Application of anti-FGL1 monoclonal antibody in preparation of medicine for treating pancreatic cancer liver metastasis

Abstract

The invention provides application of an anti-FGL1 monoclonal antibody in preparation of drugs for treating pancreatic cancer liver metastasis, and belongs to the technical field of antibody drugs. The anti-FGL1 monoclonal antibody can be combined with FGL1 protein in a high-specificity mode, the action mechanism of the anti-FGL1 monoclonal antibody in pancreatic cancer liver metastasis treatment is verified through a tumor cell model, a patient-derived tumor organ model and a mouse pancreas in-situ tumor model, and the anti-FGL1 monoclonal antibody can be used for preparing potential drugs for treating pancreatic cancer liver metastasis. Meanwhile, the anti-cancer effects of the FGL1 monoclonal antibody in normal diet mice and high fat diet mice (fatty liver models) are compared, so that the applicable population of the antibody therapy is determined, and a scientific basis is provided for the treatment of the FGL1 monoclonal antibody in pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to the use of anti-FGL1 monoclonal antibodies in the preparation of drugs for treating pancreatic cancer liver metastasis. Background Art

[0002] Pancreatic cancer is a highly malignant digestive system tumor characterized by high invasiveness and rapid proliferation. Early on, typical symptoms are absent, and over half of patients are diagnosed at the advanced stage. Up to 80% of advanced pancreatic cancers harbor distant organ metastases, with the liver being the primary site of metastasis. Pancreatic cancer cells reshape the liver's microenvironment, promoting tumor growth and immune evasion, contributing to the extremely poor prognosis of pancreatic cancer. Currently, surgery is the only curative option for pancreatic cancer. However, extensive invasion of the liver's vasculature and surrounding tissues by tumor cells makes resection difficult and associated with a high rate of postoperative complications. Therefore, surgery is not suitable for patients with pancreatic liver metastases. Furthermore, radiotherapy and chemotherapy are extremely ineffective, leaving these patients with a limited survival time of only 2-6 months. Therefore, the development of effective therapeutics for pancreatic liver metastases is an urgent clinical challenge. Summary of the Invention

[0003] To address the shortcomings of the existing technology, the inventors discovered through preliminary research that the secretory factor FGL1, which is specifically upregulated by fatty liver, binds to the pancreatic cancer cell membrane receptor LIFR and promotes LIFR acetylation modification. It then promotes its plasma-membrane translocation by upregulating GPX4 palmitoylation levels, thereby inhibiting ferroptosis of pancreatic cancer cells in the liver microenvironment and ultimately promoting pancreatic cancer liver metastasis. In response to this, the inventors conducted further in-depth research and confirmed the use of anti-FGL1 monoclonal antibodies in the preparation of drugs for treating pancreatic cancer liver metastasis. They also designed the effective concentration of FGL1 monoclonal antibodies and determined the population suitable for this antibody therapy. This is the first time that the inventors have demonstrated the mechanism of action of anti-FGL1 monoclonal antibodies on pancreatic cancer liver metastasis, providing an effective approach for the treatment of pancreatic cancer.

[0004] In order to achieve the above-mentioned purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0005] In a first aspect, the present invention provides use of an anti-FGL1 monoclonal antibody in the preparation of a drug for treating cancer liver metastasis.

[0006] In some embodiments, the cancer is pancreatic cancer.

[0007] In some embodiments, the medicament inhibits pancreatic cancer liver metastasis.

[0008] In some embodiments, the drug blocks the binding of LIFR to FGL1.

[0009] In some embodiments, the drug inhibits the growth of pancreatic cancer organoids that overexpress LIFR.

[0010] In some embodiments, the final concentration of the anti-FGL1 monoclonal antibody in the medicament is 30 ng / ml.

[0011] In some embodiments, the drug further contains pharmaceutically acceptable excipients.

[0012] In some embodiments, the drug is in the form of an injection, and specifically, the injection dosage is 0.2 mg / kg.

[0013] In a second aspect, the present invention provides a preparation for blocking the binding of LIFR to FGL1, wherein the preparation comprises an anti-FGL1 monoclonal antibody as an active ingredient.

[0014] In a third aspect, the present invention further provides a kit for detecting the content of FGL1, wherein the kit comprises an anti-FGL1 monoclonal antibody.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides the use of an anti-FGL1 monoclonal antibody in the preparation of a drug for treating pancreatic cancer liver metastasis. The antibody can specifically bind to the FGL1 protein with high affinity, regulate the tumor microenvironment, and significantly inhibit the formation and growth of pancreatic cancer liver metastases. In mice fed a normal diet and a high-fat diet, the antibody can effectively inhibit pancreatic cancer liver metastasis, and the effect is more significant in a fatty liver model. This provides a basis for clarifying the applicable population for this antibody drug, and is expected to provide more effective treatment options for pancreatic cancer patients, improve survival rates and quality of life, and provide more precise guidance for its future clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings required for use in the implementation. Obviously, the drawings described below are only some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is the affinity test result of anti-FGL1 monoclonal antibody and FGL1 purified protein.

