WO2023001258A1 - Pharmaceutical use of anti-tumor inhibin 2 antibody - Google Patents

Abstract

Provided is the use of a new anti-tumor inhibin 2 (ST2) antibody in the preparation of a drug for preventing or treating inflammatory, allergic or autoimmune diseases. The anti-ST2 antibody can bind to human ST2 with a high affinity, and can block the binding of human ST2 and human IL-33, thereby being capable of effectively inhibiting IL-33/ST2 signaling pathways. The anti-ST2 antibody has the effects of positively preventing and treating inflammatory, allergic or autoimmune diseases.

Description

Pharmaceutical uses of anti-tumor inhibin 2 antibodies

Cross References to Related Applications

This patent application claims the priority right of the Chinese invention patent application with application number CN202110824642.3 submitted on July 21, 2021, the entire content of which is hereby incorporated by reference.

technical field

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.

Background technique

Interleukin 33 (IL-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. In addition, interleukin-33 acts on its receptor and exerts its functions in vivo and in vitro by interacting with its receptor. For example, IL-33 can bind to anti-tumor inhibin 2 (suppression of tumorigenicity 2, ST2) with high affinity, trigger signaling cascade through downstream molecules, activate NFκb (nuclear factor κb) 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-1α, 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.

It can be seen that 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.

Contents of the invention

The inventors of the present invention provided a novel antibody with high affinity and high functional activity to human ST2. Studies have shown that 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.

Therefore, the object of the present invention is to provide a new pharmaceutical application of anti-ST2 antibody.

The technical scheme of the present invention is as follows.

In one aspect, 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.

According to the invention, said inflammatory, allergic or autoimmune disease is associated with activation of the IL-33/ST2 signaling pathway. Specifically, 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; And, 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. Alternatively, 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.

Preferably, 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.

Preferably, 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, and the light chain variable region (VL) comprises SEQ ID NO.7 The amino acid sequence shown in ID NO.6 or its variant.

In the context of the present invention, 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).

Therefore, with respect to 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.

Preferably, the ST2 antibody or fragment thereof further comprises a constant region. Preferably, 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.

Preferably, 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. Alternatively, for example, 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.

According to a specific embodiment of the present invention, 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. And/or, 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. As defined above, a "variant of an amino acid sequence" means having at least 75% sequence identity to said amino acid sequence.

Preferably, 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. According to a specific embodiment of the present invention, the anti-ST2 antibody of the present invention is an isolated monoclonal antibody of human IgG4/κ 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.

In another aspect, 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.

Specifically, 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; And, 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.

Preferably, 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. Alternatively, 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.

Preferably, 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.

Preferably, 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, and the light chain variable region (VL) comprises SEQ ID NO.7 The amino acid sequence shown in ID NO.6 or its variant.

Preferably, the ST2 antibody or fragment thereof further comprises a constant region. Preferably, 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.

Preferably, 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. Alternatively, for example, 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.

According to a specific embodiment of the present invention, 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. And/or, 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.

Preferably, 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. According to a specific embodiment of the present invention, the anti-ST2 antibody of the present invention is an isolated monoclonal antibody of human IgG4/κ 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.

According to the invention, said inflammatory, allergic or autoimmune disease is associated with activation of the IL-33/ST2 signaling pathway. Specifically, 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.

Compared with the prior art, the present invention provides a new anti-tumor inhibin 2 (ST2) antibody, 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.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 shows the inhibition rate of NF-κB reporter gene activity of B cell culture supernatants from different numbers of mice.

Figure 2 shows the results of the antibody inhibiting the activity of recombinant human IL-33 to promote the production of IL-6 in HUVEC cells.

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).

The best way to practice the invention

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.

The following reagents were used:

Human ST2: NP_003847.2 (Met1-Phe328)

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.

Example 1 Screening of Anti-ST2 Mouse Antibody

Using Human ST2-his as the antigen, the 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-κB 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-κB reporter gene activity, affinity determination and in vitro pharmacological research, ST2 binding experiments of different species, etc., and Compared with the control antibody CNTO7160.

