WO2023001258A1 - Utilisation pharmaceutique d'un anticorps anti-tumoral inhibine 2 - Google Patents

Utilisation pharmaceutique d'un anticorps anti-tumoral inhibine 2 Download PDF

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WO2023001258A1
WO2023001258A1 PCT/CN2022/107205 CN2022107205W WO2023001258A1 WO 2023001258 A1 WO2023001258 A1 WO 2023001258A1 CN 2022107205 W CN2022107205 W CN 2022107205W WO 2023001258 A1 WO2023001258 A1 WO 2023001258A1
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antibody
seq
amino acid
acid sequence
sequence shown
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郑斌
毛煜
梁文璐
张琳
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迈威(上海)生物科技股份有限公司
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular, the present invention relates to the use of an anti-tumor inhibin 2 antibody in preparing medicine and treating related diseases.
  • Interleukin 33 a member of the IL-1 family, is released upon cell injury or allergen stimulation, and after being processed by mast cell proteases or certain enzymatically active allergens, it directly activates local infiltrating basophils Granulocytes, mast cells, type 2 innate lymphocytes (ILC2), T cells and eosinophils induce allergic inflammation.
  • interleukin-33 acts on its receptor and exerts its functions in vivo and in vitro by interacting with its receptor.
  • IL-33 can bind to anti-tumor inhibin 2 (suppression of tumorigenicity 2, ST2) with high affinity, trigger signaling cascade through downstream molecules, activate NF ⁇ b (nuclear factor ⁇ b) and MAP (mitogen-activated protein) Kinase pathway, thereby stimulating target cells and causing target cells to produce a series of functional responses such as cytokines and chemokines.
  • Activation of the IL-33/ST2 signaling pathway promotes the expression of inflammatory cytokines, including IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, MIP-1 ⁇ , IP-10, MCP -1. TNF and GM-CSF.
  • Dysregulation or activation of the IL-33/ST2 signaling pathway has been implicated in a variety of immune-mediated diseases, including allergic asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, atopic dermatitis, allergic For rhinitis, nasal polyps, systemic sclerosis, etc., therapeutic blockade of the IL-33/ST2 pathway may help overcome the immune response.
  • the anti-ST2 antibody can block the binding of IL-33 and ST2 protein, inhibit its downstream signaling pathway, and thus play a therapeutic role in diseases related to the activation of IL-33/ST2 signaling pathway.
  • the inventors of the present invention provided a novel antibody with high affinity and high functional activity to human ST2.
  • the antibody can block the binding of human IL-33 to ST2 on cells such as mast cells, type 2 innate lymphocytes (ILC2) and basophils by specifically binding to human ST2 with high affinity, preventing IL-33 from The activation of the -33/ST2 signaling pathway achieves therapeutic effects on inflammatory and allergic diseases; further, it shows that the antibody has obvious pharmacological effects in different inflammatory and allergic disease models.
  • the object of the present invention is to provide a new pharmaceutical application of anti-ST2 antibody.
  • the present invention provides a use of an anti-tumor inhibin 2 (ST2) antibody or a fragment thereof in the preparation of a medicament for preventing, treating or improving inflammatory, allergic or autoimmune diseases.
  • ST2 anti-tumor inhibin 2
  • said inflammatory, allergic or autoimmune disease is associated with activation of the IL-33/ST2 signaling pathway.
  • the inflammatory, allergic or autoimmune diseases described in the present invention are endometriosis, heart failure, allergic dermatitis, allergic or allergic rhinitis, nasal polyps, atopic dermatitis, chronic Obstructive lung disease, asthma (eg, allergic asthma), pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-associated diarrhea , preferably, allergic dermatitis or inflammatory bowel disease.