[0019] Figure 2 This is a flow cytometric detection result showing that anti-FGL1 monoclonal antibody blocks the binding of LIFR to FGL1.

[0020] Figure 3Figure 1 is the result of the effect of anti-FGL1 monoclonal antibody on FGL1-mediated LIFR activation. In the figure: AB is the test of the effect of LIFR protein on ack-LIFR (acetylated LIFR) and E-Cad (epithelial cell adhesion molecule) after treatment with FGL1 and anti-FGL1 monoclonal antibody respectively; C is the test of whether LIFR protein interacts with GPX4 after treatment with FGL1 and anti-FGL1 monoclonal antibody respectively; D is the test of palm-GPX4 (palmitoylated GPX4) expression after treatment with FGL1 and anti-FGL1 monoclonal antibody respectively; E is the result of immunofluorescence experiment, blue is cell nucleus staining, red is GPX4, Merge is the merged picture; F is the relative level of biomarkers in the KPC tumor model after treatment with control group, FGL1 group and FGL1+LIFRi group, Lipid in the horizontal axis ROS (lipid reactive oxygen species) is related to ferroptosis, MDA (malondialdehyde) is a marker of oxidative stress, and 4-HNE (4-hydroxynonenal) is a marker of oxidative stress.

[0021] Figure 4 This is a diagram showing the expression of LIFR detected by Western blot.

[0022] Figure 5 This is a graph showing the detection results of anti-FGL1 monoclonal antibodies inhibiting the growth of pancreatic cancer organoids with high LIFR expression.

[0023] Figure 6 Schematic diagram of a mouse pancreatic orthotopic tumor model in which anti-FGL1 monoclonal antibodies inhibit pancreatic cancer liver metastasis.

[0024] Figure 7 This is a statistical analysis chart of the inhibition of pancreatic cancer liver metastasis by anti-FGL1 monoclonal antibody, wherein: A is the glucose uptake signal intensity of mice; B is the incidence rate of pancreatic cancer liver metastasis in mice.

[0025] Figure 8 This is a test result diagram of FGL1 on pancreatic cancer liver metastasis. In the figure: A is a flow chart for testing the effect of FGL1 on pancreatic cancer liver metastasis; B is a representative bioluminescent image of the liver; C is the BLI signal intensity emitted by the liver metastasis area in the bioluminescent imaging test.

[0026] Figure 9 This is the test result diagram of FGL1 promoting LIFR acetylation modification.

[0027] Figure 10This is a test result diagram of FGL1 on ferroptosis of tumor cells. In the figure: A is a volcano diagram using RNA-seq analysis to analyze the correlation between GPX4 upregulation and liver metastasis; B is a western blot test diagram; C is an immunofluorescence experiment result diagram, blue is cell nucleus staining, red is GPX4, and Merge is the merged image; D is a microscope image of the KPC tumor model after treatment with the control group, FGL1 group and FGL1+LIFRi group; E is a relative level diagram of biomarkers in the KPC tumor model after treatment with the control group, FGL1 group and FGL1+LIFRi group, in which Lipid ROS (lipid reactive oxygen species) is related to ferroptosis, MDA (malondialdehyde) is a marker of oxidative stress, and 4-HNE (4-hydroxynonenal) is a marker of oxidative stress. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0030] In the present invention, the terms "first," "second," and "third," etc., in "the first aspect," "the second aspect," "the third aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," and "third," etc., serve only as non-exhaustive enumeration and description and should be understood not to constitute closed-ended limitations on quantity.

[0031] In the present invention, the test methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] In the present invention, the “anti-FGL1 monoclonal antibody” used is a commercially available FGL1 monoclonal antibody (Bio XCell, #BE0332).

[0033] Example 1

[0034] Affinity validation of anti-FGL1 monoclonal antibodies

[0035] The affinity of the antibody to human (MCE, HY-P70737) or mouse (MCE, HY-P75188) FGL1 protein was determined by ELISA. Plates coated with human FGL1-his or mouse FGL1-his were incubated at 4°C overnight. After blocking with 5% PBS-milk, the antibody to be tested (at concentrations of 0.1, 1, 10, and 10 μg / ml, respectively) was added to the 96-well ELISA plate. 2 , 10 3 , 10 4 ng / ml) and incubated at room temperature for 1 hour. After washing three times with PBS containing 0.05% Tween, the detection antibody was used as the primary antibody and human-IgG-FC-HRP was used as the secondary antibody. TMB was added for color development, and the absorbance of each well was read at a wavelength of 450 nm.

