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WO2019007075A1 - Utilisation d'un anticorps anti-s100a4 dans la lésion immunitaire antitumorale médiée par un anticorps anti-cd137 - Google Patents

Utilisation d'un anticorps anti-s100a4 dans la lésion immunitaire antitumorale médiée par un anticorps anti-cd137 Download PDF

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WO2019007075A1
WO2019007075A1 PCT/CN2018/076455 CN2018076455W WO2019007075A1 WO 2019007075 A1 WO2019007075 A1 WO 2019007075A1 CN 2018076455 W CN2018076455 W CN 2018076455W WO 2019007075 A1 WO2019007075 A1 WO 2019007075A1
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antibody
liver
cells
monoclonal antibody
tumor
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秦志海
张金华
宋坤
王俊
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Beijing Doingtimes Institute Of Translational Medicine
Institute of Biophysics of CAS
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Beijing Doingtimes Institute Of Translational Medicine
Institute of Biophysics of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies

Definitions

  • the invention relates to the field of immunotherapy, in particular to the application of an anti-S100A4 antibody in anti-tumor immune damage mediated by anti-CD137 antibody.
  • cancer immunotherapy has received widespread attention and rapid development. They are different from traditional radiotherapy and chemotherapy and surgical treatment. They are aimed at stimulating the patient's immune system and use the immune system to kill tumors.
  • an increase in immunity may cause different degrees of side effects, that is, cause immune damage. These immune injuries are sometimes severe or even fatal. For example, it is reflected in (1) rash and mucous membrane irritation: toxicity to skin, rash and itchy skin; (2) diarrhea and colitis; (3) hepatotoxicity; (4) hypopituitarism, adrenal gland and thyroid hypofunction .
  • the costimulatory molecule CD137 (aka 4-1BB and TNFRSF9) is an inducible costimulatory signaling receptor for the tumor necrosis factor (TNF) receptor superfamily that induces expression on the surface of activated T cells and natural killer cells. It can be expressed on the surface of activated dendritic cells and tumor endothelial cells. Among them, CD137 expressed by endothelial cells can promote the infiltration of activated T cells to tumor sites with the help of intracellular adhesion molecules ICAM-1 and VCAM-1.
  • TNF tumor necrosis factor
  • the co-stimulatory molecule CD137 ligand CD137L (aka 4-1BBL and TNFSF9) can be induced to express on a variety of activated APC surfaces, such as dendritic cells, B cells, and macrophages.
  • the combination of the two not only provides co-stimulatory signals for T cell (or NK cell) activation, induces T cell proliferation and produces interferon- ⁇ and enhances its effector function, but also promotes anti-apoptotic gene Bcl- via NF- ⁇ B signaling pathway. Expression of XL and Bfl-1 inhibits activation-induced cell death.
  • the agonistic anti-CD137 antibody induces regression of tumors established in various animal models and prevents tumor recurrence in the melanoma model.
  • the signal domain of CD137 has been fused to the cytoplasmic domain of second generation CAR-T cells, allowing for optimal survival and reduced depletion.
  • modulation of CD137 expression by antigen stimulation makes it useful for isolating tumor-reactive T cells.
  • the pleiotropic nature of CD137 molecular function makes it a very attractive target for tumor immunotherapy.
  • agonistic CD137 antibodies may exhibit anti-inflammatory properties against autoimmune diseases such as lupus, experimental autoimmune encephalomyelitis and arthritis in a wide range of mouse models by inducing apoptosis in B cells or helper T cells. .
  • S100A4 protein is a protein with tissue and tissue specificity. It is expressed in liver, spleen, bone marrow, smooth muscle cells and keratinized cells of mice. Mononuclear cells and macrophages are found in humans. S100A4 protein was expressed in different degrees in polymorphonuclear granulocytes, keratinocytes, Langerhans cells and sweat gland cells, but not in tissue cells of normal lung, kidney, breast, thyroid, pancreas and colon.
