Antibody to IL-4Rα or its antigen-binding fragment, bispecific antibody and their use

Abstract

The present invention relates to the field of biomedicine. Provided are an anti-human IL-4Rα antibody or an antigen-binding fragment thereof and the use thereof. The anti-human IL-4Rα antibody comprises heavy chain complementarity determination regions HCDR1, HCDR2 and HCDR3, and light chain complementarity determination regions LCDR1, LCDR2 and LCDR3, has high affinity with human IL-4Rα, can be used for preparing an antibody drug, can be used for treating type-II inflammatory diseases, and has relatively good clinical efficacy and relatively low toxic and side effects. Further provided are a bispecific antibody against both IL-4Rα and TSLP, and the use of the antibody in the preparation of a drug for treating severe asthma, moderate to severe atopic dermatitis and chronic obstructive pulmonary disease.

Description

Anti-IL-4Rα antibody or antigen-binding fragment thereof and bispecific antibody and application thereof Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an antibody binding to human IL-4Rα, an antigen-binding fragment thereof, a bispecific antibody thereof, and applications thereof. Background Art

[0002] Interleukin-4 (IL-4) is primarily secreted by activated T cells, monocytes, basophils, mast cells, and eosinophils. Its biological effects include stimulating the proliferation and differentiation of activated B and T cells. Interleukin-13 (IL-13) is expressed by a variety of cell types. It can inhibit the release of inflammatory cytokines from monocytes, induce B cell proliferation and differentiation, promote B cell antibody secretion, and stimulate IgE expression, contributing to allergic responses. Both IL-4 and IL-13 require binding to a common functional receptor, IL-4Rα, to exert downstream regulatory effects. IL-4 exerts its downstream effects through both type I receptors (IL-4Rα / γc) and type II receptors (IL-4Rα / IL-13Rα1). The former is expressed exclusively on the surface of hematopoietic cells, while the latter is expressed on both hematopoietic and non-hematopoietic cells (such as airway epithelial cells). IL-13 exerts its downstream regulatory effects exclusively through the type II receptor. Once IL-4 or IL-13 binds to the receptor, the receptor dimerizes and further induces the phosphorylation and activation of JAKs proteins, including JAK1, JAK3 and JAK2, which correspond to IL-4Rα, γc and IL-13Rα1, respectively, and then promotes the phosphorylation of specific tyrosine residues in the intracellular domain of IL-4Rα, further phosphorylating the transcription factor STAT6, promoting gene transcription and protein expression.

[0003] Type II inflammatory diseases primarily include atopic dermatitis, asthma, allergic rhinitis, some chronic sinusitis with nasal polyps, and chronic obstructive pulmonary disease, and are typically caused by a chronic imbalance between the innate and adaptive immune systems. Environmental triggers, genetic factors, and barrier disruption continuously activate adaptive immune cells (Th2), producing cytokines such as IL-4, IL-5, and IL-13. These cytokines drive B cell class switching and IgE production, promote eosinophil differentiation and migration, and cause basophil and mast cell degranulation, exacerbating the development of inflammatory responses. IL-4Rα is a key factor in the type II immune response, mediating the functional effects of both IL-4 and IL-13 cytokines. Therefore, blocking IL-4Rα can simultaneously block both type II immune responses.

[0004] Currently, monoclonal antibody drugs targeting human IL-4Rα have demonstrated excellent therapeutic efficacy in clinical studies for multiple indications, fully demonstrating the druggability of this target. However, there is still a need to develop antibody drugs with high affinity for human IL-4Rα for the treatment of type II inflammatory diseases, which would have better clinical efficacy and lower toxic side effects, thereby providing more drug options for patients, which is of great significance.

[0005] Dupilumab, the first marketed monoclonal antibody against IL-4Rα, has demonstrated significant clinical efficacy and has been approved for multiple indications. However, limitations exist in its clinical application, including limited efficacy in patients with atopic dermatitis, low response rates, and side effects caused by increased eosinophilia in some patients during treatment. Clinical evaluation indicators of dupilumab treatment have shown limited improvement in eosinophil counts in patients treated with dupilumab, consistent with the molecular mechanism of action of IL-4Rα.

[0006] Thymic stromal lymphopoietin (TSLP) belongs to the interleukin-7 family of cytokines and is produced by epithelial cells, smooth muscle cells, stromal cells, macrophages, and monocyte-macrophage cells. Numerous studies have demonstrated that TSLP is located at the apex of multiple inflammatory cascades and is a key driver of Th2-type inflammatory responses. After epithelial cells are stimulated or damaged by allergens or pathogens, dendritic cells present antigens to naive CD4+ T cells, simultaneously releasing epithelial cytokines, TSLP, and IL-33. These co-stimulatory cytokines promote the development of allergen-specific Th2 cells, which produce cytokines such as IL-4, IL-5, and IL-13. Therefore, TSLP is generally considered to be one of the initiators of the inflammatory cascade. Inhibiting TSLP can intervene in the early stages of inflammation by preventing the release of proinflammatory cytokines by immune cells, thus being more effective than inhibiting IL-4, IL-5, and IL-13 alone. In addition, in vitro and in vivo efficacy studies suggest that TSLP can also participate in non-Th2 inflammatory responses and may promote Th1 / Th17-related autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Tezepelumab is currently the only marketed anti-TSLP monoclonal antibody drug, approved for the treatment of moderate to severe asthma in adults, and has excellent therapeutic effects. However, it also has clinical application limitations and did not meet the primary clinical study endpoint in the treatment of atopic dermatitis. A comprehensive analysis of the changes in serological indicators of patients after Tezepelumab treatment showed that after Tezepelumab treatment, the number of eosinophils in patients decreased by approximately 50%, and the levels of IL-5 and IL-13 both decreased by more than 50%. However, the improvement in IgE levels was limited, decreasing by only approximately 20%, and the regulatory effect on IL-4 was very weak.

[0007] Bispecific antibodies can simultaneously target two different antigens, expanding their range of action while retaining the high specificity of the antibody molecule, thereby enhancing clinical therapeutic efficacy. Currently, a variety of bispecific antibodies have been developed. Among them, the IgG-scFv structure retains the complete IgG form of the native monoclonal antibody, offering additional advantages in molecular design, expression, and purification, making it an ideal form of bifunctional antibody.

