-stranded DNA, 10 pmol of sequencing primer, 2 μL of 5x sequencing buffer, and double-distilled water were added to a total volume of 10 μL, and heated at 96°C for 8 minutes, and then cooled on ice for 1 minute, and centrifuged at 4°C at 10000 g for 10 seconds. 2 μL of pre-cooled labeling mixture (0.75 μmol / L of dCTP, dGTP, and dTTP, respectively), 5 μCi of α-32P-dATP, 1 μL of 0.1 mol / L DDT, and 2 U of sequencing enzyme were added, and water was added to 15 μL, and mixed well, and then placed on ice for 2 minutes, and the newly synthesized DNA strand was labeled. 3.5 μL of the labeling reaction mixture was added to the prepared microcentrifuge tube, and incubated at 37°C for 5 minutes. 4 μL of termination solution was added to each tube. The samples were heat denatured in a water bath at 80°C for 5 minutes, and 2 μL was added to each lane on the sequencing gel, and the fragments were separated by electrophoresis, and sequence information was collected.

[0048] The VH and VL sequences of the 2A6 and 56D9E10 murine antibodies are shown in Table 2. Further, the CDR sequences of the murine monoclonal antibodies were determined using the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669).

[0049] Table 2: Sequence information of murine antibodies

[0050]

[0051] Table 3 Sequence information

[0052]

[0053] Example 3 - Recombinant expression of chimeric anti-CSF3R antibodies

[0054] After the antibody gene sequence was verified, the variable region of the murine antibody was linked to the constant region of the human antibody (human IgG4 S228P), and the expression vector pTT5 containing the antibody gene was transfected into mammalian cells CHO. The supernatant of the mammalian cells containing the antibody clone grown in the culture bottle was harvested, purified using a protein A column, and the antibody protein was eluted using 100 mM acetic acid pH 3.0. The antibody protein was replaced in a PBS buffer, and the buffer was supplemented with 20% glycerol.

[0055] Example 4 - Binding activity of chimeric antibody to human and monkey CSF3R

[0056] Protein binding activity: In the screening, plating was performed by using human or monkey CSF3R protein, incubation at 4°C overnight, and the supernatant was removed the next day. The plate was washed 3 times with 200 μL of washing solution per well. 200 μL of BSA solution was added to each well, and the plate was blocked at room temperature for 2 hours. The supernatant was removed, and the plate was washed 3 times with 200 μL of washing solution per well, and the supernatant was removed. 100 μL of antibody diluted with PBS was added to each well, and incubation was performed at room temperature for 1 h, and the supernatant was removed. The plate was washed 3 times with 200 μL of washing solution per well, and the supernatant was removed. 100 μL of secondary antibody solution (HRP goat anti-human IgG, abcam catalog number ab97225) was added to each well, and incubation was performed at room temperature for 30 min, and the supernatant was removed. The plate was washed 3 times with 200 μL of washing solution per well, and the supernatant was removed. 100 μl of TMB solution was added to each well, and after color development, 100 μl of sulfuric acid stop solution was added to each well, and the machine (TECAN, model Spark 10M) was used to detect OD450nm. The binding of the chimeric antibody to the protein is shown in Table 4. Figure 1 and Table 4.

[0057] Protein affinity: The antigen was human CSF3R protein (uniprot: Q99062, aa25-332). The binding of the anti-CSF3R antibody to the human CSF3R protein was measured using the Gator non-labeled analyzer device of probe Life. The biosensor used a protein A probe, and the target antibody was loaded at a concentration confirmed by A280, and then CSF3R protein was loaded. The kinetic binding rate and dissociation rate were determined at 6-7 concentrations of CSF3R protein at pH 7.4, and the KD was determined. The KD of the chimeric antibody binding to the protein is shown in Table 4.

[0058] Cell binding activity: 200,000 CSF3R expressing cells CHOS-CSF3R, 293T-CSF3R and Baf3-CSF3R were put in FACS buffer (PBS+2% FBS) per well, and incubated with chimeric antibodies to be tested at 4 degree for 1 hour. After centrifugation to remove supernatant, cells were washed twice with FACS buffer, and secondary antibody (DyLight488 goat anti-human IgG, Abeam Cat# ab97003) was added to incubate at 4 degree for 0.5 hour. After centrifugation to remove supernatant, cells were washed twice with FACS buffer, and resuspended with FACS buffer before machine reading. Flow cytometry (BD, model C6) was used to read the experimental cells. During reading, cells were first gated according to FSC and SSC, and then analyzed according to the fluorescence channel of secondary antibody and SSC. The binding of chimeric antibodies to cells was shown in Table 4 and Table 5. Figure 2 and Table 5.