  • S100A4 is a member of the S100 calcium-binding protein family, in which the S100A4 protein is present in cells by non-covalently binding dimers, and secreted by covalently bound dimers to the outside of the cell, which exerts various biological effects in vivo. Participate in processes such as cell cycle activity, cell differentiation, tumor growth, and extracellular matrix secretion activities. In the case of tumorigenesis, multiple S100 members are abnormally expressed in tumors and are closely related to tumor invasion and metastasis. Among them, S100A4 is one of them, also known as p9Ka, calvasculin, CAPL and so on. S100A4 secreted by tumors and stromal cells is thought to play a key role in cancer cell metastasis or in affecting angiogenesis.
  • the object of the present invention is to provide an anti-S100A4 antibody for anti-tumor immune damage induced by anti-CD137 antibody, and the anti-S100A4 antibody treatment can avoid or greatly reduce the anti-CD137 antibody by using anti-CD137 antibody for treating tumor.
  • the present invention provides an anti-S100A4 antibody, a substance which causes a lack of S100A4, and a substance capable of deleting a body S100A4-positive macrophage, which is prepared by administering an anti-CD137 antibody to a therapeutic subject. Application in damaged drugs.
  • the immunological damage includes hepatotoxicity, lupus, autoimmune encephalomyelitis, and arthritis.
  • the liver toxicity includes liver collagen fiber deposition and liver fibrosis.
  • the anti-S100A4 antibody, a substance lacking S100A4, and a substance capable of deleting the body S100A4-positive macrophage do not affect the anti-tumor effect of the anti-CD137 antibody.
  • the invention also provides a composition for cancer immunotherapy, the composition comprising:
  • an anti-S100A4 antibody a substance that causes the body to lack S100A4, and a substance that can delete the body S100A4-positive macrophage.
  • the anti-CD137 antibody is an anti-CD137 monoclonal antibody, optionally an anti-mouse CD137 monoclonal antibody (clone 2A, rat IgG2a) ).
  • the anti-S100A4 antibody is an anti-S100A4 monoclonal antibody, optionally an anti-mouse S100A4 monoclonal antibody.
  • the anti-mouse S100A4 monoclonal antibody is prepared by immunizing BALB/c mice with S100A4 protein, specifically, using immune spleen
  • the cell and mouse myeloma Sp2/0 cell lines serve as fusion partners, and hybridoma cells are prepared and selected according to monoclonal antibody technology.
  • an anti-CD137 antibody and an anti-S100A4 antibody are sequentially administered to a mouse.
  • the anti-CD137 antibody is injected at a dose of 90-110 ⁇ g/time, and optionally, the anti-CD137 antibody is administered at a dose of 100 ⁇ g/time.
  • the anti-S100A4 antibody is injected at a dose of 3-5 mg/kg, alternatively, the anti-S100A4 antibody is administered at a dose of 4 mg/kg.
  • the present invention has the following beneficial effects:
  • the present invention can prevent or greatly alleviate severe hepatotoxicity caused by anti-CD137 antibody by using anti-S100A4 antibody treatment while treating tumor with anti-CD137 antibody;
  • Anti-S100A4 antibody selectively attenuates liver abnormalities, but does not affect anti-tumor immunity induced by anti-CD137 antibody treatment;
  • the present invention proposes a novel molecular mechanism of liver pathology induced by immunostimulatory antibodies, and proposes that combined immunotherapy targeting these pathways can potentially cause optimal anti-tumor immunity with minimal side effects.
  • Figure 1 is an anti-CD137 monoclonal antibody (2A) according to the present invention exhibiting antitumor activity and causing severe liver damage.
  • Figure 1A Tumor volume growth volume detection
  • Figure 1B Tumor and liver sections stained on day 21 after tumor inoculation
  • Figure 1C Mouse serum ALT (mouse serum alanine aminotransferase) level detection
  • Figure 1D Liver Sections were subjected to further Gr-1 and F4/80 staining
  • Figure 1E number of Gr-1 + cells and F4/80 + cells in the liver
  • Figure 1F Liver sections were stained with H&E and Sirius red
  • Figure 1G Experimental group (charged) Tumor) and control (non-tumor-bearing) C57BL/6 mice were treated with anti-CD137 monoclonal antibody (2A) and serum ALT levels in mice.