Finally, it was determined that the 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.

Example 2 Preparation of Humanized Monoclonal Antibody

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).

Table 1 Humanized sequence form of 5886-156 heavy and light chain variable region

With the heavy chain constant region shown in SEQ ID NO.15 and the light chain constant region shown in SEQ ID NO.16, humanized light and heavy chains derived from the same mouse antibody were constructed, combined and transfected into CHO-K1 cells, transfected After 24 hours of transfection, 10 μg/mL MSX was added for pressurized screening. After the cell density and viability recovered, inoculation was performed for Feed-batch expression. After the expression was completed, the protein was obtained from the supernatant and purified by protein A for quantification.

The collocation of the humanized antibody and its heavy and light chain variable regions is shown in Table 2, and the suffix "ix" indicates that the antibody is the corresponding chimeric antibody.

Table 2 Sequence matching of light and heavy chain variable regions of 5886-156 humanized antibody

Example 3 Activity Characterization of Humanized Monoclonal Antibody

(1) Humanized antibody inhibits IL33 binding human ST2 activity screening

Coat the human ST2-fc protein overnight, mix the diluted human IL33-his and the diluted antibody 1:1, add 50 μL/well to a 96-well plate, and incubate at room temperature for 1 hour; add histidine-tagged secondary antibody (1:2500), 50 μL/well, incubate at room temperature for 1 h; TMB color development for 10 min, 2M sulfuric acid 100 μL/well to terminate. The results are shown in Table 3.

Table 3 Humanized antibody inhibits the activity of IL33 binding to human ST2 and the relative activity compared with CNTO7160

Antibody	IC50 (μg/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%

(2) Humanized antibody inhibits IL33 and promotes KU812-IL5 production activity screening

Dilute human IL33-his to 80ng/mL with medium, and dilute antibody to 640μg/mL with medium, 4-fold dilution, a total of 11 concentrations, mix the two 1:1, add 50μL/well to a 96-well plate . The KU812 cells in the logarithmic growth phase were centrifuged, 100,000 cells/well, 50 μL/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.

The results are shown in Table 4.

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 (μg/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%

(3) Humanized antibody affinity determination

Antibody interaction experiments with human ST2-his were performed using Biacore X100.

(3-1) 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 ST2 antibody was diluted to 2 μg/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.

(3-2) Dissociation kinetic affinity experiment between human ST2 and antibody at pH 5.5:

The experimental procedure described in (6-1) was followed, except that the dissociation was performed under the condition of HBS-EP (1×) buffer at pH 5.5, and the dissociation was monitored for 600 s.

The results are shown in Table 5.

Table 5 Affinity of humanized antibody at pH7.4 and pH5.5

*Antibody Ab2 in CN104334582B

(4) Evolution of 5886-156-H1L1 humanized antibody

In order to further reduce the post-translational modification of the 5886-156-H1L1 humanized antibody and improve the degree of humanization, Q1E and R72V mutations were performed on the heavy chain variable region sequence of the original 5886-156-H1L1 humanized antibody according to the amino acid sequence to form 5886 -156H-VH-HZ2. Using the mutated heavy chain variable region and 5886-156L-VL-HZ1 (SEQ ID NO.6) to construct a monoclonal antibody of human IgG4/κ subtype, the antibody was named 5886-156-H2L1, heavy chain and light The chain sequence is as follows:

Heavy chain (SEQ ID NO.17)

(Variable region (bold): SEQ ID NO.7; H-CDR1/2/3 (bold with underline): SEQ ID NO.8/9/10; Constant region (italic with underline): SEQ ID NO .14)

Light chain (SEQ ID NO.18)

(Variable region (bold): SEQ ID NO.6; L-CDR1/2/3 (bold with underline): SEQ ID NO.11/12/13; Constant region (italic with underline): SEQ ID NO .16)

Example 4 Binding activity of 5886-156-H2L1 to recombinant human ST2 protein (Biacore method)

Interaction experiments of this antibody with human ST2 were performed using Biacore T200.