[0059] The results showed that, at the protein level, the candidate antibodies could bind to both human and monkey CSF3R proteins. At the cell level, the candidate antibodies could bind to 3 CSF3R overexpressing cells. For affinity detection, the affinity of the antibodies was in the nM level.

[0060] Table 4 Protein binding activity of antibodies to CSF3R

[0061]

[0062] Table 5 Cell binding activity of antibodies to CSF3R

[0063]

[0064] Example 5 - Antibody blocking activity of CSF3R binding to ligand G-CSF

[0065] Blocking protein binding activity: plate with human CSF3R protein, 4°C incubate overnight, remove supernatant the next day. Wash the plate 3 times with 200ul / well of wash buffer. Add 200ul / well of BSA solution, block at room temperature for 2 hours. Remove supernatant, wash the plate 3 times with 200ul / well of wash buffer, remove supernatant. Add 50ul / well of chimeric antibody diluted in PBS and 50ul / well of biotinylated ligand G-CSF, incubate at room temperature for 1 hour, remove supernatant. Wash the plate 3 times with 200ul / well of wash buffer, remove supernatant. Add 100ul / well of secondary antibody solution (HRP Streptavidin, BioLegend Cat# 405210), incubate at room temperature for 30 minutes, remove supernatant. Wash the plate 3 times with 200ul / well of wash buffer, remove supernatant. Add 100ul / well of TMB solution, after color development add 100ul / well of sulfuric acid stop solution, machine (TECAN, Model Spark 10M) detection OD450nm. Chimeric antibody blocking protein binding to ligand is shown in Table 4 and Table 5. Figure 3 and Table 6.

[0066] Blocking cell binding activity: 200,000 CSF3R expressing cells CHOS-CSF3R, 293T-CSF3R and Baf3-CSF3R (25ul) are placed in FACS buffer for use, round bottom low adsorption 96 well plate is used. Antibody samples are diluted with FACS buffer, 25ul of diluted antibody is added to the cell plate, and the corresponding negative control well is added with FACS buffer, 4°C incubate for 30 minutes, then add 50ul of biotinylated CSF3R ligand G-CSF, 4°C incubate for 1 hour, after incubation, centrifuge to remove supernatant, then wash twice with FACS buffer, add secondary antibody (APC-his antibody, Biolegend Cat# 362605) to each well, 4°C incubate for another 0.5 hours. After staining, centrifuge to remove supernatant, wash twice with FACS buffer, then add FACS buffer to each well to resuspend the cells, and then machine reading. The flow cytometer (BD, Model C6) is used to measure the cells in the experimental plate. When measuring, first define the cell position according to FSC and SSC, then select the second antibody corresponding to the fluorescence channel and SSC to analyze the cells. Chimeric antibody blocking cell binding to ligand is shown in Table 7 and Table 8. Figure 4 and Table 7.

[0067] The results show that at the protein level, the candidate antibodies can block the binding of ligand G-CSF to human CSF3R protein. For the cell level, the candidate antibodies can also block the binding of ligand G-CSF to 3 strains of CSF3R overexpressing cells.

[0068] Table 6 Antibody blocking activity of ligand G-CSF binding to CSF3R protein

[0069]

[0070] Table 7 Antibody blocking activity of ligand G-CSF binding to CSF3R expressing cells

[0071]

[0072] Example 6 - Treatment of chronic rhinosinusitis by antibodies

[0073] 1. Antibody inhibiting cell proliferation

[0074] CSF3R expressing cells Baf3-CSF3R were plated at 1 x 10 4 cells / well in 96-well white plates in RPMI 1640 medium containing 10% FBS. Antibody samples were diluted in medium, 50ul of diluted antibody was added to the cell plate, and the corresponding negative control well was added with medium. After 30 minutes incubation at 37°C, 50ul of ligand G-CSF (Sinobio, Cat#10007-HNAH) was added to the cell plate, and the plate was incubated in 37°C incubator for 96 hours. Cell proliferation was determined by CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Cat#G7571), and luminescence reading was determined by Tecan Spark 10 microplate reader. The detailed results were shown in Table 8. Figure 5

[0075] The results showed that candidate antibody 2A6 could inhibit the proliferation of Baf3 cells overexpressing CSF3R, but 56D9E10 could not inhibit the proliferation of cells, which might be because 56D9E10 itself could promote the proliferation of Baf3-CSF3R cells (data not shown).