[0008] A comprehensive analysis of the clinical efficacy of dual-target inhibition revealed limited improvement in eosinophils by dupilumab, and limited improvement in IL-4 and IgE by tezepelumab. Th2 signaling pathways are regulated by multiple cytokines, and simultaneous dual-target inhibition could theoretically significantly improve clinical response rates and expand the scope of indications. Therefore, the clinical development of new bispecific antibodies against IL-4Rα and TSLP is still needed, leveraging the unique inhibitory effects of both targets to complement each other and achieve better therapeutic outcomes and increased safety. Summary of the Invention

[0009] In response to the above situation, the present invention provides an anti-IL-4Rα antibody or its antigen-binding fragment and a bispecific antibody thereof. The antibody of the present invention has high affinity, good activity and low hydrophobicity as an anti-human IL-4Rα antibody, and can be used to prepare drugs for the treatment of allergic diseases such as atopic dermatitis and asthma.

[0010] The anti-IL-4Rα antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain complementary determining region and a light chain complementary determining region.

[0011] The heavy chain complementary determining region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 12; the HCDR2 has an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13; and the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 14; and / or

[0012] The light chain complementary determining region includes LCDR1, LCDR2 and LCDR3, wherein the LCDR1 is the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 15; the LCDR2 is the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 16; and the LCDR3 is the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO: 17.

[0013] In one embodiment of the present invention, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises:

[0014] (1) a heavy chain complementarity determining region comprising HCDR1 of the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 2, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 3; and

[0015] The light chain complementary determining region comprises LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 with the amino acid sequence shown in SEQ ID NO: 6.

[0016] In one embodiment of the present invention, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises:

[0017] (2) a heavy chain complementarity determining region comprising HCDR1 of the amino acid sequence set forth in SEQ ID NO: 1, HCDR2 of the amino acid sequence set forth in SEQ ID NO: 7, and HCDR3 of the amino acid sequence set forth in SEQ ID NO: 3; and

[0018] The light chain complementary determining region comprises LCDR1 of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 of the amino acid sequence shown in SEQ ID NO: 8.

[0019] In one embodiment of the present invention, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises:

[0020] (3) a heavy chain complementarity determining region comprising the amino acid sequences of HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 7, and HCDR3 set forth in SEQ ID NO: 9; and

[0021] The light chain complementary determining region comprises the amino acid sequence of LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 8.

[0022] In one embodiment of the present invention, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises:

[0023] (4) a heavy chain complementarity determining region comprising the amino acid sequences of HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 10, and HCDR3 set forth in SEQ ID NO: 3; and

[0024] The light chain complementary determining region comprises the amino acid sequence of LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 11.

[0025] In one embodiment of the present invention, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises:

[0026] (5) a heavy chain complementarity determining region comprising the amino acid sequences of HCDR1 set forth in SEQ ID NO: 12, HCDR2 set forth in SEQ ID NO: 13, and HCDR3 set forth in SEQ ID NO: 14; and

[0027] The light chain complementary determining region comprises the amino acid sequence of LCDR1 shown in SEQ ID NO: 15, LCDR2 shown in SEQ ID NO: 16, and LCDR3 shown in SEQ ID NO: 17.

[0028] The anti-IL-4Rα antibody or antigen-binding fragment thereof further comprises a fragment selected from the following:

[0029] A framework region (FR) sequence derived from the human germline heavy chain IGHV1-46*01+IGHJ1*01 or a backmutated sequence having at least 90% identity thereto;

[0030] The framework sequence is derived from the human germline light chain IGKV1-39*01+IGKJ2*01 or a backmutated sequence having at least 90% identity thereto.

[0031] The anti-IL-4Rα antibody or antigen-binding fragment thereof provided by the present invention is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, a diabody or a fragment thereof.

[0032] The anti-IL-4Rα antibody or antigen-binding fragment thereof provided by the present invention includes:

[0033] (1) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 29;

[0034] (2) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 30 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 31;

[0035] (3) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 33;

[0036] (4) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19; or

[0037] (5) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 21;

[0038] The anti-IL-4Rα antibody or antigen-binding fragment thereof provided by the present invention comprises an amino acid sequence with 90% or more identity.

[0039] The present invention also provides a fusion protein comprising the above-mentioned anti-IL-4Rα antibody or an antigen-binding fragment thereof.

[0040] The present invention also provides a polynucleotide encoding the anti-IL-4Rα antibody or the antigen-binding fragment thereof.

[0041] The present invention also provides an expression vector comprising the polynucleotide.

[0042] The present invention also provides a host cell comprising the above vector, wherein the host cell is a prokaryotic or eukaryotic cell.

[0043] The present invention also provides a pharmaceutical composition comprising the anti-IL-4Rα antibody or antigen-binding fragment thereof or the fusion protein and pharmaceutically acceptable excipients.

[0044] The present invention also provides a use of the anti-IL-4Rα antibody or antigen-binding fragment thereof, the fusion protein or a pharmaceutical composition thereof in the preparation of a drug for treating allergic diseases.

[0045] As one of the embodiments of the present invention, the allergic disease is selected from atopic dermatitis, asthma, allergic rhinitis, some chronic sinusitis with nasal polyps, or chronic obstructive pulmonary disease.

[0046] The present invention provides an antibody drug that is different from the prior art anti-IL-4Rα antibody or its antigen-binding fragment and has a high affinity for human IL-4Rα, and is used to treat type II inflammatory diseases, so that it has better clinical efficacy and lower toxic side effects, providing more drug options for patients, which is of great significance.

[0047] Surface plasmon resonance detection showed that the m8, m21, and m27 screened by the present invention had comparable affinities to the Dupilumab antibody, wherein the KD value of the m8 molecule was 4.37E-11M, and the KD value was below 50 pM.

[0048] The antibody molecules m21 and m27 screened by the present invention have better effects on inhibiting the proliferation of TF-1 cells induced by hIL-13 than the dupilumab antibody, and the m8 antibody molecule has a blocking activity comparable to that of dupilumab.

[0049] Hydrophobicity chromatography showed that the retention times of the m8, m21, and m27 molecules screened by the present invention were significantly lower than that of the positive antibody BMK1, and therefore their hydrophobicity was significantly lower than that of the dupilumab antibody. Antibodies with low hydrophobicity are less likely to form aggregates during process development and are less likely to bind to host proteins, resulting in poor stability, which has a significant advantage in subsequent process development.

[0050] The present invention also provides a bispecific antibody that can simultaneously resist IL-4Rα and TSLP, the bispecific antibody format is IgG-scFv, and the bispecific antibody comprises any one of the above-mentioned anti-IL-4Rα antibodies or antigen-binding fragments thereof.

[0051] The bispecific antibody of the present invention comprises immunoglobulin IgG and single-chain antibody scFv, and the bispecific antibody is selected from one of the following groups:

[0052] (1) the immunoglobulin IgG comprises an anti-TSLP antibody or an antigen-binding fragment thereof, and the single-chain antibody scFv comprises an anti-IL-4Rα antibody or an antigen-binding fragment thereof selected from any one of the above items; or

[0053] (2) The immunoglobulin IgG comprises an anti-IL-4Rα antibody or an antigen-binding fragment thereof selected from any one of the above items, and the single-chain antibody scFv comprises an anti-TSLP antibody or an antigen-binding fragment thereof.