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 μg/ml was captured in the channel of the CM5 chip with the anti-human Fc antibody for 30 seconds. After antibody capture, 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. Finally, 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.

Table 6 Affinity of 5886-156-H2L1 at pH7.4

Example 5 5886-156-H2L1 inhibits IL33 and promotes KU812-IL5 generation activity screening

Dilute human IL33-his to 80ng/mL with medium, and dilute antibody to 640μg/mL with medium, 4-fold dilution, a total of 11 concentrations, mix the two 1:1, add 50μL/well to a 96-well plate . KU812 cells in the logarithmic growth phase were centrifuged, 100,000 cells/well, 50 μL/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. The results are shown in Table 7.

Table 7 The results of humanized antibodies inhibiting IL33 to promote KU812-IL5 production and relative activity compared with CNTO7160

Antibody	IC50 (μg/mL)	relative activity	maximum inhibition rate
5886-156-H2L1	0.0343	403.79%	94.16%
CNTO7160	0.1385	the	95.21%

Example 6 Binding activity of 5886-156-H2L1 to recombinant human ST2 protein (ELISA method)

Human ST2-his was diluted to 1 μg/mL with coating buffer, added to 96-well plate at 50 μL/well, and coated overnight at 4°C; the next day, the coated plate was taken out and washed 3 times with PBST; Incubate for 1 hour, then wash with PBST for 3 times; 5886-156-H2L1 was diluted 3 times starting from 100ng/mL, 8 points, and added to 96-well plate at 50 μL/well; Add secondary antibody (1:10000) into 96-well plate at 50 μL/well; incubate at room temperature for 1 hour, wash with PBST three times, add TMB to 96-well plate at 50 μL/well, develop color in the dark for 10 minutes, stop with 100 μL/well of 2M sulfuric acid ; Read the OD450 value on a microplate reader.

The range of relative binding activity (EC50) between 5886-156-H2L1 and human ST2 recombinant protein was 16.34-17.32 ng/mL.

Example 7 Species specificity of 5886-156-H2L1 binding to ST2 (ELISA method)

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.

Human, cynomolgus monkey, rat and mouse ST2 were diluted to 1 μg/mL and coated onto ELISA plates. After blocking and washing, the Biotin-labeled 5886-156-H2L1 stock solution was diluted to 1 μg/mL, followed by 2-fold serial dilution to 0.977 ng/mL, with a total of 11 gradients. Add to the sealed ELISA plate, incubate at room temperature for about 2 hours, and wash 3 times. Add 10,000-fold diluted Streptavidin-HRP, incubate at room temperature for 30 minutes, and wash 3 times. Add the TMB solution that has been equilibrated to room temperature, and react in the dark for 8 minutes, stop the reaction, and read at 450/650nm. Fitting was performed using the software provided with the instrument. The results showed that 5886-156-H2L1 could specifically bind to human and cynomolgus monkey ST2 with similar affinity, with EC50 of 7.044ng/mL (human) and 7.588ng/mL (cynomolgus monkey), respectively. 5886-156-H2L1 does not bind rat and mouse ST2.

Example 8 Relative blocking activity of 5886-156-H2L1 on the binding of recombinant human ST2 protein and recombinant human IL-33 (ELISA method)

Coat the human ST2-fc protein overnight, mix the diluted human IL33-his with the diluted 5886-156-H2L1 1:1, add 50 μL/well to a 96-well plate, and incubate at room temperature for 1 hour; add histamine Acid-labeled secondary antibody (1:2500), 50 μL/well, incubated at room temperature for 1 h; TMB color development for 10 min, 2M sulfuric acid 100 μL/well was terminated.

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 μL 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 400μg/mL with medium, 4-fold dilution, a total of 11 concentrations, mix the two 1:1 and add 100μL/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. Specifically, 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 μg/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 μL/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.