  • the anti-ST2 antibody or fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region and comprises heavy chain CDR1 (H-CDR1), CDR2 ( H-CDR2), CDR3 (H-CDR3) and light chain CDR1 (L-CDR1), CDR2 (L-CDR2), CDR3 (L-CDR3), wherein:
  • H-CDR1 comprises the amino acid sequence shown in SEQ ID NO.8
  • H-CDR2 comprises the amino acid sequence shown in SEQ ID NO.9
  • H-CDR3 comprises the amino acid sequence shown in SEQ ID NO.10
  • L-CDR1 Contains the amino acid sequence shown in SEQ ID NO.11
  • L-CDR2 contains the amino acid sequence shown in SEQ ID NO.12
  • L-CDR3 contains the amino acid sequence shown in SEQ ID NO.13.
  • the anti-ST2 antibody or its fragments provided by the present invention can be anti-ST2 monoclonal antibody, scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv and other antibody forms.
  • the anti-ST2 antibody may be an anti-ST2 murine antibody, chimeric antibody, fully or partially humanized antibody.
  • the ST2 is mammalian ST2, preferably primate ST2, more preferably human ST2.
  • both the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-ST2 antibody or fragment thereof include the above-mentioned CDRs and spaced framework regions (framework region, FR), each structural domain
  • the arrangement is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the heavy chain variable region (VH) of the anti-ST2 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO.7 or a variant thereof
  • the light chain variable region (VL) comprises SEQ ID NO.7 The amino acid sequence shown in ID NO.6 or its variant.
  • a "variant of an amino acid sequence” means having at least 75% sequence identity (e.g. at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity, etc. >75% identity of any percentage of amino acid sequences).
  • the heavy chain variable region and the light chain variable region of an anti-ST2 antibody or fragment thereof of the present invention at most 25% of the difference in amino acid sequence resulting from said "at least 75% sequence identity" may exist in the heavy chain In any framework region in the variable region or light chain variable region.
  • the differences may result from amino acid deletions, additions or substitutions at any position, where the substitutions may be conservative or non-conservative.
  • the ST2 antibody or fragment thereof further comprises a constant region.
  • the anti-ST2 antibody or fragment thereof further comprises a human or murine heavy chain constant region (CH) and/or light chain constant region (CL), more preferably comprising a protein selected from IgG, IgA, IgM, IgD or IgE heavy chain constant region and/or kappa or lambda type light chain constant region.
  • CH human or murine heavy chain constant region
  • CL light chain constant region
  • the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain The constant region is of the kappa type.
  • the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG or IgM, such as a human subtype of IgA, IgD, IgE, IgG or IgM, more preferably a human IgG1, IgG2, IgG3 or IgG4 subtype type.
  • the anti-ST2 antibody or fragment thereof comprises a heavy chain constant region, and the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO.14 or a variant thereof.
  • the anti-ST2 antibody or fragment thereof comprises a light chain constant region, and the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO.16 or a variant thereof.
  • a "variant of an amino acid sequence" means having at least 75% sequence identity to said amino acid sequence.
  • the anti-ST2 antibody provided by the present invention comprises a heavy chain and/or a light chain, preferably two heavy chains and/or two light chains.
  • the anti-ST2 antibody of the present invention is an isolated monoclonal antibody of human IgG4/ ⁇ subtype, which comprises two identical light chains and two identical heavy chains, and the two are connected by a disulfide bond , wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO.17, and the light chain comprises the amino acid sequence shown in SEQ ID NO.18.
  • the present invention provides a method for preventing or treating inflammatory, allergic or autoimmune diseases, the method comprising administering the anti-ST2 antibody or fragment thereof of the present invention to a subject in need thereof, For example a therapeutically effective amount of said anti-ST2 antibody or fragment thereof.
  • the subject is a mammal, preferably a primate, more preferably a human or a cynomolgus monkey, more preferably a human.
  • the anti-ST2 antibody or fragment thereof of the present invention as defined above in terms of use can be used in the prophylactic or therapeutic methods provided by the present invention.