  • 2A anti-CD137 monoclonal antibody
  • Figure 2A Schematic model of chronic liver injury induced by anti-CD137 monoclonal antibody (2A);
  • Figure 2B histological features of liver fibrosis and S100A4 + cell accumulation;
  • Figure 2C quantification of the red region of Sirius in liver sections;
  • Figure 2D Liver S100A4 + cell infiltration assay;
  • Figure 2E Liver tissue double immunohistochemical staining;
  • Figure 2F-2G Flow cytometry analysis of mouse liver S100A4 + cell phenotype;
  • Figure 2H Cell culture supernatant S100A4 concentration detection.
  • Figure 3 is a graph showing that selective deletion of S100A4 + cells according to the present invention attenuates liver damage and liver fibrosis induced by anti-CD137 monoclonal antibodies.
  • Figure 3A S100A4 immunofluorescence staining of S100A4-TK + mouse liver proliferation
  • Figure 3B S100A4-TK + mouse liver section S100A4 immunohistochemical staining
  • Figure 3C Percentage of S100A4 positive cells
  • Figure 3D S100A4 -TK + mouse serum ALT level
  • Figure 3E S100A4-TK + mouse liver homogenate S100A4, MCP-1 and TNF- ⁇ protein content
  • Figure 3F S100A4-TK + mouse liver slice Sirius red staining
  • Figure 3G Quantification of the red area of Sirius in liver sections.
  • Figure 4 is a graph showing the long-term liver pathological effects of S100A4 deficiency in reducing the anti-CD137 monoclonal antibody according to the present invention.
  • FIG. 4A Schematic diagram of anti-CD137 monoclonal antibody (2A)/HCC model
  • FIG. 4B mouse serum ALT level
  • FIG. 4C representative liver images of two groups, liver tumor nodules are indicated by arrows
  • FIG. 4D- 4F shows liver tumor number, maximum tumor size and liver weight
  • Figure 4G liver tissue section H&E staining, anti-S100A4 antibody or Sirius red staining
  • Figure 4H S100A4-TK transgenic mouse anti-CD137 monoclonal antibody (2A)/HCC
  • Figure 4I Liver image
  • Figure 4J Number of liver tumors
  • Figure 4K Maximum tumor size
  • Figure 4L Representative photograph of liver weight.
  • Figure 5 is a graph showing S100A4 enhancing CD8 + T cell survival in accordance with the present invention.
  • Figure 5A FACS representative data
  • Figure 5B FACS quantitative detection of CD4 + T cells and CD8 + T cells in the liver
  • Figure 5C IHC staining of CD4 + and CD8 + in mouse liver tissue
  • Figure 5D CFSE labeling CD4 + T and CD8 + T cells in mouse spleens were analyzed for T cell proliferation by CFSE dilution
  • Figure 5E - Figure 5F mouse spleen CD4 + T and CD8 + T cells 7-AAD and Annexin V staining, FACS detection of cells Apoptosis.
  • Figure 6 is a diagram showing inhibition of CD8 + T cell apoptosis by the Akt signaling pathway by S100A4 according to the present invention.
  • Figure 6A FACS analysis of CD8 + T cell apoptosis
  • Figure 6B Western blot detection of caspase-3 and caspase-9 expression
  • Figure 6C Western blot analysis showed signal pathway
  • 6D FACS analysis of the percentage of apoptotic cells.
  • Figure 7 is a graph showing that both the CD137 and S100A4 pathways of targeted mice in accordance with the present invention reduce hepatotoxicity but retain anti-tumor immunity.
  • Figure 7A shows the tumor growth kinetics of these three groups (anti-CD137 monoclonal antibody (2A) group, RatIg group, anti-CD137 monoclonal antibody (2A) + anti-S100A4 group);
  • Figure 7B shows these three groups of serum ALT Level;
  • Figure 7C Liver tissue H&E and Sirius red staining;
  • Figure 7D FACS for statistical analysis of CD4 + T and CD8 + T cell ratios and CD8 + T in liver tissue;
  • Figure 7E FACS detection of CD4 + T in tumor tissue Statistical analysis of the ratio of cells to CD8 + T cells;
  • Figure 7F Representative IHC staining of CD8 + in tumor sections from different groups.
  • Figure 8 is a diagram showing the mechanism of action of S100A4 in anti-CD137-induced hepatotoxicity according to the present invention.