Example 11 ADCC effect experiment of 5886-156-H2L1

(1) PBMC method

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 μl of cells to each well of a 96-well cell culture plate. For dilution solution, add 100 μl 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. 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 μl of KU812 cell dilution into wells parallel to the addition of SK-BR-3 cells. Dilute the positive control (Herceptin) with assay medium to concentrations of 500ng/ml, 50ng/ml, 5ng/ml, 1.25ng/ml, 0.313ng/ml, 0.0781ng/ml, 0.00781ng/ml and 0.000781ng/ml , then take 50 μl of positive control and add to the wells where SK-BR-3 cells have been added. Dilute the 5886-156-H2L1 monoclonal antibody with the assay medium to a concentration of 1000000ng/ml, 100000ng/ml, 10000ng/ml, 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml and 0.1ng/ml, then Add 50 μl to the wells where KU812 cells have been added, add 100 μl analysis medium to each well of TSR (Target Spontaneous Release, target cell spontaneous release) and TMR (Target Maximum Release, target cell maximum release), (E+T) 50 μl assay medium was added to each well of SR wells and ESR wells. 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. Add 50 μl of PBMC cell dilution solution with a density of 2.5×10 ⁶ cells/ml into the sample well, add 25 μl of PBMC cell dilution solution with a density of 5×10 ⁶ cells/ml into the sample well of the antibody to be tested, and in (E+T) 50 μl of PBMC diluents of corresponding concentrations were added to each well of SR wells and ESR wells. After adding samples, the 96-well cell plate was placed in a CO ₂ incubator at 37°C for 4 hours. According to the Cytotoxicity LDH Assay Kit-WST kit, add 10 μl Lysis Buffer to the incubated TMR wells and VCC wells, incubate in a CO ₂ incubator at 37°C for 30 minutes, then add 100 μl Working Solution to each well, and react for 30 minutes at room temperature in the dark , and finally add 50 μl Stop Solution to each well, and read the absorbance value (OD490nm) after mixing.

Calculation formula:

%Target cell Lysis＝[(OD _ER -OD _(T+E)SR )/(OD _TMR -OD _TSR )]×100

12.2 Reporter gene method

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 μl 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. Add 15 μl of KU812 cell dilution to the wells parallel to the addition of SK-BR-3 cells. Dilute the positive control (Herceptin) with assay medium to a concentration of 1500ng/ml, 750ng/ml, 375ng/ml, 187.5ng/ml, 93.75ng/ml, 46.88ng/ml, 23.44ng/ml, 11.72ng/ml , 5.86ng/ml and 2.93ng/ml, the culture solution in the incubated SK-BR-3 cell group was aspirated, and then 30 μl of the diluted positive control was added to each well. Dilute the 5886-156-H2L1 monoclonal antibody with the assay medium to a concentration of 1000000ng/ml, 100000ng/ml, 10000ng/ml, 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01 ng/ml and 0.001ng/ml, then add 30 μl to the wells where KU812 cells have been added. Place the loaded 96-well cell plate in a CO ₂ incubator at 37°C for 30 min. 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. In the positive control Add 30 μl effector cell dilution solution with a density of 4×10 ⁶ cells/ml to the sample well, and add 15 μl 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 μl SK-BR-3 cells + 30 μl analysis medium + 30 μl effector cells with a density of 4×10 ⁶ cells/ml and 15 μl KU812 cells + 30 μl analysis medium + 15 μl with a density of 8×10 ⁶ cells/ml ml effector cells), blank control (60 μl assay medium). Place the loaded 96-well cell plate in a CO ₂ incubator at 37°C for 16-24h. After the incubation, add 60 μl/well of Bio-Glo Luciferase Assay System reagent, shake at room temperature at 900 rpm for 2 minutes in the dark, then quickly detect the light signal with a microplate reader, select the chemiluminescence mode, and complete the detection within 30 minutes.

Calculation formula:

Background mean value: mean value of RLU of blank control wells; mean value of negative control: mean value of RLU of negative control wells.

The experimental results showed that 5886-156-H2L1 had no obvious ADCC effect in two different detection systems.

