Composition containing anti-FGL-1 antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a composition containing an anti-FGL-1 antibody and application of the composition, the composition is used for research and development of a corresponding protein detection reagent or a therapeutic antibody, and the composition is composed of the anti-FGL-1 antibody and an anti-LAG3 antibody; the composition provided by the invention is used for a detection reagent, a medicine or a medicine composition and is applied to tumor treatment. According to the technical scheme, the anti-FGL-1 antibody and the anti-LAG3 antibody reduce the growth of tumors by influencing secretion of cell factors and enhancing activation of T cells, and the synergistic effect of the two antibodies is stronger than the anti-tumor effect of the two antibodies independently used.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a composition comprising an anti-FGL-1 antibody and an application thereof, which are used for the research and development of corresponding protein detection reagents or therapeutic antibodies. Background Art

[0002] According to the latest data from the World Health Organization's International Agency for Research on Cancer (IARC), breast cancer has surpassed lung cancer to become the most common malignant tumor in women worldwide. It is also one of the main causes of cancer death in women, seriously threatening women's survival and health. Although breast cancer has made certain progress in detection and comprehensive treatment in recent years, the prognosis of patients has not been significantly improved, and the overall survival rate is still very low. Therefore, the study of new breast cancer molecular markers and therapeutic targets is of great significance for the early diagnosis, post-metastasis treatment and high drug resistance of breast cancer.

[0003] FGL-1 is a protein secreted by hepatocytes and belongs to the fibrinogen family. Under normal physiological conditions, its function is to participate in hepatocyte mitosis, energy metabolism, lipid metabolism and blood sugar regulation, and promote hepatocyte regeneration. Recent studies have found that FGL-1 is upregulated in lung cancer, prostate cancer, melanoma, colorectal cancer, breast cancer and brain tumors, while downregulated in pancreatic cancer, liver cancer and head and neck cancer. Further studies have found that FGL-1 plays a regulatory role in inflammation, cancer and immune regulation. LAG3 is a member of the IG superfamily and has a negative regulatory effect on T cells. It is mainly expressed on the surface of activated natural killer cells and T lymphocytes. LAG3 can maintain the body's immune homeostasis under physiological conditions, and inhibiting LAG3 function can enhance the anti-tumor effect of specific CD8+T cells. MHC II is a classic ligand of LAG3. Cancer cells reduce T cell activity by binding to LAG3. However, in some subsequent studies, it was found that monoclonal antibodies that did not block the binding of MHC II to LAG3 still showed the ability to activate T cells, confirming that MHC II is not an immunosuppressive ligand of LAG3. More valuable ligands may restart immune brakes by binding to LAG3. Therefore, FGL-1 has become a newly discovered ligand with high affinity for LAG3. The interaction between them is stable, specific, and conserved across species. In melanoma studies, blocking the interaction between LAG3 and FGL-1 with monoclonal antibodies increased intratumoral T cell responses, resulting in a reduction in tumor volume in a mouse melanoma model, which provides a new mechanism for targeted immunotherapy. Summary of the invention

[0004] The object of the present invention is to provide a composition comprising an anti-FGL-1 antibody and its application, so as to solve the problems existing in the above-mentioned background technology.

[0005] To achieve the above objectives, this application is implemented through the following technical solutions:

[0006] A composition comprising an anti-FGL-1 antibody, wherein the composition consists of an anti-FGL-1 antibody and an anti-LAG3 antibody.

[0007] Furthermore, the composition is used to inhibit the binding of FGL-1 to LAG3.

[0008] Furthermore, the composition is used for detecting a reagent, a drug or a drug composition.

[0009] Furthermore, the detection reagent, drug or pharmaceutical composition also includes an acceptable carrier.

[0010] Furthermore, the drug or pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the dosage forms of the drug or pharmaceutical composition include injections, tablets, capsules, granules, suspensions, emulsions, solutions, sols, lyophilized powder injections and sustained-release preparations.

[0012] An application, comprising the use of any of the above-mentioned compositions comprising anti-FGL-1 antibodies on tumors.

[0013] Furthermore, the tumor includes but is not limited to triple-negative breast cancer.

[0014] Furthermore, the anti-FGL-1 antibody and the anti-LAG3 antibody in the composition comprising the anti-FGL-1 antibody reduce tumor growth by affecting the secretion of cytokines and enhancing the activation of T cells.

[0015] Furthermore, blocking FGL-1 directly prevents tumor growth and metastasis.

[0016] The beneficial effects of the present invention are:

[0017] The anti-FGL-1 antibody and anti-LAG3 antibody of the present technical solution reduce tumor growth by affecting the secretion of cytokines and enhancing the activation of T cells, and the synergistic effect of the two antibodies is stronger than the anti-tumor effect of the two antibodies used alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of FGL-1 expression in breast cancer tissues, where: Figure 1 A shows that the expression of FGL-1 in breast cancer tissue is significantly higher than that in adjacent tissues. Figure 1 B shows that the expression of FGL-1 in poorly differentiated breast cancer is higher than that in moderately differentiated breast cancer.

[0019] Figure 2The figure is a schematic diagram of the CCK8 experimental results for samples numbered MDA-MB-468 and MDA-MB-231. The results show that compared with the untransfected triple-negative breast cancer cells MDA-MB-468 and MDA-MB-231, silencing FGL-1 led to a significant inhibition of the proliferation of these two triple-negative breast cancer cells.