[0054] As one embodiment, the bispecific antibody of the present invention, wherein the anti-TSLP antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein

[0055] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 has the amino acid sequence shown in SEQ ID NO: 48; the HCDR2 has the amino acid sequence shown in SEQ ID NO: 49; and the HCDR3 has the amino acid sequence shown in SEQ ID NO: 50;

[0056] The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has an amino acid sequence as shown in SEQ ID NO: 51; the amino acid sequence of the LCDR2 is DDS; and the LCDR3 has an amino acid sequence as shown in SEQ ID NO: 52.

[0057] As one embodiment, the bispecific antibody of the present invention, wherein the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein,

[0058] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3, wherein the HCDR1 has the amino acid sequence shown in SEQ ID NO: 1; the HCDR2 has the amino acid sequence shown in SEQ ID NO: 2; and the HCDR3 has the amino acid sequence shown in SEQ ID NO: 3;

[0059] The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein the LCDR1 has the amino acid sequence shown in SEQ ID NO: 4; the LCDR2 has the amino acid sequence shown in SEQ ID NO: 5; and the LCDR3 has the amino acid sequence shown in SEQ ID NO: 6.

[0060] In the present invention, as one embodiment, the anti-TSLP antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0061] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 42, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 43, or,

[0062] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 34, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 36.

[0063] In the present invention, as one embodiment, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:

[0064] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 44, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 45, or

[0065] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 29.

[0066] In the present invention, as one embodiment, the immunoglobulin IgG and the single-chain antibody scFv are connected by a linker, and the linker is (GGGGS)n, where n is a positive integer from 1 to 5, preferably an amino acid sequence as shown in SEQ ID NO: 46. As an exemplary illustration, n can be 1, 2, 3, 4 or 5.

[0067] In the present invention, as one embodiment, the single-chain antibody scFv further contains a linker connecting the heavy chain variable region and the light chain variable region of the scFv, and the linker is (GGGGS)n, where n is a positive integer from 1 to 5, preferably an amino acid sequence as shown in SEQ ID NO: 47. As an exemplary illustration, n can be 1, 2, 3, 4 or 5.

[0068] As one embodiment, the bispecific antibody of the present invention is selected from any one of the following groups:

[0069] (1) the immunoglobulin IgG comprises a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 34, a heavy chain constant region with an amino acid sequence as set forth in SEQ ID NO: 35, a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 36, and a light chain constant region with an amino acid sequence as set forth in SEQ ID NO: 37, and the single-chain antibody scFv portion has a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 44 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 45; or

[0070] (2) The immunoglobulin IgG comprises a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 28, a heavy chain constant region as shown in SEQ ID NO: 40, a light chain variable region as shown in SEQ ID NO: 29, and a light chain constant region as shown in the amino acid sequence of SEQ ID NO: 41, and the single-chain antibody scFv portion has a heavy chain variable region as shown in the amino acid sequence of SEQ ID NO: 42 and a light chain variable region as shown in the amino acid sequence of SEQ ID NO: 43.

[0071] As one embodiment, the bispecific antibody of the present invention is selected from any one of the following groups:

[0072] (1) the bispecific antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 57 and a light chain with an amino acid sequence as shown in SEQ ID NO: 58; or

[0073] (2) The bispecific antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 59 and a light chain having an amino acid sequence as shown in SEQ ID NO: 60.

[0074] The present invention also provides a pharmaceutical composition comprising the above-mentioned bispecific antibody or antigen-binding fragment thereof and pharmaceutically acceptable excipients.

[0075] The present invention also provides the use of any of the above-described bispecific antibodies or pharmaceutical compositions in the preparation of a medicament for treating moderate to severe asthma, moderate to severe atopic dermatitis, allergic rhinitis, some chronic sinusitis with nasal polyps, and chronic obstructive pulmonary disease.

[0076] The bispecific antibodies MTM and MMT provided herein have comparable affinities for hIL-4Rα to dupilumab and comparable affinities for hTSLP to tezepelumab. The bispecific antibodies MTM and MMT provided herein can effectively block the activity of hIL-13 and hIL-4, with comparable activity to dupilumab. The ability of the bispecific antibodies to block hIL-13 and hIL-4 was evaluated using a GFP reporter gene assay targeting IL-4Rα, and the results showed that MTM and MMT can effectively block the activity of hIL-13 and hIL-4.

[0077] The bispecific antibodies MTM and MMT provided by the present invention can effectively block the activity of TSLP, with activity comparable to that of tezepelumab. The ability of the bispecific antibodies to block hTSLP was evaluated using a luciferase reporter gene assay for TSLP activity, and the results showed that MTM and MMT can effectively block hTSLP activity.

[0078] The bispecific antibody MTM provided by the present invention exhibits excellent stability. The MTM molecule can be concentrated to concentrations exceeding 100 mg / mL in a 20 mM sodium acetate + 100 mM L-proline buffer, pH 5.0. Stability studies conducted at 4°C, 25°C, and 40°C, using monomer analysis by SEC-HPLC as an evaluation metric, demonstrated excellent stability, facilitating subsequent process development. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1A: FACS analysis of the activity of the first batch of mouse antibody molecules binding to CHO-K1-hIL-4Rα;

[0080] Figure 1B: FACS assay of the second batch of mouse antibody molecules binding to CHO-K1-hIL-4Rα;

[0081] Figure 2A: Reporter gene assay for the inhibitory effect of the first group of candidate mouse antibody molecules on hIL-13;

[0082] Figure 2B: Reporter gene assay for the inhibitory effect of the second group of candidate mouse antibodies on hIL-13;

[0083] Figure 3: Reporter gene assay for the inhibitory effect of affinity-matured mAbs on hIL-4 activity;

[0084] Figure 4: Efficacy results of m8 and m21 mAb molecules in the B-hIL4 / IL4Rα humanized mouse asthma model;

[0085] Figure 5: SEC-HPLC method to detect the molecular purity of m8 and m21 monoclonal antibodies;

[0086] Figure 6: Detection of the hydrophobicity of m8 and m21 mAbs by HIC-HPLC;

[0087] Figure 7: Purity of the bispecific antibody was detected by SDS-PAGE.