Example 12 CDC effect experiment of 5886-156-H2L

Centrifuge the Raji cells at 300×g for 5 minutes, remove the supernatant, add CDC analysis medium to resuspend, take samples and count and dilute the cell density to 1×10 ⁶ cells/mL. KU812 cells in the logarithmic growth phase were collected, sampled and counted, centrifuged at 300 g for 5 min, resuspended in the analysis medium and diluted to a cell density of 1×10 ⁶ cells/ml. Two cell dilutions were prepared by adding 50 μl to each well of different 96-well cell culture plates. Dilute the positive control (MabThera) with assay medium to a concentration of 10000ng/ml, 5000ng/ml, 2500ng/ml, 1250ng/ml, 625ng/ml, 312.5ng/ml, 156.3ng/ml, 78.1ng/ml, 39.1 ng/ml and 19.5ng/ml, take 50 μl of the diluted positive control and add it to the wells with Raji cells. Dilute the 5886-156-H2L1 monoclonal antibody with the assay medium to a concentration of 1000000ng/ml, 100000ng/ml, 10000ng/ml, 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01 ng/ml and 0.001ng/ml, then 50 μl was added to the wells where KU812 cells had been added. Place the added 96-well cell plate in a CO ₂ incubator and incubate at 37°C for 30min. After the incubation, 50 μl of assay medium containing 24% complement was added to each well, and the cell plate was placed in a CO ₂ incubator at 37° C. for 2 h. During this period, no complement control, no antibody control and blank control should be set. After adding 10 μl of Resasurin to each well of the 96-well plate, place it in a constant temperature shaker and shake it at room temperature for 1-2 minutes, continue to incubate in the incubator for 16-20 hours, and then take out the plate for reading.

Calculation formula: CDC%=100×(E-S)/(M-S)

E: signal of experimental well; S (spontaneous killing): signal of no antibody control; M (maximum killing): signal of blank control.

The experimental results show that 5886-156-H2L1 has no obvious activity of activating the complement system to mediate the CDC effect.

Example 13 Study on the efficacy of 5886-156-H2L1 in allergic dermatitis induced by Ascaris suum egg extract

In this example, the allergic dermatitis model of cynomolgus monkeys induced by Ascaris suum egg extract (Ascaris. preventive or therapeutic effect.

Divide 24 cynomolgus monkeys naturally sensitive to Ascaris suum egg extract into 4 groups, 6 in each group, followed by vehicle control group, positive control group (diphenhydramine), 5886-156-H2L1 low-dose group (10mg /kg), 5886-156-H2L1 high-dose group (30mg/kg). Among them, for the two 5886-156-H2L1 groups, 5886-156-H2L1 diluted by normal saline was injected intravenously in advance (-4h); for the positive control group, diphenhydramine 2mg/ kg orally; for the vehicle control group, normal saline (2 mL/kg) was injected intravenously in advance (-4h).

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 swelling area of the stimulation point was measured at 0 min and 20 min after stimulation with normal saline, histamine and A.suum, and the swelling response of the skin was reflected by the ratio of the two (Ratio=20 min area/0 min area).

The results showed that compared with the vehicle control group, the 5886-156-H2L1 high-dose group (30mg/kg) could significantly reduce the skin swelling reaction caused by intradermal injection of A.suum (p<0.01); compared with the vehicle control group , 5886-156-H2L1 low-dose group and high-dose group (10mg/kg and 30mg/kg) preventive administration can significantly reduce the skin swelling reaction caused by intradermal injection of histamine. The result is shown in Figure 4.

Example 14 Pharmacodynamic study of 5886-156-H2L1 in irinotecan-induced intestinal mucositis model

In this example, 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.

Divide 9 cynomolgus monkeys into 3 groups (G1 group, G2 group, G3 group), with 3 monkeys in each group. In the three days from Day 0 to 2, 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, and 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.

Compared with the normal control group (G1), intravenous 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.

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention, as long as they do not depart from the spirit of the present invention, all should belong to the scope of the appended claims of the present invention.