[0076] Table 8 Antibody inhibiting cell proliferation

[0077]

[0078] 2. Antibody inhibiting G-CSF induced neutrophil survival

[0079] ​Neutrophils were isolated from whole blood using EasySep™ Human Neutrophil Isolation Kit (Stemcell, Cat# 17957) according to the manufacturer's instruction, and the purity of isolated neutrophils was determined using CD11b-APC (Biolegend, Cat# 101212) and CD49d-FITC (Biolegend, Cat# 304316), neutrophils were identified by high side scatter (SSC) and CD11b+CD49d-phenotype. Neutrophils were plated at 1 x 10 5 antibody samples were diluted in culture medium, 50ul of diluted antibody was added to the cell plate, and the corresponding negative control wells were added with culture medium, incubated at 37°C for 30 minutes, then 50ul of ligand G-CSF (Sinobio, Cat# 10007-HNAH) was added, and incubated in 37°C incubator for 48 hours, the viability of neutrophils was determined using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Cat# G7571), and the luminescence reading of cell plate was determined using Tecan Spark10 microplate reader. The specific test results are shown in Table 9 and Table 10. Figure 6

[0080] From the results, it can be seen that the candidate antibodies can inhibit the survival of G-CSF-induced neutrophils.

[0081] Table 9 Antibody inhibits the survival ability of neutrophils

[0082]

[0083] 3. Antibody blocking pSTAT3 signaling ability

[0084] Neutrophils (purification steps are the same as Example 7, and the subsequent use of neutrophils is pure neutrophils, which will not be described below) were blocked using Human TruStain FcX™ (Biolegend, Cat# 422302), then 2 x 10 5 ​Cells were seeded per well in 96-well round-bottom plates and incubated with 50 μL of diluted antibody at 37°C for 20 minutes. Then, 50 μL of human G-CSF (Sinobio, catalog number 10007-HNAH) was added for stimulation for 15 minutes. Subsequently, cells were fixed with 4% paraformaldehyde (BD Biosciences) and permeabilized with methanol. After incubation, the cells were centrifuged to remove the supernatant, washed twice with FACS buffer, stained with anti-pSTAT3-PE (BD, catalog number 612569) for 30 minutes, washed twice, and analyzed by flow cytometry. Cell readings were performed using a flow cytometer (BD, model C6). Cell locations were first delineated using FSC and SSC, and then the corresponding fluorescence channel and SSC were selected for analysis. The ability of the chimeric antibody to block pSTAT3 signal transduction is shown below. Figure 7 As shown in Table 10.

[0085] The results showed that the candidate antibody could block the pSTAT3 signaling ability in neutrophils.

[0086] Table 10. Antibody's ability to block pSTAT3 signal transduction

[0087]

[0088] 4. Antibodies block the migration ability of neutrophils.

[0089] Neutrophils at 1.5 × 10 5 Cells were seeded per well in 96-well round-bottom plates and pretreated with 50 μL of diluted chimeric antibody or culture medium alone for 30 min, followed by activation with 50 μL of recombinant G-CSF for 20 h, with blank culture medium as a control. Additional cells for the standard curve were cultured overnight in IMDM medium containing 10% FBS.

[0090] The treated cells (upper chamber) were incubated with macrophage inflammatory protein-2 (MIP-2) and CXCL8 (lower chamber) in Transwell plates for 30 minutes. Cell migration was then measured using the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, catalog number G7571). The cell plates were read using a Tecan Spark10 microplate reader. Readings were compared to a standard curve generated using known cell numbers and to migration comparisons with cells incubated without chemical inducers. Specific results are as follows: Figure 8 As shown.

[0091] The results showed that the candidate antibody could block the migration of neutrophils to chemokines MIP-2 and CXCL8.

[0092] 5. Antibody inhibits G-CSF induced increase in expression of CXCR1 and CXCR2

[0093] Neutrophils were blocked with Human TruStain FcX™ (Biolegend, Cat# 422302) at 2x10 5 Cells were plated in 96-well round bottom plates at 2x10

[0094] Supernatant was removed by centrifugation and cells were washed twice with FACS buffer. PerCP / Cyanine 5.5-CXCR1 (Biolegend, Cat# 320622) and APC-CXCR2 (Biolegend, Cat# 320710) antibodies were added and incubated at 4 degrees Celsius for 0.5 hours. After staining, cells were centrifuged to remove supernatant and washed twice with FACS buffer. Cells were resuspended with FACS buffer and read on the machine. The flow cytometer (BD, Model C6) was used to read the cells. The cells were gated based on FSC and SSC, then the fluorescence channel corresponding to the staining antibody and SSC were selected for analysis. The results are shown in Figure 9

[0095] From the results, it can be seen that G-CSF can enhance the surface expression of CXCR1 and CXCR2 of neutrophils (data not shown), and the addition of the candidate antibody can inhibit the increase in expression of CXCR1 and CXCR2 induced by G-CSF.