  • CD8 + T cells mainly memory T cells
  • IFN-g IFN-g
  • Blocking antibodies with S100A4 targeting S100A4 affects CD8 + T cell survival and causes minimal hepatotoxicity.
  • a similar effect can also be obtained by deletion of S100A4 or deletion of S100A4-positive macrophages.
  • Example 1 Anti-tumor effect of anti-CD137 monoclonal antibody simultaneously causes severe liver damage
  • FIG. 1A Tumor growth volume of tumor-bearing mice treated with anti-CD137 antibody (clone 2A, rat IgG2a), RatIg (Sigma-Aldrich, St. Louis, MO) and PBS It was measured once every 2-3 days with an electronic caliper and multiplied by the vertical diameter. It can be seen from Figure 1A that the increase in tumor volume after treatment with anti-CD137 monoclonal antibody (2A) was only on day 30. One third of the increase in tumor volume after treatment with RatIg and PBS. (3) Tumor and liver section staining (Fig.
  • Fig. 1C Monitoring of serum ALT (serum alanine aminotransferase) levels
  • Fig. 1C experimental group (tumor-bearing mice) and control group (non-tumor-bearing mice) were treated with anti-CD137 antibody (2A) and RatIg and PBS.
  • Serum ALT levels were measured using a commercial kit (Biosino, Beijing).
  • serum ALT levels after treatment with anti-CD137 monoclonal antibody (2A) were at least 5 times higher than those treated with RatIg or PBS.
  • Fig. 1C Further staining of liver sections
  • liver serial sections were stained with H&E and Sirius red (Fig. 1F): 7 ⁇ m thick liver tissue sections were prepared and stained with H&E or with saturated picric acid containing 0.1% Sirius red and 0.1% Fast Green after routine treatment.
  • Collagen deposition assay liver sections were evaluated under microscope (DP71, OLYMPUS) to monitor liver collagen fiber deposition and liver fibrosis.
  • Figure 1F shows liver collagen fiber deposition and liver fibrosis after anti-CD137 monoclonal antibody treatment. It is much higher than collagen fiber deposition and fibrosis of liver slices after treatment with RatIg.
  • mice were treated with anti-CD137 monoclonal antibody (2A) to detect serum ALT levels in mice (Fig. 1G), from Figure 1G. It can be seen that the ALT level of the serum of the experimental group (tumor-bearing) C57BL/6 mice treated with anti-CD137 monoclonal antibody (2A) and the control group (non-tumor-bearing) C57BL treated with anti-CD137 monoclonal antibody (2A). ALT levels in /6 mouse sera were comparable, and at least 12 times the ALT level in the serum of control (non-tumor) C57BL/6 mice treated with RatIg.
  • 2A anti-CD137 monoclonal antibody
  • Example 2 Anti-CD137 monoclonal antibody treatment induces infiltration of a large number of S100A4 + macrophages (Fig. 2)
  • Embodiments (1) Anti-CD137 monoclonal antibody and RatIg-treated mice (Fig. 2A): 6-week-old male wild-type C57BL/6 mice were intraperitoneally injected once a week with 100 ⁇ g of anti-CD137 monoclonal antibody or RatIg. 5 weeks. Liver tissue was harvested at the time points specified at weeks 1, 3, and 5 for further observation. (2) Liver section staining and collagen deposition and S100A4 + cell quantitative analysis (Fig. 2B-2D): 7 ⁇ m thick frozen liver tissue sections were prepared. After routine treatment, liver sections were stained with H&E or with 0.1% Sirius Red and 0.1%.
  • FIG. 2C after treatment with anti-CD137 monoclonal antibody (2A) for 5 weeks, the Sirius red region was at least the control RatIg group. Sirius red region of 10 times, can be seen in FIG. 2D, after 5 weeks of treatment with anti-CD137 monoclonal antibody (2A), S100A4 + cell number is at least in mouse liver in liver control group RatIg S100A4 + 10 times the number of cells. (3) Liver tissue double immunofluorescence staining (Fig.