[0088] Figure 8A: Biacore 8k detection of the ability of MMT molecules to simultaneously bind to hIL4Rα and hTSLP antigens;

[0089] Figure 8B: Biacore 8k detection of the ability of MTM molecules to simultaneously bind to hIL4Rα and hTSLP antigens;

[0090] Figure 9: ELISA assay for the level of CCL17 secreted by human PBMCs;

[0091] Figure 10A: MTM dual antibody inhibits OVA-specific IgE expression in the B-hIL4 / IL4Rα humanized mouse asthma model;

[0092] Figure 10B: MTM dual antibody inhibits eosinophil expression in the B-hIL4 / IL4Rα humanized mouse asthma model;

[0093] Figure 11A: MTM dual antibody inhibits IgE expression in the B-hTSLP / TSLPR / IL7Rα humanized mouse asthma model;

[0094] Figure 11B: MTM dual antibody inhibits eosinophil expression in the B-hTSLP / TSLPR / IL7Rα humanized mouse asthma model. DETAILED DESCRIPTION

[0095] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0096] The experimental methods in the examples of the present invention were all performed according to conventional conditions such as the Cold Spring Harbor Laboratory Manual of Antibody Technology, the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturers. All reagents were purchased commercially.

[0097] The BMK1 described in the present invention is a positive control molecule (Dupilumab antibody), the sequence of which is derived from SEQ ID ID.162 and SEQ ID ID.164 of patent application number CN200980143007.6. The preparation method refers to the document doi:10.1016 / j.ejbt.2019.07.002.

[0098] Example 1: Preparation and sequencing of anti-hIL-4Rα mouse antibodies

[0099] 1.1 Animal immunization

[0100] CHO-K1 cells (approximately 10 7 Four BALB / c mice were immunized by subcutaneous injection in the abdomen after the initial immunization. After the first immunization, booster immunization was performed every two weeks. After two immunizations, blood was collected from the tail and the serum titer of the mice was detected by ELISA. The titer was greater than 2×10 4 The spleen cells of the mice were used for subsequent hybridoma cell fusion.

[0101] 1.2 Preparation of hybridoma cell library

[0102] Take titer greater than 2×10 4 The spleen cells of BALB / c mice and SP2 / 0 mouse myeloma cells were electroporated at a ratio of 1:1.2, and the fused hybridoma cells were expressed as 1×10 4 Cells / well were plated into 96-well plates and cultured at 37°C for 10 days before screening.

[0103] 1.3 Subclone sorting and preliminary screening of positive clones binding to hIL-4Rα by ELISA

[0104] The 30 hybridoma cell pools obtained by screening were subcloned and sorted using a semi-solid culture medium method to obtain monoclonal cells. Subsequently, the positive clones binding to the hIL-4Rα protein were screened using the ELISA method.

[0105] Streptavidin protein (Jackson, Cat: 016-000-113) was diluted to 0.25 μg / ml in coating buffer (200 mM Na2CO3 / NaHCO3, pH 9.2), mixed thoroughly, and added to a 384-well plate at 30 μl / well. Coating was allowed to proceed overnight at 4°C. The plates were washed once with 100 μl / well of 1× PBS-T (0.05% Tween 20). 80 μl of blocking solution (2% BSA / 1× PBS) was added to each well, incubated at 25°C for 1 hour, and washed three times with 100 μl of 1× PBS-T (0.05% Tween 20) per well. IL-4Rα-biotin was diluted to 0.05 μg / ml in 2% BSA buffer (200 mM Na2CO3 / NaHCO3, pH 9.2). After mixing, 30 μl / well of the culture supernatant was added to a 384-well plate and washed three times with 100 μl / well of 1× PBS-T (0.05% Tween20). The supernatant of the sorted subclones was added to each well of the plate and incubated at 25°C for 2 h. The plates were washed three times with 100 μl / well of 1× PBS-T (0.05% Tween20). Then, 30 μl of goat anti-mouse IgG Fc-HRP (1:5000 dilution) (Bethyl, Cat: A90-231P) was added to each well and incubated at 25°C for 1 h. The plates were washed six times with 100 μl / well of 1× PBS-T (0.05% Tween20). 30 μl of TMB substrate was added to each well and reacted at 25°C for 20 min. 30 μl of stop solution (2 M HCl) was then added to each well to terminate the reaction, and the absorbance was measured at 450 nm.

[0106] 1.4 Flow cytometry fluorescence sorting technology FACS further screened positive clones binding to hIL-4Rα

[0107] Based on the absorbance values ​​from the ELISA, the 60 molecules with the highest absorbance values ​​were selected for FACS analysis (CHO-K1.IL-4Rα cells). CHO-K1.IL-4Rα cells overexpressing hIL-4Rα were harvested in 50 ml centrifuge tubes and added to a 96-well plate at a concentration of 1E5 cells / well. Centrifuge at 1500 rpm for 4 minutes, and the supernatant discarded. 100 μl / well of the culture supernatant from the sorted subclones was added to a 96-well plate, incubated at 4°C for 1 hour, and washed twice with 200 μl / well of 1% BSA / 1× PBS. PE-labeled goat anti-mouse IgG Fc (1:100) (Jackson, Cat:115-115-164) was added to 100 μl / well and incubated at 4°C for 0.5 h. The cells were washed twice with 80 μl / well 1% BSA / 1×PBS, and then resuspended with 30 μl / well 1% BSA / 1×PBS. Finally, the mean fluorescence intensity was detected by flow cytometry.

[0108] 1.5 Screening of positive clones that can inhibit hIL-4 activity using reporter gene method (HEK-293T.STAT6-GFP cells)

[0109] Based on the ELISA results, the 60 molecules with the highest absorbance values ​​were selected for single-concentration reporter gene detection (CHO-K1.IL-4Rα cells). HEK-293T.STAT6-GFP cells were prepared and resuspended in TransDetect BrightFluore DMEM + 10% FBS medium. The cell density was adjusted to 5E5 cells / ml and added to a black 96-well plate at 100μl / well. Incubate at 37°C for 24h. hIL-4 was diluted to 1.0ng / ml with TransDetect BrightFluore DMEM + 10% FBS medium, 20μl was added to each well, and then 80μl of the subclone culture supernatant was added to the well, gently mixed, and cultured at 37°C for another 24h. The fluorescence signal intensity was detected using a microplate reader with an excitation wavelength of 488nm and an emission wavelength of 520nm.

[0110] 1.6 Expression, purification and activity evaluation of candidate molecules

[0111] Based on the results obtained from the above three evaluation methods, the 10 best mouse monoclonal cells were selected for amplification culture. The mouse antibodies obtained after protein A purification of the culture medium were evaluated by FACS and reporter gene method.