  • the administered anti-ST2 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region and comprises heavy chain CDR1 (H-CDR1), CDR2 (H-CDR2), CDR3 (H-CDR3) and light chain CDR1 (L-CDR1), CDR2 (L-CDR2), CDR3 (L-CDR3), wherein:
  • H-CDR1 comprises the amino acid sequence shown in SEQ ID NO.8
  • H-CDR2 comprises the amino acid sequence shown in SEQ ID NO.9
  • H-CDR3 comprises the amino acid sequence shown in SEQ ID NO.10
  • L-CDR1 Contains the amino acid sequence shown in SEQ ID NO.11
  • L-CDR2 contains the amino acid sequence shown in SEQ ID NO.12
  • L-CDR3 contains the amino acid sequence shown in SEQ ID NO.13.
  • the anti-ST2 antibody or fragment thereof may be in the form of anti-ST2 monoclonal antibody, scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv and other antibody forms.
  • the anti-ST2 antibody may be an anti-ST2 murine antibody, chimeric antibody, fully or partially humanized antibody.
  • the ST2 is mammalian ST2, preferably primate ST2, more preferably human ST2.
  • both the heavy chain variable region (VH) and the light chain variable region (VL) of the anti-ST2 antibody or fragment thereof include the above-mentioned CDRs and spaced framework regions (framework region, FR), each structural domain
  • the arrangement is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the heavy chain variable region (VH) of the anti-ST2 antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO.7 or a variant thereof
  • the light chain variable region (VL) comprises SEQ ID NO.7 The amino acid sequence shown in ID NO.6 or its variant.
  • the ST2 antibody or fragment thereof further comprises a constant region.
  • the anti-ST2 antibody or fragment thereof further comprises a human or murine heavy chain constant region (CH) and/or light chain constant region (CL), more preferably comprising a protein selected from IgG, IgA, IgM, IgD or IgE heavy chain constant region and/or kappa or lambda type light chain constant region.
  • CH human or murine heavy chain constant region
  • CL light chain constant region
  • the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain The constant region is of the kappa type.
  • the antibody is an immunoglobulin, specifically IgA, IgD, IgE, IgG or IgM, such as a human subtype of IgA, IgD, IgE, IgG or IgM, more preferably a human IgG1, IgG2, IgG3 or IgG4 subtype type.
  • the anti-ST2 antibody provided by the present invention comprises a heavy chain and/or a light chain, preferably two heavy chains and/or two light chains.
  • the anti-ST2 antibody of the present invention is an isolated monoclonal antibody of human IgG4/ ⁇ subtype, which comprises two identical light chains and two identical heavy chains, and the two are connected by a disulfide bond , wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO.17, and the light chain comprises the amino acid sequence shown in SEQ ID NO.18.
  • said inflammatory, allergic or autoimmune disease is associated with activation of the IL-33/ST2 signaling pathway.
  • the inflammatory, allergic or autoimmune diseases described in the present invention are endometriosis, heart failure, allergic dermatitis, allergic or allergic rhinitis, nasal polyps, atopic dermatitis, chronic Obstructive lung disease, asthma (eg, allergic asthma), pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-associated diarrhea , preferably, allergic dermatitis or inflammatory bowel disease.
  • the present invention provides a new anti-tumor inhibin 2 (ST2) antibody
  • ST2 anti-tumor inhibin 2
  • experiments prove that the antibody can bind to human ST2 with high affinity, block the binding of human IL-33 and human ST2, This effectively prevents the activation of the IL-33/ST2 signaling pathway; in addition, the antibody has no obvious ADCC and CDC effects.
  • Pharmacodynamic experiments further prove that the anti-ST2 antibody of the present invention has positive preventive and therapeutic effects on inflammatory, allergic or autoimmune diseases.
  • Figure 1 shows the inhibition rate of NF- ⁇ B reporter gene activity of B cell culture supernatants from different numbers of mice.
  • Figure 3 shows the results of the antibody inhibiting the activity of recombinant human IL-33 to promote the production of IL-8 in HMC-1 cells.
  • Fig. 4 shows the change ratio (Ratio) of skin swelling area of cynomolgus monkeys detected in Example 13, wherein 4A: physiological saline challenge; 4B: histamine challenge; 4C: challenge with Ascaris suum egg extract. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, G2-G4 vs G1 (One-way ANOVA/Dunnett's).