Claims (15)

1. Use of an anti-tumor inhibin 2 (ST2) antibody or a fragment thereof in the preparation of medicines for preventing, treating or improving inflammatory, allergic or autoimmune diseases.
2. The use according to claim 1, characterized in that the inflammatory, allergic or autoimmune diseases are related to the activation of IL-33/ST2 signaling pathway.
3. The application according to claim 1 or 2, characterized in that, the inflammatory or allergic diseases are endometriosis, heart failure, allergic dermatitis, allergic or allergic rhinitis, nasal polyps, atopic Dermatitis, chronic obstructive pulmonary 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, is atopic dermatitis or inflammatory bowel disease.
4. The use according to any one of claims 1 to 3, wherein the anti-ST2 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region and the variable region of the heavy chain and the light chain can be The variable region contains heavy chain CDR1 (H-CDR1), CDR2 (H-CDR2), CDR3 (H-CDR3) and light chain CDR1 (L-CDR1), CDR2 (L-CDR2), CDR3 (L-CDR3) ,in:

   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; And, 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.
5. The application according to any one of claims 1 to 4, wherein the anti-ST2 antibody or its fragment is an anti-ST2 monoclonal antibody, scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab' , F(ab') ₂ or Fv antibody formats;

   Alternatively, the anti-ST2 antibody is an anti-ST2 murine antibody, chimeric antibody, fully or partially humanized antibody.
6. The application according to any one of claims 1 to 5, wherein the heavy chain variable region (VH) of the anti-ST2 antibody comprises the amino acid sequence shown in SEQ ID NO.7 or variants thereof , the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO.6 or variants thereof.
7. The use according to any one of claims 1 to 6, wherein the ST2 antibody or fragment thereof further comprises a constant region;

   Preferably, 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 a protein selected from IgG, IgA, IgM, IgD or IgE heavy chain constant region and/or light chain constant region of kappa or lambda type;

   Preferably, 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.
8. The use according to any one of claims 1 to 7, wherein the anti-ST2 antibody comprises a heavy chain and/or a light chain, preferably two heavy chains and/or two light chains;

   Preferably, the anti-ST2 antibody is a monoclonal antibody of human IgG4/κ subtype, which contains two identical light chains and two identical heavy chains;

   Further preferably, the heavy chain of the antibody 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.
9. A method for preventing or treating an inflammatory, allergic or autoimmune disease comprising administering an anti-ST2 antibody or fragment thereof to a subject in need thereof.
10. The method according to claim 9, characterized in that, the subject is a mammal, preferably a primate, more preferably a human or a cynomolgus monkey, more preferably a human.
11. The method according to claim 9 or 10, wherein the anti-ST2 antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, and in the heavy chain variable region and the light chain variable region respectively Contains 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; And, 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;

    Preferably, the anti-ST2 antibody or fragment thereof is in the form of an anti-ST2 monoclonal antibody, scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv antibody; or, the Anti-ST2 antibodies are murine antibodies, chimeric antibodies, fully or partially humanized antibodies against ST2.
12. The method according to any one of claims 9 to 11, wherein the heavy chain variable region (VH) of the anti-ST2 antibody comprises the amino acid sequence shown in SEQ ID NO.7 or a variant thereof , the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO.6 or variants thereof.
13. The method according to any one of claims 9 to 12, wherein the ST2 antibody or fragment thereof further comprises a constant region;

    Preferably, 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 light chain constant region of kappa or lambda type;

    Preferably, 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.
14. The method according to any one of claims 9 to 13, wherein the anti-ST2 antibody comprises a heavy chain and/or a light chain, preferably two heavy chains and/or two light chains;

    Preferably, the anti-ST2 antibody is a monoclonal antibody of human IgG4/κ subtype, which contains two identical light chains and two identical heavy chains;

    Further preferably, the heavy chain of the antibody 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.
15. The method according to any one of claims 9 to 14, wherein the inflammatory, allergic or autoimmune disease is associated with activation of the IL-33/ST2 signaling pathway;

    Preferably, the inflammatory, allergic or autoimmune disease is endometriosis, heart failure, atopic dermatitis, allergic or allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary 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, For atopic dermatitis or inflammatory bowel disease.

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