[0020] Figure 3 The figure is a schematic diagram of the flow cytometry results for samples numbered MDA-MB-468 and MDA-MB-231. The results show that compared with the untransfected triple-negative breast cancer cells MDA-MB-468 and MDA-MB-231, silencing FGL-1 led to a significant increase in the apoptosis level of these two triple-negative breast cancer cells.

[0021] Figure 4 This is a bar graph showing the results of a cell migration experiment for samples numbered MDA-MB-468 and MDA-MB-231. The experiment showed that silencing FGL-1 significantly inhibited the migration of MDA-MB-468 and MDA-MB-231 triple-negative breast cancer cells.

[0022] Figure 5 To establish the co-culture system, 4T1 cells after antibody blocking FGL-1 were co-cultured with CD8+ T cells and divided into NC group, FGL-1 blocking group, LAG-3 blocking group, and FGL-1 / LAG-3 co-blocking group. Figure 5 The left picture in A shows the unactivated T cell state, where the T cells are densely arranged and their morphology has not changed. The right picture shows the activated T cell state, where the T cells become larger and grow in clusters. Figure 5 B shows the expression and location of FGL-1 and LAG-3 in the co-culture system of 4T1 breast cancer cell line and activated T cells. Green is FITC-labeled FGL-1 antibody, blue is DAPI-stained cell nucleus, and red is Cy3-labeled LAG3 antibody. Merge shows that FGL-1 and LAG3 are both expressed in the co-culture system, and the two are co-localized in the co-culture system. Figure 5 C shows that after FGL-1 antibody was added to 4T1 cells, 20 ng / mL of FGL-1 antibody had a significant inhibitory effect on the proliferation of 4T1 cells at 6 h.

[0023] Figure 6 To establish a co-culture system change map, Figure 6 A shows the overall proliferation changes of each group after co-culture of 4T1 cells and activated T cells; Figure 6 B shows the proliferation changes among groups after co-culture of 4T1 cells and activated T cells; Figure 6 C shows the migration of 4T1 cells after co-culture with activated T cells; Figure 6 D shows the statistical graph of 4T1 cell migration after co-culture of 4T1 cells with activated T cells (*P<0.05, **P<0.01, ***P<0.0001).

[0024] Figure 7 The figure is a schematic diagram of the tumor changes in the Balb / c mouse 4T1 subcutaneous tumor model with different injection doses. The mice were divided into different treatment groups (n=3), and PBS, Anti-FGL-1 6 mg / kg, and Anti-FGL-1 30 mg / kg were injected intraperitoneally every 2 days. Figure 7 A shows the histological diagram of mouse 4T1 tumor; Figure 7 B shows the changes in mouse body weight during treatment; Figure 7 C shows the growth curve of mouse 4T1 tumor.

[0025] Figure 8 Schematic diagram of the establishment of the 4T1 subcutaneous tumor model in Balb / c female mice, which were divided into different treatment groups (n=3) and intraperitoneally injected with PBS, Anti-FGL-1 (6 mg / kg), and Anti-FGL-1 / LAG3 (Anti-LAG3 5 mg / kg), respectively, once every 2 days; Figure 8 A shows the histological diagram of mouse 4T1 tumor; Figure 8 B shows the changes in mouse body weight during treatment; Figure 8 C shows the growth curve of 4T1 tumor in mice (*P<0.05, **P<0.01).

[0026] Fig. 9 For detection Figure 7 and Figure 8 Comparison of various parameters of tumor tissues in the three groups of mice. Fig. 9 A is the difference in the expression of Caspase 3 and FGL-1 proteins in tumor tissues of three groups of mice; Fig. 9 B and Fig. 9 Group C shows the relative expression ratio of Caspase 3 and FGL-1 proteins. Fig. 9 D. Fig. 9 E and Fig. 9 F shows the changes in the levels of different cytokines in the blood. The concentration of IL-2 (Figure D) was the highest in the Anti-FGL-1 6 mg / kg group, and the concentration of IL-10 (Figure E) was the lowest in the Anti-FGL-1 6 mg / kg group. The concentration of TNF-α (Figure F) was the highest in the Anti-FGL-1 6 mg / kg group (*P<0.05, **P<0.01). DETAILED DESCRIPTION

[0027] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings. The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as limiting the technical solution of the present invention.

[0028] 1. Expression level of FGL-1 in breast cancer:

[0029] Methods Fifty paraffin tissue samples from breast cancer patients were collected from the Department of Pathology of the Second Affiliated Hospital of Shandong First Medical University. HE staining and immunohistochemical staining were used to observe the expression differences of FGL-1 and LAG3 proteins in breast cancer tissues and adjacent tissues, and their correlation was analyzed.

[0030] Four human breast cancer cell lines, MDA-MB-468, MDA-MB-231, MCF-7 and T-47D cells, were selected, and the expression of FGL-1 protein was detected by immunofluorescence staining and Western Blot experiments, and the expression differences were analyzed. According to the expression level of FGL-1, siRNA transfection was used to knock down the FGL-1 gene in breast cancer cells, and its knockdown rate was demonstrated by western blot. CCK-8 cell proliferation experiment, cell scratch experiment and flow cytometry technology were used to detect the effect of FGL-1 knockdown on the proliferation, migration and apoptosis of breast cancer cells, and the effect of FGL-1 on the biological characteristics of breast cancer cells was studied.