  • Figure 5 shows the disease-related symptoms and severity of cynomolgus monkeys detected in Example 14, wherein X+2A: body weight change; X+2B: diarrhea score; X+2C: activity score; X+2D: appetite score; X+2E: AUC of severity. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, G1, G3 vs G2 (Two-way ANOVA/Dunnett's).
  • Human ST2-his Human ST2 with 6 histidine tags fused at the C-terminus
  • Human IL33 NP_254274.1 (Ser112-Thr270)
  • Human IL33-his Human IL33 with 6 histidine tags fused to the C-terminus
  • human ST2-fc Human ST2 with human IgG1 Fc tag fused to the C-terminus
  • Control antibody CNTO7160 the heavy chain is shown in SEQ ID NO.19, and the light chain is shown in SEQ ID NO.20.
  • mice were immunized by conventional immunization procedures, and then the serum of the immunized mice was tested by ELISA with the antigen, and the spleens of mice with positive serum reactions were obtained.
  • the antigen was used for panning and culturing of B cells.
  • the culture supernatant of B cells was taken, and ELISA screening was performed again against the antigen Human ST2-his; in addition, KU812 NF- ⁇ B reporter gene screening was performed.
  • the inhibition rate of each clone was calculated according to the values of the negative control and the positive control, and the results of some B cell clones are shown in Figure 1.
  • the mRNA of B cells is extracted, reverse-transcribed into cDNA, and the antibody sequence is amplified by PCR.
  • the light and heavy chains from the same B cell clone were transfected into CHO-K1 cells, and the mouse antibody was obtained from the supernatant after the expression was completed. Take the obtained mouse antibody and carry out the screening of binding human ST2 activity, the screening of inhibiting IL33 binding human ST2 activity, the screening of inhibiting IL33 activation of KU812 NF- ⁇ B reporter gene activity, affinity determination and in vitro pharmacological research, ST2 binding experiments of different species, etc., and Compared with the control antibody CNTO7160.
  • mouse anti-"5886-156H+L was selected for humanization.
  • the mouse anti-"5886-156H+L comes from the 5886-156 cell clone, its heavy chain is 5886-156H, and its light chain is 5886-156L.
  • the heavy and light chain variable region sequences of the 5886-156H+L murine antibody were compared to the human germline sequence using a blast search of the IMGT database. Redundant genes as well as those with unpaired cysteines were removed from the set of human germline genes. The remaining closest matching human germline genes were selected as recipient human frameworks in both framework and CDR regions. FR-4 was selected based on sequence similarity to the IGHJ/IGJK germline genes.
  • Table 1 is the mouse antibody of 5886-156H+L and its humanized sequence form, in which the HZ0 version represents only CDR transplantation, and the HZ1 version introduces a back mutation. The specific sequence after humanization is shown in Table 1, wherein the corresponding CDRs are shown underlined (according to the definition of enhanced Chothia/AbM CDR).
  • Antibody IC50 ( ⁇ g/mL) relative activity maximum inhibition rate 5886-156-H1L0 1.217 103.12% 89.24% 5886-156-H1L1 1.004 125.00% 88.05% CNTO7160 the the 92.55%
  • the KU812 cells in the logarithmic growth phase were centrifuged, 100,000 cells/well, 50 ⁇ L/well were added to the above-mentioned 96-well plate, and incubated for 48 hours.
  • the concentration of IL5 in the cell culture supernatant was determined according to the instructions of the Human IL-5 DuoSet ELISA kit.
  • Table 4 The results of humanized antibodies inhibiting the activity of IL33 to promote KU812-IL5 production and the relative activity compared with CNTO7160
  • Antibody IC50 ( ⁇ g/mL) relative activity maximum inhibition rate 5886-156-H1L0 0.03539 181.12% 98.07% 5886-156-H1L1 0.02893 221.57% 94.95% CNTO7160 the the 97.55%
  • the anti-human ST2 antibody was diluted to 2 ⁇ g/mL and captured on the surface of the Protein A chip for 60 s. After antibody capture, human ST2-his in the form of a solution (initial concentration 20 nM, 2-fold serial dilution of 6 concentrations) was injected. Association was monitored for 180 s, dissociation was monitored for 700 s, and regeneration of the sensor surface was achieved by injection of a glycine solution at pH 2.0. Kinetic data were analyzed using a 1:1 binding model.