[0112] 1.7 FACS screening of molecules binding to hIL-4Rα protein

[0113] The 10 best-performing mouse monoclonal antibodies (see Table 1) were selected for full FACS screening. CHO-K1.IL-4Rα cells overexpressing hIL-4Rα were harvested in 50 ml centrifuge tubes. 1E5 cells / well were added to a 96-well plate and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded. The purified antibody was diluted to 200 nM in 1% BSA / 1× PBS solution, serially diluted 1:4 through 11 concentrations. The plates were incubated at 4°C for 1 hour and washed twice with 200 μl / well of 1% BSA / 1× PBS. PE-conjugated goat anti-mouse IgG Fc (1:100) was added in 100 μl / well and incubated at 4°C for 0.5 hour. The plates were washed twice with 80 μl / well of 1% BSA / 1× PBS, and the cells were resuspended in 30 μl / well of 1% BSA / 1× PBS. Finally, the mean fluorescence intensity was detected by flow cytometry, and the four-parameter curve was fitted to calculate the EC50 value. The results shown in Table 1 and Figures 1A and 1B were obtained, where BMK1 was used as a positive control and human IgG4 ctrl was used as a negative control.

[0114] 1.8 Screening for molecules that block hIL-13 activity using reporter gene assay

[0115] The 10 best-performing mouse antibodies (see Table 1) were selected for the complete reporter gene assay. HEK-293T.STAT6-GFP cells were prepared and resuspended in TransDetect BrightFluore DMEM + 10% FBS medium. The cell density was adjusted to 5E5 cells / ml. 100 μl was added to a black 96-well plate at a concentration of 100 μl / well and incubated at 37°C for 24 hours. hIL-13 was diluted to 20.0 ng / ml in TransDetect BrightFluore DMEM + 10% FBS medium. The antibody was diluted with the diluted hIL-13 solution in a 1:4 serial dilution series of 11 concentrations. 100 μl was added to each well of the 96-well plate, gently mixed, and incubated at 37°C for an additional 24 hours. The fluorescence signal intensity was detected by a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. A four-parameter curve was fitted to calculate the IC50 value. The results shown in Table 1 and Figures 2A and 2B were obtained, where BMK1 was used as a positive control and human IgG4 ctrl was used as a negative control.

[0116] 1.9 Affinity detection

[0117] Based on the above data analysis, the two most active molecules were selected for affinity testing. The binding affinities (KD) of the two antibodies for hIL-4Rα were determined using real-time surface plasmon resonance analysis (Cytiva, Biacore 8K) at 25°C. Anti-mouse Fc IgG antibodies were first coupled to a CM5 chip to form an antibody capture surface. The two selected antibodies were then further coupled to anti-mouse Fc IgG antibodies at a flow rate of 10 μl / min for 30 s. The analyte (hIL-4Rα.ECD) was diluted in a 1:2 dilution series of six concentrations and assayed sequentially. The association time was 180 s, the dissociation time was 1800 s, and the flow rate was 10 μl / min. Finally, kinetic analysis was performed using BIA analysis software to determine the equilibrium binding constants (Ka), equilibrium dissociation constants (Kd), and affinity constants (KD). The results are shown in Table 1. Both molecules displayed high affinity for hIL-4Rα.

[0118] Table 1 Activity and affinity data of molecules

[0119] 1.10 Sequencing of variable regions of candidate mouse antibodies

[0120] The above two mouse monoclonal antibody molecules (1.34.4-mouse, 2.46.13-mouse) were sequenced to confirm their sequence information and perform sequence analysis. The sequence information is shown in Table 2.

[0121] Table 2 CDR sequence list of mouse monoclonal antibody molecules

[0122] 1.34.4- The sequence of the heavy chain variable region of the murine monoclonal antibody molecule is:

[0123] 1.34.4- The sequence of the light chain variable region of the mouse monoclonal antibody molecule is:

[0124] 2.46.13- The sequence of the heavy chain variable region of the murine monoclonal antibody molecule is:

[0125] 2.46.13- The sequence of the light chain variable region of the mouse monoclonal antibody molecule is:

[0126] Example 2: Antibody humanization and affinity evaluation

[0127] The variable region sequences of two mouse monoclonal antibodies, 1.34.4-mouse and 2.46.13-mouse, were aligned with human germline antibody sequences to identify sequences with high homology for CDR transplantation. The heavy chain CDR region of 1.34.4-mouse was transplanted into the framework region of IGHV1-46*01+IGHJ1*01, and the light chain CDR region was transplanted into the framework region of IGKV1-39*01+IGKJ2*01. The heavy chain CDR region of 2.46.13-mouse was transplanted into the framework region of IGHV3-21*01+IGHJ6*01, and the light chain CDR region was transplanted into the framework region of IGKV1-39*01+IGKJ2*01. The specific sequences are shown in Tables 3-1 and 3-2. Computer-based homology modeling was then performed to analyze the amino acid sequences of the CDR and framework regions, predicting key amino acid sites within the sequence that may interact with the hIL-4Rα protein and stabilize its spatial structure. Based on this, reverse mutation sites were designed and screened for reverse mutations. The humanized variable region sequence after the designed reverse mutations was linked to the human IgG4 constant region. Antibody expression (expression methods refer to the literature doi:10.1016 / j.ejbt.2019.07.002) and affinity evaluation were then performed using the same methods as in 1.9. Based on the affinity data, the 1.34.4-z18 humanized antibody molecule, which underwent reverse mutations, was ultimately selected for affinity maturation. The sequence of 1.34.4-z18 is shown in Tables 3-1 and 3-2, and the affinity data are shown in Table 4.

[0128] Table 3-1 CDR sequence list of humanized monoclonal antibody molecules

[0129] Table 3-2 Sequence list of variable regions of humanized monoclonal antibody molecules

[0130] Table 4 Affinity data of humanized antibody molecules

[0131] Example 3: Affinity Maturation

[0132] 3.1 Single-site saturation mutation

[0133] Using saturation mutagenesis, every CDR site of the 1.34.4-z18 humanized antibody was saturated to create a Fab single-point mutation library (>5,000 molecules). This library was screened for binding activity using competitive ELISA. The top 15 Fab-positive clones were then subjected to single-point affinity testing and sequencing. Mutants with a 2-10-fold reduction in dissociation constant were selected for subsequent combinatorial mutagenesis.

[0134] 3.2 Combinatorial Mutant Molecular Affinity Screening

[0135] After confirming the sites with the most significant affinity improvement, these sites were subjected to combinatorial mutation screening. The resulting molecular dissociation constants were all less than 1.00E-05 (1 / s), which was below the instrument detection limit, and the affinities were all less than 9.43E-11 (M), indicating that all affinity-matured molecules can bind to the hIL-4Rα protein with high affinity. Finally, the m8, m21, and m27 molecules were screened for cell activity and affinity evaluation. The specific sequences are shown in Table 5.