[0031] 1. Paraffin embedding and sectioning:

[0032] 1) Fixation: After taking out the freshly excised tissue in the shortest possible time, cut it into small pieces and then quickly put it into 4% paraformaldehyde fixative for 24 hours. The purpose of fixation is to make the tissue have a certain hardness and try to maintain the original tissue structure and chemical composition. Then trim the fixed tissue into small pieces of 1.0 cm × 1.0 cm × 0.5 cm and put it into a dehydration box with a pre-labeled name.

[0033] 2) Rinse: Place the fixed tissue block under the faucet and rinse the tissue block with running water for 24 hours.

[0034] 3) Dehydration: Place the dehydration box in gradient anhydrous ethanol for dehydration, 70% (2 h) → 80% (2 h) → 90% (1 h) → 95% (1 h) → 100% I (45 min) → 100% II (45 min) to replace the water in the cells.

[0035] 4) Transparency: After dehydration, use forceps to gently pick up the tissue block to avoid any damage to the tissue block. Place it on filter paper to absorb the excess ethanol on the surface of the tissue. After the ethanol on the surface of the tissue block is fully absorbed, put it into xylene for transparency. Xylene I (10 min) → Xylene II (10 min). Observe the transparency every 5 minutes, and put it into wax when the tissue becomes transparent.

[0036] 5) Wax immersion: Turn on the wax dissolving machine in advance, place the dehydration box into 58℃~60℃ liquid paraffin, take it out after 3 hours, and maintain the temperature of the wax.

[0037] 6) Embedding: First, take out the embedding box and inject melted paraffin. Then, use the heated tweezers to gently pick up the wax-soaked tissue block and place it in the paraffin in the embedding frame. Pay special attention to the cut surface of the tissue facing down and placing it flat to ensure that the subsequent sectioning operation can proceed smoothly. After placing the tissue block, wait for the paraffin to cool and solidify. After the paraffin is completely solidified, gently open the embedding frame and remove the wax block.

[0038] 7) Sectioning: Finely trim the wax block and place it on a paraffin slicer. After sectioning, gently spread the slices on a warm water slicer set at 40°C to avoid folding or sticking. Then use a clean slide to gently lift the slices from the water surface. Finally, place the slide with the slices on a 60°C baking machine. By baking the slices, the moisture on the slices is quickly evaporated, making the slices smoother and easier to use in subsequent experiments.

[0039] 2. HE staining:

[0040] 1) Dewaxing: Place sections in xylene I (15 min) → xylene II (15 min).

[0041] 2) Rehydration: Place the dewaxed sections in gradient anhydrous ethanol in sequence, starting with 100% anhydrous ethanol and decreasing the concentration step by step: 100% I (10 min) → 100% II (10 min) → 95% (5 min) → 90% (5 min) → 80% (5 min) → 70% (5 min) → distilled water (5 min).

[0042] 3) Staining: Place the slices in hematoxylin for 2-3 minutes and rinse with running water. Then place them in hydrochloric acid differentiation solution for 3 seconds and rinse with running water to terminate differentiation. Finally, place them in eosin staining solution for 5 minutes and rinse with running water.

[0043] 4) Dehydration: Dehydrate in gradient anhydrous ethanol, 70% (5 s) → 80% (5 s) → 90% (5 s) → 95% (2 min) → 100% (2 min) → 100% II (2 min).

[0044] 5) Sealing: Use neutral gum to seal the slide and observe under a microscope.

[0045] 3. Immunohistochemistry:

[0046] 1) Slice baking: Mark the slices with the indicators to be tested and put them into the slice baking machine at 56℃ / 30 min.

[0047] 2) Dewaxing and rehydration: Place the sections in xylene I (20 min) → xylene II (20 min) for dewaxing. Then place the dewaxed sections in gradient anhydrous ethanol, starting with 100% anhydrous ethanol, and gradually reduce the concentration from 100% I (10 min) → 100% II (10 min) → 95% (10 min) → 90% (10 min) → 80% (10 min) → 70% (10 min). After rinsing with running water, place them in PBS I (5 min) → PBS II (5 min).

[0048] 3) Antigen repair: Set the temperature of the water bath to 100°C and place the pre-prepared Tris-EDTA antigen repair solution (PH=9.0) in the water bath. Completely immerse the slices to be processed in the EDTA solution to ensure that each slice is fully exposed to the repair solution. After 20 minutes, remove the slices from the water bath and let them cool naturally to room temperature. Wash with PBS 3 times, 3 minutes each time. After washing, circle the outline of the tissue with an immunohistochemistry pen to avoid waste of reagents and deviation of experimental results. Drop 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes. After incubation, wash with PBS for 3 minutes × 3 times and shake off excess liquid on the slices.

[0049] 4) Blocking: Block with 5% bovine serum albumin, incubate at room temperature for 1 hour, do not wash.

[0050] 5) Primary antibody: After blocking, use 5% bovine serum albumin to prepare the primary antibody. After dropping the antibody onto the slice, place it in a wet box and keep it in a refrigerator at 4°C overnight.

[0051] 6) Secondary antibody: The next day, take the wet box out of the 4°C refrigerator, return to room temperature, recover the primary antibody, wash the sections in PBS for 3 min × 3 times, add secondary antibody, and incubate at room temperature for 1 h.