[0036] The results are as follows Figure 1 As shown, the EC50 of the binding activity of the anti-FGL1 antibody to human FGL1 is between 26.78-40.68 ng / ml; the EC50 of the binding activity of the anti-FGL1 antibody to mouse FGL1 protein is between 18.54-27.21 ng / ml. It can be seen that the anti-FGL1 antibody has good binding affinity.

[0037] Example 2

[0038] Analysis of the blocking effect of anti-FGL1 monoclonal antibodies on FGL1 / LIFR binding

[0039] 1. A lentiviral vector carrying human LIFR (Ref. Registration No.: NM_001127671) was transduced into the CHO-K1 cell line to generate the huLIFR-CHO-K1 stable cell line. Anti-FGL1 monoclonal antibody and anti-mouse IgG (Abcam, ab131368) were incubated with human FGL1-mFc (Sinobio, Cat: 13484-H38H, final concentration 0.6 μg / ml) at 4°C for 1 h. The cells were added to a flat-bottomed plate, centrifuged to remove the supernatant, and 100 μL of antibody and huFGL1-mFc dilution was added to the cells. The cells were incubated at 4°C for 1 hour, washed three times with FACS buffer, and incubated with anti-FGL1 detection antibody (Abcam, ab275091) (primary antibody) and PE anti-mouse IgG Fc (Abcam, ab98742) antibody (secondary antibody) in sequence. The primary antibody reaction conditions were 4°C for 1 hour, and the secondary antibody reaction conditions were 4°C for 30 minutes. Finally, the cells were washed five times with FACS buffer and analyzed using a flow cytometer.

[0040] 2. Treatment of pancreatic cancer cells with anti-FGL1 monoclonal antibodies:

[0041] Pancreatic cancer cells were treated with FGL1 recombinant protein (20 ng / ml) for 24 hours before being treated with FGL1 monoclonal antibodies (final concentration 30 ng / ml). The cells were cultured in medium containing FGL1 monoclonal antibodies for 24 hours. Subsequently, cells from the control and treatment groups were collected and analyzed for protein expression by immunoblotting, protein immunoprecipitation to analyze LIFR binding to GPX4, acyl-biotin displacement assay to detect palmitoylated GPX4, immunofluorescence staining to determine the intracellular distribution of GPX4, and cellular oxidation markers to assess cellular antioxidant status (i.e., ferroptosis sensitivity).

[0042] In this embodiment, related experiments include but are not limited to references to the patentee's published literature:

[0043] J Clin Invest.2023Jul 17;133(14):e164428.doi:10.1172 / JCI164428; NatCommun.2021Dec 1;12(1):7006.doi:10.1038 / s41467-021-27348-8; Cancer Res. 2022Nov15;82(22):4191-4205.doi:10.1158 / 0008-5472.CAN-22-1203.

[0044] In this example, the antibodies and kits involved were obtained from the following sources:

[0045] anti-LIFR (Abcam, ab300551), anti-Acetylated-Lysine (CST, #9681), anti-E-Cadherin (CST, #3195), anti-GPX4 (Abcam, ab125066), anti-GAPDH (CST, #2118), protein palmitoyl immunoblotting kit (Aimsmass, AM10313), lipid peroxidation detection kit (BODIPY 581 / 591C11) (Biyuntian, S0043), lipid peroxidation (MDA) detection kit (MCE, HY-K0319), 4-HNE ELISA kit (Abcam, ab287803).

[0046] Depend on Figure 2 、 3It can be seen that anti-FGL1 monoclonal antibodies significantly inhibited the acetylation modification of LIFR mediated by FGL1, and at the same time inhibited the binding of LIFR to GPX4, reduced the membrane distribution of GPX4, and increased the sensitivity of tumor cells to ferroptosis, which was manifested as a significant increase in the level of lipid peroxidation of cells under the conditions of ferroptosis inducer treatment, proving that anti-FGL1 monoclonal antibodies can block the binding of LIFR to FGL1 and inhibit LIFR activation.

[0047] Example 3

[0048] Analysis of the effect of anti-FGL1 monoclonal antibodies on organoids from pancreatic cancer patients with high LIFR expression

[0049] First, the expression level of LIFR in liver metastasis biopsy tissues of pancreatic cancer patients was detected by Western blotting (eg Figure 4 As shown in the figure), liver metastasis tissues with high LIFR expression were selected to construct pancreatic cancer liver metastasis organoids for anti-FGL1 treatment sensitivity testing.