[0136] Table 5 CDR sequences of monoclonal antibody molecules after affinity maturation

[0137] The heavy chain variable region sequence of the affinity matured m8 molecule is:

[0138] The light chain variable region sequence of the affinity matured m8 molecule is:

[0139] The heavy chain variable region sequence of the affinity matured m21 molecule is:

[0140] The light chain variable region sequence of the affinity matured m21 molecule is:

[0141] The heavy chain variable region sequence of the affinity matured m27 molecule is:

[0142] The light chain variable region sequence of the affinity matured m27 molecule is:

[0143] 3.3 Reporter gene assay to detect antibodies that block hIL-4 binding to hIL-4Rα protein

[0144] The aforementioned molecules were further tested for cellular activity using a reporter gene assay. HEK-293T.STAT6-GFP cells were prepared and resuspended in TransDetect BrightFluore DMEM + 10% FBS medium at a cell density of 5E5 cells / ml. 100 μl / well of the solution was added to a black 96-well plate and incubated at 37°C for 24 hours. hIL-4 was diluted to 1.0 ng / ml in TransDetect BrightFluore DMEM + 10% FBS medium. Antibodies were diluted with the diluted hIL-4 solution in a 1:4 dilution series of 11 concentrations, and 100 μl / well of the solution was added to the 96-well plate. Mixing was performed gently, and the cells were incubated at 37°C for another 24 hours. Fluorescence signal intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Four-parameter curve fitting was used to obtain the results shown in Figure 3 and Table 6. BMK1 was used as a positive control. All the molecules showed significant inhibition of hIL-4 activity.

[0145] Table 6 Reporter gene assay to detect the activity of hIL-4

[0146] 3.4 Surface Plasmon Resonance Detection of the Affinity of Candidate Molecules for hIL-4Rα Protein

[0147] First, an anti-human Fc IgG antibody was coupled to a CM5 chip to form an antibody capture surface. The screened antibodies were then further coupled to the anti-human Fc IgG antibody at a flow rate of 10 μl / min for 30 seconds. The analyte (hIL-4Rα.ECD) was diluted in six 1:2 dilutions and assayed sequentially. The association time was 180 seconds, the dissociation time was 1800 seconds, and the flow rate was 10 μl / min. Finally, kinetic analysis was performed using BIA analysis software to determine the equilibrium binding constant, ka, equilibrium dissociation constant, kd, and affinity constant, KD. The results are shown in Table 7. BMK1 was used as a positive control molecule, and the candidate molecules had comparable affinities to the control molecule.

[0148] Table 7 Surface plasmon resonance assay for the affinity of candidate molecules to hIL-4Rα protein

[0149] 3.5 TF-1 cell proliferation inhibition assay to detect the ability of candidate molecules to inhibit hIL-13 activity

[0150] m8, m21, and m27 were further evaluated using a proliferation inhibition assay. TF-1 cells were adjusted to a density of 5E5 cells / ml using RPMI1640 medium with 5% FBS. 100 μl / well of the solution was added to a 96-well plate. hIL-13 was diluted to 30 ng / ml using culture medium. The antibody was diluted with the diluted hIL-13 solution in a 1:4 dilution series of 11 concentrations, and 50 μl / well of the solution was added to the 96-well plate. Mix gently and incubate at 37°C for 72 hours. The 96-well plate was removed and 100 μl / well of CellTiter-Glo colorimetric solution was added. The reaction was allowed to proceed at room temperature for 15 minutes. Luminescence intensity was measured using a microplate reader. The results are shown in Table 7. BMK1 was used as a positive control. The m21 and m27 antibodies showed superior inhibition of hIL-13-induced TF-1 cell proliferation compared to BMK1. The m8 antibody exhibited comparable blocking activity to BMK1.

[0151] Table 8 Data on blocking hIL-13 activity detected by proliferation inhibition assay

[0152] Example 4: Effects of m8 and m21 on OVA-induced B-hIL4 / IL-4Rα mouse asthma model

[0153] OVA (ovalbumin) was used to induce B-hIL4 / IL-4Rα mice to establish a mouse asthma model. D0 was the first day of model establishment. On D0 and D14, 10 μg OVA was injected intraperitoneally for sensitization, and 5% OVA was challenged by nasal drops from D21 to D24. The experimental mice were divided into 4 groups, including a negative control group NC, a positive control group BMK1, and experimental groups m8 and m21, with 8 mice in each group. The dose was 25 mg / kg, and the administration method was subcutaneous injection. The mice were given on D7, D10, D14, D17, D21, and D24. During the period, the weight changes of mice in each group were measured, and on D25, the blood, bronchoalveolar lavage fluid (BALF) and lung tissue of mice in each group were collected for index detection.

[0154] The experimental results are shown in Figures 4A and 4B. Compared with the NC control group, the number of BALF-derived lymphocytes and eosinophils in the experimental group and the positive control group was significantly reduced, with m8 and m21 decreasing more. At the same time, the OVA-specific IgE content in plasma was detected. As shown in Figure 4C, the IgE content in the experimental group and the positive control group was significantly reduced compared with the NC control group.

[0155] Example 5: Analysis of molecular physicochemical properties

[0156] The purity of the samples obtained by one-step Protein A purification was detected by size exclusion method. The results are shown in Figure 5. After integration treatment, the monomer content was greater than 98%, indicating that the above molecules had good stability during the expression and purification process and were not easy to form aggregates. The hydrophobicity of the m8 molecule was evaluated by hydrophobic chromatography. The results are shown in Figure 6. The control is a low-hydrophobic antibody molecule with a faster peak elution and a shorter retention time. The retention time of the m8 molecule is significantly lower than that of the positive antibody BMK1. Therefore, the hydrophobicity is significantly lower than that of the positive antibody BMK1, which has a great advantage in subsequent process development.

[0157] Example 6: Preparation of anti-TSLP antibody Tezepelumab

[0158] The sequence of the marketed anti-TSLP monoclonal antibody Tezepelumab is derived from Chinese patent CN101809035A, and the nucleic acid sequence was commissioned to be synthesized by GenScript.

[0159] The amino acid sequence of the Tezepelumab heavy chain variable region (TezeHv) is:

[0160] The amino acid sequence of the Tezepelumab heavy chain constant region is:

[0161] The amino acid sequence of the Tezepelumab light chain variable region (TezeLv) is:

[0162] The amino acid sequence of the tezepelumab light chain constant region is:

[0163] The amino acid sequence of the Tezepelumab heavy chain (Tezepelumab-HC) is:

[0164] The amino acid sequence of the tezepelumab light chain (Tezepelumab-LC) is:

[0165] The heavy and light chain cDNA sequences of tezepelumab were cloned into the pKS001 vector to generate recombinant expression plasmids expressing tezepelumab. The recombinant plasmids were transiently transfected into ExpiCHOS cells (Thermo Fisher Scientific), and the resulting culture medium was purified by Protein A affinity chromatography and then tested.