[0052] 7) Color development: Wash in PBS for 3 min × 3 times, shake off PBS, add DAB color development solution, incubate at room temperature for 5-8 min (observe under microscope), rinse with distilled water to terminate DAB color development. Then stain with hematoxylin for 2 min, pass through hydrochloric acid differentiation solution for 3 s, and rinse with running water.

[0053] 8) Dehydration, transparency and sealing: Dehydration in gradient anhydrous ethanol, 70% (2 min) → 80% (2 min) → 90% (2 min) → 95% (2 min) → 100% I (2 min) → 100% II (2 min) → xylene I (2 min) → xylene II (2 min) → neutral gum sealing - observation under microscope.

[0054] 4. Cell Immunofluorescence:

[0055] 1) Take out the required cell slides in advance, soak them in anhydrous ethanol, and keep them in a 4°C refrigerator overnight.

[0056] 2) Place the required complete culture medium, trypsin, sterile PBS and other experimental reagents in a 37℃ water bath to rewarm. Take the cell slide out of the 4℃ refrigerator and place it in a 24-well plate using tweezers on the operating table. Wash the slide three times with anhydrous ethanol and once with PBS, then open the cover and sterilize with ultraviolet light for 1 hour.

[0057] 3) Take the cells out of the incubator, discard the culture medium, and slowly rinse with sterile PBS. Add 1 mL of trypsin and digest in the incubator for 2-3 minutes. Observe the cell state under a microscope. When the digestion state is reached, immediately add an equal amount of trypsin and complete culture medium to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and add 1 mL of complete culture medium to the centrifuge tube to prepare a single cell suspension.

[0058] 4) Clean the cell counting plate and cover glass, take 10 μL of the mixed single cell suspension and drop it on the edge of the counting pool. After the cell suspension enters the counting pool, let it stand for a while to evenly distribute in the counting pool. Then move the counting plate stably under the microscope for counting. The counting principle is: record the top but not the bottom, and record the left but not the right; the number of cells in the single cell suspension = (number of counts / 4) × dilution factor × 104.

[0059] 5) The number of cells required for each well of cell immunofluorescence is 4×10 4 , add the counted single cell suspension into the well plate, shake the well plate to make the cells evenly distributed, observe under the microscope, spray with 75% alcohol and place in the incubator for 24 hours.

[0060] 6) The next day, carefully observe the growth of cells under a microscope. When the cells grow to about 40% coverage, discard the cell culture medium, slowly rinse with PBS three times, and then add 4% paraformaldehyde to fix for 30 minutes. Fix the cells at room temperature. After fixation, wash with PBS three times, 3 minutes each time. Then, after absorbing all the PBS, add 5% BSA and block at room temperature for 2 hours. After blocking, discard the blocking solution and directly add 200 μL of primary antibody diluted with 1% BSA and store in a refrigerator at 4℃ overnight.

[0061] 7) The next day, take the plate out of the 4℃ refrigerator and let it stand for 30 minutes to return to room temperature. Recover the primary antibody and wash it with PBS three times, 3 minutes each time. Add fluorescent secondary antibody and incubate at room temperature for 1 hour under light-proof conditions. After washing with PBS, add DAPI staining solution and incubate in the dark for 3 minutes to stain the cell nuclei. After the excess DAPI is washed with PBS, use curved tweezers and a needle to carefully remove the cell slide from the plate. After absorbing the liquid on the slide with absorbent paper, turn it upside down on a slide with anti-fluorescence quencher sealing solution. Finally, observe and collect images under a fluorescence microscope.

[0062] 5. Total protein extraction:

[0063] For cells in the logarithmic growth phase or after transient transfection, discard the complete medium, slowly rinse with 4°C pre-cooled PBS three times, and carefully discard the PBS with a pipette. Add cell lysis buffer (PIPA:PMSF=100:1), and after a short lysis, use a cell scraper and a pipette to collect the lysed cell fragments into a 1.5 mL centrifuge tube, and immediately place it on ice for lysis for 30 min. During this period, shake the centrifuge tube once every 10 min to promote the full lysis of cell fragments. After the lysis is completed, place the centrifuge tube in a 4°C pre-cooled centrifuge and centrifuge at 12000 rpm / min for 15 min. After the centrifugation is completed, carefully remove the supernatant, mark the name and date, and store it in a -80°C refrigerator.

[0064] 6. BCA protein concentration determination:

[0065] 1) Take 10 μL of BSA standard and dilute it to 100 μL with PBS to make the final concentration 0.5 mg / mL. Add 0, 2, 4, 6, 8, 12, 16, 20 μL of the standard to the protein standard wells of the 96-well plate and add PBS to make up to 20 μL.

[0066] 2) Dilute the sample appropriately with PBS (2-fold, 4-fold, 8-fold dilution) and add 20 μL to the sample wells of a 96-well plate.

[0067] 3) According to the number of standards and samples, prepare BCA working solution by adding 1 volume of Cu reagent to 50 volumes of BCA reagent (50:1) and mix thoroughly.

[0068] 4) Add 200 μL of BCA working solution to each well and place at 37°C for 15-30 min. Measure A562 nm with an ELISA reader and calculate the protein concentration based on the standard curve.

[0069] 5) After calculating the concentration of the extracted protein, adjust the concentration of each sample, add 4× protein loading buffer according to the amount of protein, mix well, put into a metal bath, and cook at 100℃ for 10 min. After returning to room temperature, store in a -20℃ refrigerator.