  • liver non-parenchymal cells Cells were isolated from the liver using a two-stage collagenase perfusion technique. The filtered cells were centrifuged at 50 g for 2 minutes to remove hepatocytes. The remaining non-parenchymal cells were collected, washed and isolated using a 40% and 70% non-linear Percoll (GE Healthcare biosciences, Pittsburgh, PA) gradient system.
  • Liver NPC single cell suspension stained with the following directly labeled mouse-specific monoclonal antibodies: Percp/Cy5.5-labeled anti-CD11b (clone M1/70), AP-labeled anti-Ly6C (clone HK1.4), PE Labeled anti-F4/80 (clone BM8), all of which were purchased from Biolegend at a concentration of 0.2 [mu]g/mL.
  • S100A4 + CD11b + and S100A4 - CD11b + cell culture supernatants were assayed for S100A4 concentration by ELISA. From Figure 2H, S100A4 + CD11b + cell culture supernatant was found. The S100A4 + level is at least 4 times the level of S100A4 + in the S100A4 - CD11b + cell culture supernatant.
  • anti-CD137 monoclonal antibody treatment induces infiltration of a large number of S100A4 + macrophages.
  • Example 3 Selective deletion of S100A4 + cells attenuated liver damage and liver fibrosis induced by anti-CD137 monoclonal antibodies (Fig. 3)
  • Embodiments (1) Establishment of a mouse model: S100A4-TK mice (Eric G. Nielsen, Northwestern University, Feinberg College Medicine) that selectively deleted S100A4 + cells were given on the first day of the week. 100 ⁇ g of anti-CD137 monoclonal antibody was administered intraperitoneally, and 50 mg/kg of GCV (ganciclovir, ganciclovir) (Hubei Keyi Pharmaceutical Co., Ltd., China) was given on days 1, 3, 4, 6 and 7 of each week. The PBS was intraperitoneally injected and the treatment was repeated for 4 weeks.
  • GCV ganciclovir, ganciclovir
  • FIG. 3A Mouse liver section staining: liver slices treated with anti-CD137 monoclonal antibody in S100A4-TK mice were labeled with S100A4 (Abeam, Cambridge, UK) and Ki67 (proliferating cell-associated nuclear antigen, cells in the proliferative cycle) ) antibody staining (FIG. 3A), can be seen in Figure 3A, S100A4 + Ki67 antigen expression in cells indicates that these cells are S100A4 + proliferation. Liver sections of S100A4-TK mice treated with anti-CD137 monoclonal antibody and GCV or PBS were stained with S100A4 antibody (Fig. 3B) and quantitative analysis (Fig. 3C). As can be seen from Fig.
  • liver collagen fibers treated with anti-CD137 monoclonal antibody (2A) and PBS were much higher than that of liver collagen fibers deposited by anti-CD137 monoclonal antibody (2A) and GCV (Sirius red staining), which can be seen from Figure 3G. It was seen that the Sirius red region treated with anti-CD137 monoclonal antibody (2A) and PBS was at least 3 times that of the Sirius red region treated with anti-CD137 monoclonal antibody and GCV.
  • ALT levels in serum treated with anti-CD137 monoclonal antibody (2A) and PBS were at least treated with anti-CD137 monoclonal antibody (2A) and GCV.
  • the ALT level in serum is 1.5 times.
  • the S100A4 content in the liver treated with anti-CD137 monoclonal antibody (2A) and PBS is at least the liver treated with anti-CD137 monoclonal antibody (2A) and GCV. 2 times the amount of S100A4, the MCP1 content in the liver treated with anti-CD137 monoclonal antibody (2A) and PBS is at least twice the MCP1 content in the liver treated with anti-CD137 monoclonal antibody (2A) and GCV.
  • the amount of TNF- ⁇ in the liver treated with anti-CD137 monoclonal antibody (2A) and PBS was at least 1.5 times that of the liver treated with anti-CD137 monoclonal antibody (2A) and GCV.
  • selective deletion of S100A4 + cells attenuated liver damage and liver fibrosis induced by anti-CD137 monoclonal antibodies.