[0166] Example 7: Preparation of anti-IL-4Rα antibody Dupilumab

[0167] The amino acid sequence of the dupilumab heavy chain (Dup-HC) is:

[0168] The amino acid sequence of dupilumab light chain (Dup-LC) is:

[0169] The heavy and light chain cDNA sequences of dupilumab were cloned into the pKS001 vector to generate recombinant expression plasmids expressing dupilumab. The recombinant plasmids were transiently transfected into ExpiCHOS cells (Thermo Fisher Scientific), and the resulting culture medium was purified by Protein A affinity chromatography and then tested.

[0170] Example 8: Preparation of anti-IL-4R antibody m8

[0171] The amino acid sequence of the heavy chain variable region (m8Hv) of the m8 molecule is:

[0172] The amino acid sequence of the heavy chain constant region of the m8 molecule is:

[0173] The amino acid sequence of the light chain variable region (m8Lv) of the m8 molecule is:

[0174] The amino acid sequence of the light chain constant region of the m8 molecule is:

[0175] The amino acid sequence of the heavy chain (m8-HC) of the m8 molecule is:

[0176] The amino acid sequence of the light chain (m8-LC) of the m8 molecule is:

[0177] The heavy and light chain cDNA sequences of m8 were cloned into the pKS001 vector to generate recombinant expression plasmids expressing m8. The recombinant plasmids were transiently transfected into ExpiCHOS cells (Thermo Fisher Scientific), and the resulting culture medium was purified by Protein A affinity chromatography and then tested.

[0178] Example 9: Sequence design of bispecific antibodies

[0179] 1. Sequence Design

[0180] The bispecific antibody structure of the present invention is an IgG-scFv model, in which the C-termini of the two heavy chains of a complete IgG antibody are connected to the scFv fragment of another antibody. The main components of the heavy chain and light chain are shown in Table 9.

[0181] Based on molecular stability and activity considerations, mutations were introduced into the light and heavy chain variable regions of Tezepelumab and m8 molecules. The specific sequences are as follows:

[0182] The mutated heavy chain variable region sequence of Tezepelumab (MtezeHv) is:

[0183] The mutated light chain variable region sequence of Tezepelumab (MtezeLv) is:

[0184] The sequence of the heavy chain variable region of m8 after mutation (Mm8Hv) is:

[0185] The sequence of the light chain variable region of m8 after mutation (Mm8Lv) is:

[0186] The amino acid sequence of Linker1 is GGGGSGGGGS; (SEQ ID NO: 46)

[0187] The amino acid sequence of Linker2 is GGGGSGGGGSGGGGSGGGGS; (SEQ ID NO: 47)

[0188] Table 9-1 Amino acid sequences of the complementarity determining regions of the immunoglobulin portion of MTM and MMT

[0189] Table 9-2 Composition of heavy and light chains of MTM and MMT

[0190] The amino acid sequence of the MTM heavy chain (MTM-HC) is:

[0191] The amino acid sequence of the MTM light chain (MTM-LC) is:

[0192] The amino acid sequence of the MMT heavy chain (MMT-HC) is:

[0193] The amino acid sequence of the MMT light chain (MMT-LC) is:

[0194] 2. Antibody Expression and Purification

[0195] The heavy and light chain cDNA sequences of the above bispecific antibody sequences were cloned into the pKS001 vector to generate recombinant expression plasmids expressing the bispecific antibody. The recombinant plasmids were transiently transfected into ExpiCHOS cells (Thermo Fisher Scientific), and the resulting culture medium was purified by Protein A affinity chromatography and then tested.

[0196] 3. Antibody testing

[0197] The antibody purity was detected by SDS-PAGE. The specific results are shown in Figure 7. Both molecules can reach an electrophoretic purity of more than 95%.

[0198] Example 10: Affinity constant determination

[0199] 1. Affinity Determination of Four Bisspecific Antibodies to hIL-4Rα

[0200] The binding affinities (KD) of the two antibodies for hIL-4Rα were determined using real-time surface plasmon resonance analysis (Biacore 8K) at 25°C. Anti-mouse Fc IgG antibodies were first coupled to a CM5 chip to form an antibody capture surface. The antibodies were then further coupled to anti-mouse Fc IgG antibodies at a flow rate of 10 μL / min for 30 s. The analyte (hIL-4Rα.ECD) was diluted in a 1:2 dilution series of six concentrations and assayed sequentially. The association time was 180 s, the dissociation time was 1800 s, and the flow rate was 30 μL / min. Kinetic analysis was performed using BIA analysis software to determine the equilibrium association constants (Ka), equilibrium dissociation constants (Kd), and affinity constants (KD). The results are shown in Table 10. The affinities of the two constructed bispecific antibodies for hIL-4Rα were comparable to those of the control molecules m8 and dupilumab, demonstrating their high specificity.

[0201] Table 10 Affinity data of antibodies to hIL-4Rα

[0202] 2. Determination of the affinity of the bispecific antibody molecule to hTSLP

[0203] The binding affinities (KD) of the two antibodies for hTSLP were determined using real-time surface plasmon resonance analysis (Biacore 8K) at 25°C. Anti-mouse Fc IgG antibodies were first coupled to a CM5 chip to form an antibody capture surface. The antibodies were then further coupled to anti-mouse Fc IgG antibodies at a flow rate of 10 μL / min for 30 s. The analyte (hTSLP) was diluted in a 1:2 dilution series of six concentrations and assayed sequentially. The association time was 180 s, the dissociation time was 1800 s, and the flow rate was 30 μL / min. Kinetic analysis was performed using BIA analysis software to determine the equilibrium binding constants Ka, Kd, ​​and KD. The results are shown in Table 11. The two bispecific antibodies maintained comparable affinity to the control molecule, tezepelumab.