[0070] 7. Western Blot:

[0071] 1) Gel preparation: Align the cleaned glass plates and insert them into the gel preparation rack to ensure that they fit tightly without gaps. Add 1 mL of ddH2O between the glass plates and carefully observe whether there is any leakage. Take an equal volume of 2.5 mL of the lower gel solution and 2.5 mL of the lower gel buffer, mix them together, add 50 μL of the improved coagulant to the mixture, and gently mix again. Add the prepared lower gel solution to the gel preparation plate to avoid bubbles. Add an appropriate amount of anhydrous ethanol to flatten the separation gel. After waiting for 30 minutes, the lower gel gradually solidifies. Pour out the upper layer of anhydrous ethanol and gently absorb the excess ethanol with filter paper. Take an equal volume of 0.75 mL of the upper gel solution and 0.75 mL of the colored upper gel buffer, also add 15 μL of the improved coagulant, and mix well. Pour the prepared upper gel solution onto the solidified lower gel, and then slowly insert the comb to ensure that there are no bubbles between the comb and the gel surface. After the upper gel solidifies, the electrophoresis experiment can be carried out.

[0072] 2) Electrophoresis: Insert the prepared gel and glass plate into the electrophoresis tank, making sure they are tightly combined. After adding the electrophoresis solution, slowly pull out the comb, and add the pre-prepared marker and protein sample into the electrophoresis tank in turn. Connect the electrophoresis tank to the power supply according to the specified electrodes. Use 80 V for electrophoresis for 15 min. When bromophenol blue has been electrophoresed to the lower separation gel, change to 200 V for electrophoresis until the protein sample is completely electrophoresed to the bottom of the gel.

[0073] 3) Transfer: Before transfer, cut a suitable PVDF membrane and place it in methanol for 2 min to activate the membrane. Then place the sponge and activated PVDF membrane in the fast transfer solution. After the electrophoresis is completed, remove the glass plate, pry it open slowly, and cut the target band according to the position of the marker band based on the molecular weight of the target protein. Then open the transfer clamp and clamp it in the order of "black gel and white film" and place it in the transfer tank. Pay attention to connecting the positive and negative electrodes, adjust the current to 400 mA and transfer for 30 min.

[0074] 4) Blocking: Before transfer, use TBST to prepare 5% skim milk powder blocking solution. After transfer, place the PVDF membrane in a protein incubation box pre-filled with blocking solution and block at room temperature for 1 hour.

[0075] 5) Apply primary antibody: After blocking, discard the blocking solution and wash the membrane 3 times with TBST, 10 min each time. Prepare the primary antibody according to the ratio. After washing the membrane, add the primary antibody and place the antibody incubation box on a low-speed shaker in a 4°C refrigerator overnight.

[0076] 6) Apply secondary antibody: The next day, take the antibody incubation box out of the 4°C refrigerator in advance, restore it to room temperature and recover the primary antibody. After washing the membrane three times with TBST, add the prepared secondary antibody and incubate at room temperature for 1 hour.

[0077] 7) Development: Prepare ECL luminescent solution in the ratio of A solution: B solution = 1:1. After washing the membrane, place the PVDF in the tray of the developer and drip the luminescent solution. After exposure, save the development results and finally use ImageJ software to analyze the development results.

[0078] 8. FGL-1 si-RNA transfection of breast cancer cells:

[0079] 1) Cell plating (taking transfection of 6-well plates as an example): Observe the density and growth status of cells under a microscope before transfection, digest with trypsin, and inoculate 150,000 ± 5,000 cells into 6-well plates after centrifugation (ensuring that the cell density reaches 50% to 70% during transfection). Culture in a 37°C, 5% CO2 incubator for 24 h. The optimal transfection state is achieved when the cell mass reaches 60% to 80%.

[0080] 2) Preparation of transfection complex: First, after centrifugation, remove the Oligo RNA, add 125 μL of DEPCH2O, resuspend the Oligo RNA, and mix gently to ensure that 1 OD of si-RNA is fully dissolved into a 20 μm sample. Prepare two 1.5mL sterile centrifuge tubes for preparing two mixtures. In the first centrifuge tube, add 200 μL of OPTI-MEM and 6 μL of si-RNA-Mate transfection reagent, mix gently with a pipette, and let stand at room temperature for 5 min. In the second centrifuge tube, add 200 μL of OPTI-MEM and 7.5 μL of Oligo RNA, also mix gently with a pipette, and let stand at room temperature for 5 min. The purpose of these two steps is to allow the transfection reagent and RNA to fully combine to form a stable complex. Finally, add the GP-transfect-Mate-culture medium mixture dropwise to the RNA oligo / DNA-culture medium mixture, mix well, and let stand at room temperature for 15-20 min to further stabilize the complex. After the resting time, the complexes were immediately added to the cells for transfection.

[0081] 3) Transfection: While the complex is resting, take out the 6-well plate and add 1.6 mL of OPTI-MEM medium restored to room temperature to each well. Then, add 400 μL of the rested transfection complex to the plate, so that the final system in each well reaches 2 mL. After adding the complex, gently shake the plate to evenly distribute the transfection complex in the plate. Place the cells in the incubator and culture them. After 4 to 6 hours, change to complete culture medium. After 48 to 96 hours, use Western Blot to detect protein knockdown levels, such as Figure 1 shown.