[0096] 6. Antibody epitope detection

[0097] ​The detection antigen is human CSF3R protein (uniprot: Q99062, aa25-332). The epitope of different anti-CSF3R antibodies is measured by using the Gator non-labeled analyzer equipment of probeLife. The Anti-histidine probe is used, the CSF3R protein is loaded first, so that the binding response value is about 0.5 nm, after the probe is balanced with the buffer, the antibody 1 (each antibody to be detected) is loaded until the binding response reaches saturation, for the competitive step, the antibody 2 (all antibodies to be detected) is loaded, and the curve binding response value is observed. If the antibody 2 binding curve does not rise, it is determined that the antibody 1 and the antibody 2 are the same epitope, and if the antibody curve rises, the antibody 1 and the antibody 2 may be different epitopes or partially overlapping epitopes, which are quantified as inhibition rates here. The inhibition rate less than 30% is strong competition, between 30%-70% is partial competition, and greater than 70% is no competition. The inhibition rates of different antibodies are shown in Table 11.

[0098] From the results, it can be seen that 56D9E10 and CSL324 are strong competition and are the same epitope, and 2A6 is different from 56D9E10 and CSL324.

[0099] Table 11 Inhibition rate of antibody epitope determination

[0100]

[0101] From the above, it can be seen that the anti-CSF3R antibody 2A6 and 56D9E10 of the application can block the interaction of CSF3R with the ligand G-CSF, and then inhibit G-CSF signaling, neutrophil survival and migration, and the effect is equivalent to that of the reference antibody CSL324. Neutrophils are one of the key inflammatory cells of chronic sinusitis, and by blocking their activation or migration to the inflammatory site, the symptoms of chronic sinusitis can be relieved, and the therapeutic effect on chronic sinusitis can be achieved.

[0102] Obviously, the above embodiments are only examples for clearly illustrating, but not limitation to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. An anti-CSF3R antibody, characterized in that: The anti-CSF3R antibody comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region VH comprises VHCDR1, VHCDR2 and VHCDR3, and the light chain variable region VL comprises VLCDR1, VLCDR2 and VLCDR3; the sequence of VHCDR1 is shown as SEQ ID NO: 3, the sequence of VHCDR2 is shown as SEQ ID NO: 4, the sequence of VHCDR3 is shown as SEQ ID NO: 5, the sequence of VLCDR1 is shown as SEQ ID NO: 6, the sequence of VLCDR2 is shown as SEQ ID NO: 7, and the sequence of VLCDR3 is shown as SEQ ID NO: 8; or the sequence of VHCDR1 is shown as SEQ ID NO: 11, the sequence of VHCDR2 is shown as SEQ ID NO: 12, the sequence of VHCDR3 is shown as SEQ ID NO: 13, the sequence of VLCDR1 is shown as SEQ ID NO: 14, the sequence of VLCDR2 is shown as SEQ ID NO: 15, and the sequence of VLCDR3 is shown as SEQ ID NO:

16.

2. The anti-CSF3R antibody of claim 1, wherein: the sequence of the heavy chain variable region VH is shown as SEQ ID NO: 1, and the sequence of the light chain variable region VL is shown as SEQ ID NO: 2; or the sequence of the heavy chain variable region VH is shown as SEQ ID NO: 9, and the sequence of the light chain variable region VL is shown as SEQ ID NO:

10.

3. The anti-CSF3R antibody of claim 1, wherein: The anti-CSF3R antibody is a murine antibody.

4. A nucleotide molecule, characterized in that: The nucleotide molecule encodes the anti-CSF3R antibody according to any one of claims 1-3.

5. An expression vector, characterized by: The expression vector contains the nucleotide molecule according to claim 4.

6. A host cell, characterized in that: The host cell contains the expression vector according to claim 5 or the nucleotide molecule according to claim 4 is integrated into the genome of the host cell.

7. Use of the anti-CSF3R antibody according to any one of claims 1-3 in the preparation of a medicament for treating chronic rhinosinusitis.

8. Use according to claim 7, characterized in that: The anti-CSF3R antibody achieves the therapeutic effect of treating chronic rhinosinusitis by blocking the interaction of CSF3R with the ligand G-CSF, thereby inhibiting G-CSF signaling, neutrophil survival and migration.

9. A medicament for treating chronic rhinosinusitis, characterized by: The medicament comprises the anti-CSF3R antibody according to any one of claims 1-3.

10. The medicament according to claim 9, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier or excipient.

Images