[0050] The specific steps include:

[0051] A portion of fresh tumor tissue was placed in liquid nitrogen for quick freezing, then the tissue was broken up by a tissue homogenizer to release the intracellular proteins, then an appropriate amount of RIPA lysis buffer (Proteintech, PR20035) was added to prepare a protein solution, and the protein concentration was detected by BCA kit (ThermoFisher, A55864). Equal amounts of protein from different patients were used for Western blotting, and anti-LIFR antibody (Abeam, ab300551) was used to detect the expression level of LIFR in tumor tissue. On this basis, samples with high expression of LIFR were selected, and the corresponding fresh tumor tissue was digested with 2 mg / ml collagenase (#C9407, Sigma Aldrich) at 37°C for 20 minutes, and the isolated cells were resuspended in 50% basement membrane extract (BME, #3432-001-01, R&D Systems). After the BME-cell suspension solidified in a 48-well plate, 200 μl of PDAC-specific organoid medium (Accurate international biotech) was immediately added to establish the PDAC organoid model, and the medium was changed every 2-3 days. Subsequently, the organoids were passaged approximately every 7-14 days. The organoids were cultured to 3-5 passages, and the activity of the organoid cells was measured by CellTiter-Glo 3D Cell Viability Assay Kit (#G9683, Promega). The organoids were treated with FGL1 recombinant protein (20 ng / ml) for 24 hours, then blocked with FGL1 monoclonal antibody, i.e. cultured continuously with organoid medium containing FGL1 monoclonal antibody (final concentration 30 ng / ml) with medium changed every other day, and the growth of pancreatic cancer organoids was detected.

[0052] As shown in FIG. 1, the anti-FGL1 monoclonal antibody of the present application can significantly inhibit the growth of pancreatic cancer organoids with high expression of LIFR. Figure 5

[0053] Example 4

[0054] Analysis of the effect of anti-FGL1 monoclonal antibody on pancreatic cancer liver metastasis

[0055] Pancreatic orthotopic tumors were constructed using fatty liver mice and control mice, and each mouse was inoculated with 1 x 10 6 After 2 days of in vivo inoculation of pancreatic cancer cells, anti-FGL1 monoclonal antibody treatment was started (intraperitoneal injection, 0.2 mg / kg, twice a week for 5 weeks), and after the end of the treatment, small animal PET / CT was used to detect the distribution of tumors, and the incidence of liver metastasis in the treatment group and the control group of mice was counted.

[0056] As shown in FIG. 2, the anti-FGL1 monoclonal antibody of the present application can significantly inhibit the growth of pancreatic cancer organoids with high expression of LIFR. Figure 6 , 7 ​As shown, the incidence of liver metastasis in the anti-FGL1 monoclonal antibody-treated KPC mice and PANCO2 mice was much lower than that in the control group. In particular, the incidence of liver metastasis in the KPC mice treated with the anti-FGL1 monoclonal antibody was 0%, indicating that the anti-FGL1 monoclonal antibody of the present invention can significantly inhibit pancreatic cancer liver metastasis.

[0057] In summary, the inventors found through preliminary research that Figure 8-10 As shown, the secretory factor FGL1, which is specifically upregulated by fatty liver, binds to the pancreatic cancer cell membrane receptor LIFR and promotes LIFR acetylation modification, and then promotes its plasma-membrane translocation by upregulating the palmitoylation level of GPX4, thereby inhibiting the ferroptosis of pancreatic cancer cells in the liver microenvironment and ultimately promoting pancreatic cancer liver metastasis. In response to this, the present invention uses tumor cell models, patient-derived tumor organoid models, and mouse pancreatic in situ tumor models to verify the mechanism of action of anti-FGL1 monoclonal antibodies in the treatment of pancreatic cancer liver metastasis, and compares its anti-cancer effects in mice fed a normal diet and mice fed a high-fat diet (fatty liver model), thereby determining the applicable population for this antibody therapy, providing a scientific basis for the treatment of pancreatic cancer with anti-FGL1 monoclonal antibodies, and also providing new directions and strategies for the clinical treatment of pancreatic cancer liver metastasis.

[0058] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Application of anti-FGL1 monoclonal antibodies in the preparation of drugs for the treatment of cancer liver metastasis.

2. The use according to claim 1, characterized in that The cancer is pancreatic cancer.

3. The use according to claim 1, characterized in that The drug inhibits pancreatic cancer liver metastasis.

4. The use according to claim 1, characterized in that The drug blocks the binding of LIFR to FGL1.

5. The use according to claim 1, characterized in that The drug inhibits the growth of pancreatic cancer organoids that overexpress LIFR.

6. The use according to claim 5, characterized in that The final concentration of the anti-FGL1 monoclonal antibody in the drug is 30 ng / ml.

7. The use according to claim 1, characterized in that The medicine also contains pharmaceutically acceptable excipients.

8. The use according to claim 1, characterized in that The dosage form of the medicine is an injection form.

9. A preparation for blocking the binding of LIFR and FGL1, characterized in that The preparation uses anti-FGL1 monoclonal antibody as an active ingredient.

10. A kit for detecting the content of FGL1, characterized in that: The kit includes an anti-FGL1 monoclonal antibody.

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