[0082] 9. CCK-8 cell proliferation assay:

[0083] 1) Take cells in the logarithmic growth phase, wash with PBS, digest with trypsin, centrifuge and prepare a single-cell suspension.

[0084] 2) Count the cells using a cell counting plate and adjust the cell density to 7 × 10 3 / well, inoculated in a 96-well plate. Set up a blank control group, set up 3 replicate wells in each group, and culture in an incubator for 24 h.

[0085] 3) The next day, when the cell density reached 60% to 80% under the microscope, the transfection complex was prepared according to the instructions of Genema Gene, and further transfection was performed. Then, the cells were placed in an incubator and cultured for 0 to 72 hours.

[0086] 4) Take out the 96-well plate from the incubator, add 10 μL / well of CCK-8 reagent in the dark on the operating table, and incubate it in the incubator again for 1 to 4 hours. Finally, use an ELISA reader to measure the absorbance (OD) at 450 nm, record the experimental data, and calculate the cell inhibition rate. Cell inhibition rate = [(experimental group - blank group average) / (control group - blank group) × 100%, see the results Figure 2 shown.

[0087] 10. Scratch test:

[0088] 1) Take cells in the logarithmic growth phase and prepare a single cell suspension. Count the cells using a cell counting plate and adjust the cell density to 2×10 5 / well, inoculated into a 6-well plate and cultured in an incubator for 24 h.

[0089] 2) The next day, use the tip of a pipette to draw a vertical line in the 6-well plate by touching the cover of the 6-well plate, rinse the cells with PBS, and add serum-free medium. Prepare the transfection complex according to the instructions of Genentech and perform further transfection. Then place the 6-well plate in an incubator for culture.

[0090] 3) Take pictures of cell scratches at 0, 24, 48, and 72 hours under a microscope, and finally use ImageJ to process the pictures and calculate the scratch area. Cell scratch area = (initial scratch area - scratch area at time T) / initial scratch area.

[0091] 11. Detection of cell apoptosis by flow cytometry

[0092] 1) Take cells in the logarithmic growth phase and digest them with EDTA-free trypsin to prepare a single-cell suspension. Carefully remove the supernatant, add 1 mL of 4°C pre-cooled PBS, resuspend the cells, centrifuge again to precipitate the cells, and aspirate the supernatant.

[0093] 2) Dilute the binding buffer 1:3 with deionized water, and then resuspend the cells in 1× binding buffer to adjust the concentration to 1×10 6 ~5×10 6 / mL.

[0094] 3) Take 100 μL of cell suspension and place it into a 5 mL flow cytometry tube, then add 5 μL of Annexin V / PE reagent and mix thoroughly. Then, incubate the mixture at room temperature in the dark for 5 min.

[0095] 4) Add 10 μL of 7AAD (20 μg / mL) and immediately add 400 μL of PBS for flow cytometry detection. The results are shown in Figure 3 shown.

[0096] 2. Effects of FGL-1 / LAG-3 blockade on breast cancer:

[0097] In order to further explore the effects of FGL-1 and LAG3 on breast cancer, the expression of FGL-1 in mouse breast cancer cells 4T1 was detected by immunofluorescence, and the effects of silencing FGL-1 expression on the proliferation and migration of mouse breast cancer cells were further detected by cell proliferation assay and cell scratch assay.

[0098] 4T1 cells after blocking FGL-1 were co-cultured with primary activated CD8+T cells, and cell proliferation and cell migration experiments were used to detect the changes in the ability of CD8+T cells to kill breast cancer 4T1 cells after blocking FGL-1 / LAG3 in the co-culture system.

[0099] A 4T1 breast cancer mouse model was constructed, and Western blot and Elisa were used to explore the effects of FGL-1 / LAG3 on tumor cell apoptosis, T cell infiltration, and changes in inflammatory factors.

[0100] 1. Co-culture of tumor cells and CD8+T cells:

[0101] 1) 6- to 8-week-old female Balb / c mice were selected and killed with chloral hydrate. The mice were immersed in anhydrous ethanol for 5 min and the spleen was removed from the abdominal cavity.

[0102] 2) Place the removed spleen in ice-cold PBS, chop it into small pieces in a 6-well plate, and then place the chopped tissue pieces on a 40 μm nylon mesh. Place a 50 mL centrifuge tube under the nylon mesh and use a 5 mL disposable syringe piston to rotate and grind the spleen. After grinding, collect the suspension containing spleen cells and transfer it to a 15 mL centrifuge tube. In order to remove impurities and unnecessary components in the suspension, place the centrifuge tube in a pre-cooled 4°C centrifuge and centrifuge at 300 g for 10 min.

[0103] 3) After centrifugation, discard the supernatant, add 1 mL of cell separation buffer (PBS + 3% fetal bovine serum + 10 mM EDTA) and 3 mL of red blood cell lysis buffer, mix with a pipette, place on ice for 15 min, vortex twice during the period, and then centrifuge at 450 g in a precooled 4°C centrifuge for 10 min.

[0104] 4) Add 200 μL of buffer [PBS (PH7.2) + 2 mM EDTA + 0.5% BSA] to resuspend the cells. Count the cells and count every 10 7 Add 40 μL buffer per cell and add buffer according to the number of cells after cell counting.