  • Dissociation kinetic affinity experiment between Human ST2 and antibody at pH 7.4 All experiments were performed in HBS-EP (1 ⁇ ) buffer (pH 7.4) at 25°C.
  • the anti-human Fc antibody was coupled to the CM5 chip, and the 5886-156-H2L1 antibody diluted to 5.0 ⁇ g/ml was captured in the channel of the CM5 chip with the anti-human Fc antibody for 30 seconds.
  • different concentrations of Human ST2 antigen were injected (initial concentration 16nM, 2-fold ratio dilution to 7 concentrations).
  • the monitoring binding time was 180s and the dissociation time was 700s.
  • the chip was regenerated using glycine at pH 1.5. Kinetic data were analyzed using a 1:1 binding model. The results are shown in Table 6.
  • Example 5 5886-156-H2L1 inhibits IL33 and promotes KU812-IL5 generation activity screening
  • Table 7 The results of humanized antibodies inhibiting IL33 to promote KU812-IL5 production and relative activity compared with CNTO7160
  • Antibody IC50 ( ⁇ g/mL) relative activity maximum inhibition rate 5886-156-H2L1 0.0343 403.79% 94.16% CNTO7160 0.1385 the 95.21%
  • the range of relative binding activity (EC50) between 5886-156-H2L1 and human ST2 recombinant protein was 16.34-17.32 ng/mL.
  • the 5886-156-H2L1 stock solution was replaced into PBS buffer, and Sulfo-NHS-LC-LC-Biotin reagent was added, and the molar ratio of Biotin to 5886-156-H2L1 molecules was 10:1. After mixing, react at room temperature for 30 minutes. The labeled solution was then ultrafiltered into PBS buffer to remove excess labeling reagent. Aliquot and store in ultra-low temperature freezer.
  • the range of 5886-156-H2L1 blocking IL-33 and ST2 binding ability (IC50) was determined to be 869.6-994.9 ng/mL.
  • Example 9 5886-156-H2L1 inhibits the activity of recombinant human IL-33 to promote the production of IL-6 in HUVEC cells (ELISA method)
  • HUVECs expressing ST2 in the membrane were used as target cells, and the blocking activity of 5886-156-H2L1 antibody was evaluated indirectly by detecting the production of IL-6. Specifically, 10,000 HUVEC cells/well were incubated in 100 ⁇ L of the system at 37° C. and 5% CO 2 for 18-24 hours. Dilute human IL33-his to 10ng/mL with medium, 5886-156-H2L1 to 400 ⁇ g/mL with medium, 4-fold dilution, a total of 11 concentrations, mix the two 1:1 and add 100 ⁇ L/well to Incubate in the above 96-well plate at 37° C. and 5% CO 2 for 18-24 hours.
  • the concentration of IL6 in the cell culture supernatant was determined according to the instructions of the Human IL-6 DuoSet ELISA kit.
  • the experimental results show that 5886-156-H2L1 has a significant dose-effect curve and has a good biological activity of blocking the combination of IL-33 and ST2. The results are shown in Figure 2.
  • Example 10 5886-156-H2L1 inhibits the activity of recombinant human IL-33 to promote the production of IL-8 in HMC-1 cells (ELISA method)
  • HMC-1 membrane expressing ST2 was used as target cells, and the blocking activity of 5886-156-H2L1 antibody was evaluated indirectly by detecting the production of IL-8.
  • human IL33-his was diluted to a final concentration of 1000 ng/mL, and 5886-156-H2L1 was diluted 4 times according to the initial concentration of 640 ⁇ g/mL, with a total of 11 concentrations. The two were mixed 1:1 and added to 96 in the orifice plate.
  • 50,000 HMC-1 cells in the logarithmic growth phase were added to the above-mentioned 96-well plate at 50 ⁇ L/well, and incubated for 18-24 hours at 37° C. and 5% CO 2 .