  • Example 4 S100A4 deficiency reduces long-term liver pathogenesis of anti-CD137 monoclonal antibodies ( Figure 4)
  • Embodiments (1) Hepatic cell carcinoma model of S100A4 -/- mice and WT male C57BL/6 mice induced by diethylnitrosamine (DEN)/anti-CD137 monoclonal antibody (Fig. 4A): 15 days old S100A4 -/- mice (S100A4 -/- mice purchased from Jackson Laboratory Bar Harbor, ME, USA) or WT male C57BL/6 mice, DEN (Sigma-Aldrich) dissolved at 50 ⁇ g / g body weight at 0.1 mL PBS, single intraperitoneal injection on day 1, after one month of DEN injection, mice received an intraperitoneal injection of 100 ⁇ g of anti-CD137 monoclonal antibody or RatIg once a week for two consecutive months.
  • DEN diethylnitrosamine
  • Fig. 4A 15 days old S100A4 -/- mice (S100A4 -/- mice purchased from Jackson Laboratory Bar Harbor, ME, USA) or WT male C57BL/6 mice, DEN (S
  • mice were sacrificed 8 months after DEN treatment for further analysis.
  • Fig. 4B Monitoring of serum ALT levels in mice (Fig. 4B), it can be seen from Fig. 4B that ALT levels of WT male C57BL/6 mice are at least twice that of S100A4 -/- mice.
  • Liver tumor nodule monitoring in mice Fig. 4C
  • Fig. 4D tumor number
  • Fig. 4E tumor maximum diameter
  • Fig. 4F tumor weight
  • WT male C57BL/ can be seen by Fig. 4C-4F. 6 mice had larger liver tumor nodules, more tumors, larger tumor diameters, and heavier tumors than S100A4 -/- mice.
  • Fig. 4C Monitoring of mouse liver cancer tissue S100A4 + cell infiltration (Fig.
  • Fig. 4I number of liver tumors (Fig. 4J), maximum tumor size (Fig. 4K) and liver weight (Fig. 4L).
  • Fig. 4I-4L compared with the control group WT, the number and size of hepatocellular carcinoma (HCC) tumors after GCV treatment in S100A4-TK mice were about 1/3 of that of the control group, and the liver weight was about 7/10 of the control group.
  • HCC hepatocellular carcinoma
  • Example 5 S100A4 enhances CD8 + T cell survival (Figure 5)
  • Embodiments (1) Analysis of CD4 + and CD8 + T cell infiltration in mouse liver: S100A4 -/- and WT mice were treated with anti-CD137 monoclonal antibody for 4 weeks, and CD4 + T in liver was detected by flow cytometry.
  • Cell and CD8 + T cell content (Fig. 5A and 5B), it can be seen from Figures 5A and 5B that the CD4 + T cell content in the liver of WT mice is at least the CD4 + T cell content in the liver of S100A4 -/- mice. 1.5 times, the CD8 + T cell content in the liver of WT mice is at least 2.5 times that of the CD8 + T cells in the liver of S100A4 -/- mice.
  • Liver NPC single cell suspension was stained using the following directly labeled mouse-specific monoclonal antibodies: Percp-labeled anti-CD4 + (clone GK1.5) and APC-labeled anti-CD8 + (clone 53-6.7). These antibodies were purchased from Biolegend at a concentration of 0.2 [mu]g/mL. Cells were harvested on a FACS Calibur (BD Biosciences, San Diego, CA) and analyzed by FlowJo software (TreeStar, Ashland, OR). IHC staining of CD4 + and CD8 + in mouse liver tissue (Fig. 5C).
  • Example 6 S100A4 inhibits CD8 + T cell apoptosis via Akt signaling pathway
  • Embodiments (1) Effect of S100A4 on apoptosis of CD8 + T cells (Fig. 6A): CTLL-2 cells (by Chinese Academy of Sciences, Wu Peijun) with or without S100A4 (1 ⁇ g/mL) and S100A4+ anti-S100A4 monoclonal antibody ( 6 ⁇ g/mL) was cultured for 3 days in combination, and apoptosis was analyzed by FACS. Specifically: CTLL-2 cells were deprived of IL-2 and then treated with or without S100A4 and S100A4+ anti-S100A4 antibodies. After three days, each group of cells was collected and stained with PI and Annexin V-fluorescein isothiocyanate (FITC).