[0105] 5) To label and isolate CD8+ T cells, calculate the amount of Biotin-Antibody Cocktail required based on the number of cells. 7 Add 10 μL of Biotin-Antibody Cocktail to each cell. Mix thoroughly by pipetting carefully, and then incubate in a 4°C refrigerator for 5 min. 7 cells, add 30 μL of buffer. 7 cells, added 20 μL of Anti-Biotin Micro Beads, ensured that the magnetic beads and cells were fully mixed, and then placed in a 4°C refrigerator for incubation for 10 min.

[0106] 6) Place the LS column in the magnetic field of the MACS separator. Wash the column with 3 mL of buffe first, then add the cell suspension to the column, and finally rinse the column with 3 mL of buffe. The cells that flow into the 15 mL centrifuge tube are the sorted CD8+T cells. After centrifugation at 300 g for 10 min, add 2-3 mL of complete medium for CD8+T cells (10% fetal bovine serum inactivated at 56°C + 2mM L-glutamine + 1% penicillin-streptomycin + RPMI 1640 medium) and count the cells.

[0107] 7) Before the experiment, use a 12-well plate with antibody coating. Dilute the CD3 antibody to 5 μg / mL with sterile PBS, 1 mL per well, set up a control, and coat the control well with PBS. Place the culture plate in a 4°C refrigerator and incubate for 12 h.

[0108] 8) Count the sorted cells according to the number of cells, 3×10 5 ~4×10 5 Transfer to a 12-well plate, add 1 mL per well, add 2 μg / mL CD28 antibody (diluted with PBS) and 10 ng / mL IL-2, and do not add to the control well. Seal the plate and culture in an incubator for 24 to 72 hours.

[0109] 9) After observing the T cells growing in clusters and increasing in size under a microscope, centrifuge the T cells and count the cells. After counting the cells, observe their proliferation.

[0110] 10) The activated CD8+T cells were directly co-cultured with the tumor cells 4T1, and FGL-1 and LAG3 antibodies were added to the co-culture system to form a system of blocking FGL-1, blocking LAG3, and jointly blocking FGL-1 / LAG3 to detect its effect on breast cancer.

[0111] 2. Co-culture Transwell migration experiment:

[0112] CD8+T and 4T1 cell migration assays were performed using a 6-well plate with a diameter of 8 μm. The cells were divided into NC group, FGL-1 blocking group, LAG3 blocking group, and FGL-1 / LAG3 co-blocking group. The 4T1 cells in the FGL-1 blocking group and the co-blocking group were first treated with FGL-1 antibody for 6 h, resuspended in serum-free medium in the upper chamber of Transwell, and CD8+T cells cultured in complete medium were added to the lower chamber. Subsequently, LAG3 antibody was added to the LAG3 blocking group and the co-blocking group. After the cells were cultured for 24 h, the medium was discarded, the cells were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. Cell migration images were collected under a microscope, as shown in Figure 2. Figure 4 , Figure 5 , Figure 5 As shown in A.

[0113] 3. Subcutaneous tumor:

[0114] 1) Cell preparation: Take the mouse tumor cells 4T1 in logarithmic phase. The culture medium of all cultured cells was replaced the day before inoculation to ensure that the cells are in the best growth environment. On the day of inoculation, take out the cultured cells from the incubator, discard the culture medium, add trypsin for digestion, centrifuge and discard the supernatant, add PBS to make a single cell suspension, count the cells, and adjust the concentration to 5×10 per 200 μL. 5 Place the cells on ice and complete the inoculation within 15 to 30 min.

[0115] 2) Tumor model construction: 6-8 week old Balb / c female mice were selected. Before the experiment, the right armpit of the mice was depilated to ensure that the subcutaneous injection area was hairless. After weighing, the skin on the back of the right armpit of the mice was disinfected with 75% alcohol. A 1 mL syringe was used to extract 0.2 mL of cell suspension, which contained 5×10 cells. 5 The position of the middle and outer side of the axilla near the head was selected as the near needle point. The mouse was first pierced obliquely into the subcutaneous tissue at an angle of 45 degrees, and then kept parallel to the skin, penetrating about 1 to 1.5 cm deep, and then subcutaneously injected. The injection site should be ensured to be in the subcutaneous tissue layer to avoid injecting the cell suspension into the inner layer of the skin. After the injection is completed, the injection site is disinfected again and pressed lightly with a sterile cotton swab for 10 s to prevent the cell suspension from leaking out of the needle hole. After the injection, the mice were fed normally, the mice were observed every 2 days, and the weight and tumor volume of the mice were recorded. Tumor calculation formula: tumor volume = ab2 / 2 (a is the long diameter and b is the short diameter).

[0116] 3) Intraperitoneal administration: Record the tumor volume of mice. We selected mice with tumors of 80 mm in size. 3The drug was administered at about 1:10 pm. First, the tumor-bearing mice were divided into three groups (n=3 in each group) to study the optimal therapeutic concentration of FGL-1 for tumor-bearing mice. Then, the optimal concentration of FGL-1 antibody was used together with LAG3 antibody to treat tumor-bearing mice to study the inhibitory ability of FGL-1 and LAG3 combined blockade on tumors. The dosage was intraperitoneally injected according to the mouse body weight × 10 μg. The drug was administered every two days for a total of 6 times. The mouse body weight and tumor volume were measured before each administration. The mice were killed two days after the last administration, and the mouse body weight and tumor volume change curves were plotted. See Figure 2. Figure 6 and Figure 6 As shown in A.