  • the concentration of IL8 in the cell culture supernatant was determined according to the instructions of the Human IL-8 DuoSet ELISA kit.
  • the experimental results show that 5886-156-H2L1 has a significant dose-effect curve and has a good biological activity of blocking the combination of IL-33 and ST2. The results are shown in Figure 3.
  • ADCC analysis medium Digest SK-BR-3 cells and resuspend them in ADCC analysis medium. After sampling and counting, adjust the cell density to 5 ⁇ 104 cells/mL, and add 100 ⁇ l of cells to each well of a 96-well cell culture plate. For dilution solution, add 100 ⁇ l ADCC analysis medium to the wells of ESR (Effector Spontaneous Release, effector cells spontaneously released) wells, CMB (Culture Medium Background, blank medium) wells and VCC (Volume Correction Control, volume correction) wells, and add The sample 96-well cell plate was placed at 37°C in a CO 2 incubator and incubated for 18 ⁇ 2h.
  • ESR Effective Rector Spontaneous Release, effector cells spontaneously released
  • CMB Culture Medium Background, blank medium
  • VCC Volume Correction Control, volume correction
  • KU812 cells in the logarithmic growth phase were collected, sampled and counted, centrifuged at 1000 rpm for 5 min, resuspended in the analysis medium and diluted to a cell density of 2 ⁇ 10 5 cells/ml. Add 25 ⁇ l of KU812 cell dilution into wells parallel to the addition of SK-BR-3 cells.
  • the 96-well cell plate with the sample added was placed in a microplate constant temperature shaker, and mixed at 100 rpm for 30 min. Centrifuge the PBMC cells at 400 ⁇ g for 10 minutes, discard the supernatant and resuspend in the analysis medium for counting. Take an appropriate amount of the cell suspension to adjust the cell density to 2.5 ⁇ 10 6 cells/ml and 5.0 ⁇ 10 6 cells/ml.
  • %Target cell Lysis [(OD ER -OD (T+E)SR )/(OD TMR -OD TSR )] ⁇ 100
  • SK-BR-3 cells were digested and resuspended in ADCC analysis medium. After sampling and counting, the cell density was adjusted to 2 ⁇ 10 5 cells/mL, and 100 ⁇ l of cells were added to each well of a 96-well cell culture plate for dilution. solution, put the 96-well cell plate with the sample at 37°C in a CO 2 incubator and incubate for 22 ⁇ 2h. KU812 cells in the logarithmic growth phase were collected, sampled and counted, centrifuged at 1000 rpm for 5 min, resuspended in the analysis medium and diluted to a cell density of 1.33 ⁇ 10 6 cells/ml.
  • effector cells During the preparation of effector cells, the effector cells Jurkat-ADCC12 cells were centrifuged at 400 ⁇ g for 5 minutes, then resuspended in the analysis medium and counted, and then the cell density was adjusted to 4 ⁇ 10 6 cells/ml and 8 ⁇ 10 6 cells/ml.
  • the positive control Add 30 ⁇ l effector cell dilution solution with a density of 4 ⁇ 10 6 cells/ml to the sample well, and add 15 ⁇ l effector cell dilution solution with a density of 8 ⁇ 10 6 cells/ml to the 5886-156-H2L1 monoclonal antibody sample well.
  • Negative controls were set during this period (30 ⁇ l SK-BR-3 cells + 30 ⁇ l analysis medium + 30 ⁇ l effector cells with a density of 4 ⁇ 10 6 cells/ml and 15 ⁇ l KU812 cells + 30 ⁇ l analysis medium + 15 ⁇ l with a density of 8 ⁇ 10 6 cells/ml ml effector cells), blank control (60 ⁇ l assay medium). Place the loaded 96-well cell plate in a CO 2 incubator at 37°C for 16-24h.
  • E signal of experimental well
  • S spontaneous killing
  • M maximum killing
  • the allergic dermatitis model of cynomolgus monkeys induced by Ascaris suum egg extract Ascaris. preventive or therapeutic effect.