  • FITC Annexin V-fluorescein isothiocyanate
  • CTLL-2 cells were cultured for 48 hours or 72 hours in deprived IL-2 and in medium with or without S100A4 and anti-S100A4 monoclonal antibodies. The cells were then collected, washed with PBS, and lysed. Anti-caspase-9, anti-caspase-3 (both from Cell Signaling, Danvers, MA) was used. HRP-conjugated goat anti-mouse or goat anti-rabbit IgG was used as the secondary antibody. As can be seen from Figure 6B, the expression of caspase-3 and caspase-9 in CTLL-2 cells was significantly decreased after S100A4 treatment. (3) S100A4 signaling pathway affecting CD8 + T cell apoptosis (Fig.
  • CTLL-2 cells were stimulated with 1 ⁇ g/mL S100A4, and samples were collected at different time points for Western Blotting to detect the main survival-related signaling pathways. .
  • CTLL-2 cells were cultured in serum-free medium for 6 hours with deprivation of IL-2, then added to S100A4 for 5 minutes, 15 minutes, 30 minutes, 45 minutes or 60 minutes, and the cells were collected and washed in PBS. And lyse.
  • Anti-Erk, anti-p-Erk, anti-Akt, anti-p-Akt, anti-STAT3, anti-p-STAT3, and anti-P65 and antip-P65 primary antibodies were used.
  • CTLL-2 cells were deprived of IL-2 and then treated with S100A4 alone or with anti-S100A4 antibody, Erk inhibitor or Akt inhibitor. After three days, each group of cells was collected and stained with PI and Annexin V-fluorescein isothiocyanate (FITC). Cells were tested on a FACS Calibur (BD Biosciences, San Diego, CA) and analyzed by FlowJo software (TreeStar, Ashland, OR). As can be seen from Figure 6D, Erk inhibitors have little effect, but Akt inhibitors can Reversal of S100A4 inhibition of T cell apoptosis, thus demonstrating that S100A4 inhibits apoptosis of CD8+ T cells via the Akt pathway.
  • FITC Annexin V-fluorescein isothiocyanate
  • Example 7 Targeting mouse CD137 and S100A4 pathways all reduced hepatotoxicity, but retained anti-tumor immunity ( Figure 7)
  • liver collagen deposition was greatly reduced by intraperitoneal injection of anti-CD137 monoclonal antibody (2A) plus anti-S100A4 monoclonal antibody;
  • T cell infiltration monitoring in liver and tumor CD4 in liver and tumor + T cell and CD8 + T cell infiltration ratios were analyzed by FACS ( Figures 7D and 7E), as described above, as can be seen by Figure 7D and Figure 7E, by using anti-CD137 monoclonal antibody (2A) plus anti-S100A4 single The clonal antibody was injected intraperitoneally.
  • CD8 + T cells in the liver were reduced by about 50%, but the combined treatment had no effect on the CD8 + T cell content in the tumor.
  • CD8 + cells in tumor sections were stained with immunohistochemistry (Fig. 7F).
  • Fig. 7F CD8 + T cells induced by anti-CD137 antibody were infiltrated into the tumor without the addition of anti-S100A4 antibody.

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Abstract

La présente invention se rapporte à l'utilisation d'un anticorps anti-S100A4, une substance provoquant une déficience en S100A4 dans le corps et une substance capable de supprimer les macrophages positifs pour l'anti-S100A4 dans le corps, dans la préparation d'un médicament destiné au traitement d'une lésion immunitaire induite par l'administration d'un anticorps anti-CD137 à un patient. L'invention concerne également une composition pour l'immunothérapie anticancéreuse, la composition comprenant un anticorps anti-CD137 et un anticorps anti-S100A4, une substance provoquant une déficience en S100A4 dans le corps et une substance capable de supprimer les macrophages positifs pour l'anti-S100A4 dans le corps.
PCT/CN2018/076455 2017-07-06 2018-02-12 Utilisation d'un anticorps anti-s100a4 dans la lésion immunitaire antitumorale médiée par un anticorps anti-cd137 Ceased WO2019007075A1 (fr)

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CN201710547960.3A CN107375923A (zh) 2017-07-06 2017-07-06 抗s100a4抗体在抗cd137抗体介导抗肿瘤免疫损伤中的应用
CN201710547960.3 2017-07-06

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