[0117] 4. ELISA:

[0118] 1) Collect blood from mouse eyeballs, let it agglutinate for more than 30 min at room temperature, and centrifuge at 1,000 g for 10 min. Detect serum samples immediately after collection, or aliquot and store in a -80℃ refrigerator.

[0119] 2) Before testing, ensure that all reagents and samples have been equilibrated to room temperature to ensure the accuracy and stability of the experiment. Take out the ELISA plate and add 300 μL 1× washing solution, soak for 30 seconds to fully wash the residue on the ELISA plate. Place the ELISA plate on absorbent paper and tap it several times to ensure that the excess liquid in the microplate is completely absorbed.

[0120] 3) After washing, add 2-fold diluted standards and samples, and do not let the microplate dry.

[0121] 4) Add 50 μL of detection antibody to each well, and add samples continuously within 15 minutes without interruption. Seal the plate with a sealing film. Oscillate at 100-300 rpm / min and incubate at room temperature for 1.5 hours. Then discard the liquid, add 300 μL of washing solution to each well, and wash the plate 6 times.

[0122] 5) Add 100 μL of "streptavidin working solution" to each well, seal the ELISA plate with a new sealing film, and incubate it at room temperature for 30 minutes to allow streptavidin to fully react with the sample. After the incubation, wash the ELISA plate, add 100 μL of substrate color solution to each well under light-proof conditions, and after 5 to 30 minutes, allow the substrate to react with the target substance in the sample. After the color reaction is completed, add 100 μL of stop solution to each well. At this time, the originally blue reaction solution will quickly turn yellow.

[0123] 6) Within 30 minutes, use a microplate reader to perform dual wavelength detection (450 nm, 630 nm). The calibrated OD value is the value measured at 450 nm minus the value measured at 630 nm, see Figure 7shown.

[0124] The present application provides a composition comprising an anti-FGL-1 antibody, wherein the composition is composed of an anti-FGL-1 antibody and an anti-LAG3 antibody. In the composition of the present application, the ratio of the anti-FGL-1 antibody to the anti-LAG3 antibody is not specified in the present application, and can be finally confirmed for different populations based on actual experimental data. However, the amount of the anti-FGL-1 antibody is usually less than the amount of the anti-LAG3 antibody.

[0125] In the present application, the composition is used to inhibit the binding of FGL-1 to LAG3. Anti-FGL-1 antibodies alone can also inhibit the binding of FGL-1 to LAG3, but the effect is obviously not obvious.

[0126] In the present application, the composition is used for detecting a reagent, a drug or a drug composition.

[0127] In the present application, the detection reagent, drug or pharmaceutical composition also includes an acceptable carrier, and the drug or pharmaceutical composition also includes a pharmaceutically acceptable excipient. In the present application, the excipient and the carrier may be the same or different.

[0128] In the present application, the dosage forms of the drugs or pharmaceutical compositions include injections, tablets, capsules, granules, suspensions, emulsions, solutions, sols, lyophilized powder injections and sustained-release preparations, and may also include any other applicable dosage forms.

[0129] The present application also relates to an application, namely, the application of any of the above-mentioned compositions comprising anti-FGL-1 antibodies on tumors, including but not limited to triple-negative breast cancer. The specific types of tumors can be obtained by those skilled in the art through experiments based on the technical ideas and routes of the present application.

[0130] In the present application, the anti-FGL-1 antibody and the anti-LAG3 antibody in the composition comprising the anti-FGL-1 antibody reduce tumor growth by affecting the secretion of cytokines and enhancing the activation of T cells.

[0131] In the present application, tumor growth and metastasis are directly prevented by blocking FGL-1.

[0132] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition comprising an anti-FGL-1 antibody, characterized in that The composition consists of an anti-FGL-1 antibody and an anti-LAG3 antibody.

2. The composition comprising an anti-FGL-1 antibody according to claim 1, characterized in that The composition is used to inhibit the binding of FGL-1 to LAG3.

3. The composition comprising an anti-FGL-1 antibody according to claim 1, characterized in that The composition is used for detecting a reagent, a drug or a pharmaceutical composition.

4. The composition comprising an anti-FGL-1 antibody according to claim 3, characterized in that The detection reagent, drug or pharmaceutical composition also includes an acceptable carrier.

5. The composition comprising an anti-FGL-1 antibody according to claim 4, characterized in that The medicament or pharmaceutical composition further includes pharmaceutically acceptable excipients.

6. The composition comprising an anti-FGL-1 antibody according to claim 3, characterized in that The dosage forms of the medicine or pharmaceutical composition include injection, tablet, capsule, granule, suspension, emulsion, solution, sol, lyophilized powder injection and sustained-release preparation.

7. An application, characterized in that: Use of the composition comprising an anti-FGL-1 antibody according to any one of claims 1 to 6 for treating tumors.

8. The use according to claim 7, characterized in that: The tumor includes but is not limited to triple-negative breast cancer.

9. The use according to claim 7, characterized in that: The anti-FGL-1 antibody and the anti-LAG3 antibody in the composition comprising the anti-FGL-1 antibody reduce tumor growth by affecting the secretion of cytokines and enhancing the activation of T cells.

10. The use according to claim 7, characterized in that: Directly inhibit tumor growth and metastasis by blocking FGL-1.

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