  • Skin swelling reaction was stimulated after administration: the animals in each group were intradermally injected with 1 point of normal saline, 1 point of histamine and 3 points of A.suum in the abdomen.
  • the cynomolgus monkey mucositis model induced by irinotecan (CPT-11, irinotecan hydrochloride, Hubei Shengtian Hengchuang Biotechnology Co., Ltd.; Cas number: 136572-09-3) was used to study 5886-156- Preventive or therapeutic effects of H2L1 on irinotecan-induced intestinal mucositis.
  • G1 group was intravenously injected with CPT-11 vehicle ( ⁇ 3% DMSO)
  • G2 and G3 groups were intravenously injected with CPT-11 (30mg/kg), once a day; 8.
  • Group G2 was given intravenous injection of normal saline
  • group G3 was given intravenous injection of 5886-156-H2L1 (diluted with normal saline) (10 mg/kg); Day 12 animals were dissected to obtain intestinal tissues for pathological examination.
  • irinotecan induced mucositis manifested as diarrhea, decreased appetite, decreased body weight, decreased intestinal inflammation and mucus secretion.
  • 5886-156-H2L1 showed efficacy in alleviating disease symptoms (diarrhea, weight loss, loss of appetite and lack of activity) compared to the saline group (G2).
  • the AUCs for disease-related symptom change and severity are shown in Figure 5.

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  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne l'utilisation d'un nouvel anticorps anti-tumoral inhibine 2 (ST2) dans la préparation d'un médicament pour la prévention ou le traitement de maladies inflammatoires, allergiques ou auto-immunes. L'anticorps anti-ST2 peut se lier à la ST2 humaine avec une affinité élevée, et peut bloquer la liaison de la ST2 humaine et de l'IL-33 humaine, ce qui permet d'inhiber de manière efficace les voies de signalisation d'IL-33/ST2. L'anticorps anti-ST2 a pour effet de prévenir et de traiter des maladies inflammatoires, allergiques ou auto-immunes.
PCT/CN2022/107205 2021-07-21 2022-07-21 Utilisation pharmaceutique d'un anticorps anti-tumoral inhibine 2 WO2023001258A1 (fr)

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WO2013173761A2 (fr) * 2012-05-18 2013-11-21 Amgen Inc. Protéines de liaison à l'antigène st2
CN103845731A (zh) * 2012-12-05 2014-06-11 复旦大学 抗st2/il-1r4抗体在制备防治瘙痒药物中的应用
CN106199002A (zh) * 2016-07-19 2016-12-07 河南华程氏生物科技股份有限公司 一种用于检测st2的化学发光免疫分析试剂盒、制备方法及应用
CN110357963A (zh) * 2019-07-31 2019-10-22 北京智仁美博生物科技有限公司 抗人st2抗体及其用途
CN113214395A (zh) * 2020-01-21 2021-08-06 迈威(上海)生物科技股份有限公司 抗st2抗体及其应用

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WO2013173761A2 (fr) * 2012-05-18 2013-11-21 Amgen Inc. Protéines de liaison à l'antigène st2
CN103845731A (zh) * 2012-12-05 2014-06-11 复旦大学 抗st2/il-1r4抗体在制备防治瘙痒药物中的应用
CN106199002A (zh) * 2016-07-19 2016-12-07 河南华程氏生物科技股份有限公司 一种用于检测st2的化学发光免疫分析试剂盒、制备方法及应用
CN110357963A (zh) * 2019-07-31 2019-10-22 北京智仁美博生物科技有限公司 抗人st2抗体及其用途
CN113214395A (zh) * 2020-01-21 2021-08-06 迈威(上海)生物科技股份有限公司 抗st2抗体及其应用

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KELSEN, S. G. ET AL.: "Astegolimab (Anti-ST2) Efficacy and Safety in Adults with Severe Asthma: A Randomized Clinical Trial", JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, vol. 148, no. 3, 16 April 2021 (2021-04-16), XP086762505, DOI: 10.1016/j.jaci.2021.03.044 *

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