[0204] Table 11 Affinity data of antibodies to hTSLP

[0205] 3. MTM and MMT antibodies simultaneously bind to hIL-4Rα and hTSLP antigens

[0206] Real-time biosensor surface plasmon resonance analysis (Biacore 8K) was used to investigate the ability of MTM and MMT molecules to simultaneously bind to hIL-4Rα and hTSLP antigens, respectively, at 25°C. Anti-mouse Fc IgG antibodies were first coupled to a CM5 chip to form an antibody capture surface. MTM and MMT antibodies were then further coupled to the anti-mouse Fc IgG antibodies at a flow rate of 10 μL / min for 30 seconds. Subsequently, the analytes hIL-4Rα and hTSLP were sequentially flowed through the chip for binding ability testing. The binding time was 180s. The binding spectrum determined that the two antibody molecules retained the ability to bind to hTSLP after binding to the hIL-4Rα antigen. The spectrum is shown in Figure 8. Figure 8A shows that after coupling with the MMT molecule, hIL-4Rα and hTSLP molecules were continuously injected, and MMT was able to bind. Figure 8B shows that after coupling with the MTM molecule, hIL-4Rα and hTSLP molecules were continuously injected, and MTM was able to bind. This indicates that the MTM and MMT antibody molecules can simultaneously bind to the hIL-4Rα and hTSLP antigens.

[0207] Example 11: Evaluation of IL-4 and IL-13 Cell Activity

[0208] 1. Reporter gene assay (HEK-293T.STAT6-GFP cells) to evaluate the ability of bispecific antibodies to inhibit IL-4 activity

[0209] HEK-293T.STAT6-GFP cells were prepared and resuspended in TransDetect BrightFluore DMEM + 10% FBS medium, adjusting the cell density to 5E5 cells / mL. 100 μL / well of the solution was added to a black 96-well plate and incubated at 37°C for 24 hours. hIL-4 was diluted to 1.0 ng / mL in TransDetect BrightFluore DMEM + 10% FBS medium. The antibody was diluted with the diluted hIL-4 solution in a 1:4 dilution series of 11 concentrations, and 100 μL / well of the solution was added to the 96-well plate. Gently mix and incubate at 37°C for another 24 hours. Fluorescence signal intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The results shown in Table 12 show that MTM and the positive control molecule m8 exhibit comparable inhibitory activity against hIL-4.

[0210] Table 12 Data on inhibition of hIL-4 activity by reporter gene method

[0211] 2. Reporter gene assay (HEK-293T.STAT6-GFP cells) to evaluate the ability of bispecific antibodies to inhibit IL-13 activity

[0212] HEK-293T.STAT6-GFP cells were prepared and resuspended in TransDetect BrightFluore DMEM + 10% FBS medium to a cell density of 5E5 cells / mL. 100 μL / well of the solution was added to a black 96-well plate and incubated at 37°C for 24 hours. hIL-13 was diluted to 30 ng / mL in TransDetect BrightFluore DMEM + 10% FBS medium. The antibody was diluted with the diluted hIL-4 solution in a 1:4 dilution series of 11 concentrations, and 100 μL / well of the solution was added to the 96-well plate. Mix gently and incubate at 37°C for another 24 hours. Fluorescence signal intensity was measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The results shown in Table 13 show that MMT and MTM exhibit comparable inhibitory activity against hIL-13 as the positive control molecules m8 and dupilumab.

[0213] Table 13 Data on inhibition of hIL-13 activity by reporter gene method

[0214] Example 12: Evaluation of the Ability of Bispecific Antibodies to Inhibit TSLP Activity by Reporter Gene Assay (HEK-293T-hTSLPR-IL7Ra-STAT5-Luciferase Cells)

[0215] Prepare HEK-293T-hTSLPR-IL7Ra-STAT5-Luciferase cells and resuspend them in DMEM + 10% FBS medium to adjust the cell density to 5E5 cells / mL. Add 100 μL / well to a black 96-well plate and incubate at 37°C for 24 hours. Dilute hTSLP to 5 ng / mL in DMEM + 10% FBS medium. Dilute the antibody molecule with the diluted hTSLP solution in a 1:3 dilution series of 11 concentrations. Add 100 μL / well to the 96-well plate, gently mix, and incubate at 37°C for another 24 hours. Remove the luminescent substrate and place it at room temperature. After the sample incubation is complete, remove the 96-well plate and place it at room temperature for 10 minutes. Then, add 100 μL of the luminescent substrate to each well and incubate at room temperature for 2 minutes in the dark. Luminescent signal intensity is measured using a microplate reader. The results shown in Table 14 show that MTM and MMT molecules have comparable inhibitory effects on hTSLP activity as the positive control molecule, tezepelumab.

[0216] Table 14 Data on inhibition of hTSLP activity by reporter gene method

[0217] Example 13: Ability of MTM Antibodies to Inhibit Chemokine 17 (CCL17) Expression in Human PBMC Cells

[0218] Human PBMC cells were purchased from Shanghai Aoneng Biotechnology Co., Ltd. Human PBMC cells were revived using RPMI1640 + 10% FBS medium, and then cultured at 37 ° C for 2 hours. After cell activation was completed, the cells were washed twice with RPMI1640 medium without FBS, and then resuspended with RPMI1640 medium without FBS and counted. 96-well plates were plated at 100,000 cells per well, with 100 μL per well. IL-13 and TSLP were diluted with RPMI1640 medium without FBS to a concentration of 20 ng / mL. The antibody molecules were diluted to an equimolar concentration with diluent, and then the drug solution was added to the corresponding wells. The cells were then cultured for 48 hours at 37 ° C. The cell culture supernatant was collected and the CCL17 content in the supernatant was detected by sandwich ELISA. The test results are shown in Figure 9. PBMC cells can induce different degrees of CCL17 secretion under the stimulation of IL-13 and TSLP, respectively. The stimulation effect is the strongest when IL-13 and TSLP are added at the same time. When IL-13 and TSLP are stimulated at the same time, Tezepelumab (Teze) and Dupilumab (Dup) are administered respectively. Compared with the control group without antibody addition, the monoclonal antibodies can partially inhibit the secretion of CCL17. Simultaneous administration of Tezepelumab (Teze) and Dupilumab (Dup), or Tezepelumab (Teze) and m8 can significantly inhibit the secretion of CCL17. The administration of MTM dual antibody alone can achieve an inhibitory effect that is even better than that of the combined administration of monoclonal antibodies.

[0219] Example 14: In vivo efficacy evaluation of MTM antibody in B-hIL4 / IL4R humanized mouse asthma model

[0220] OVA (chicken ovalbumin, Sigma, catalog number A5503) was used as an inducer to establish an asthma model in B-hIL4 / IL4R humanized mice (doi.org / 10.1016 / j.imbio.2020.151998). The experimental mice were divided into 4 groups, 4 animals in G1 (non-modeling group), and 6 animals in each group of G2-G4. Among them, the G1 group was the blank control group, the G2 group was the isotype control group, the G3 group was the m8 control group, and the dosage was 25 mg / kg. The G4 group was the MTM test group, and the dosage was 34 mg / kg. The administration method was intraperitoneal administration. The administration started on the 13th day of modeling, and the administration time points were 13, 16, 20 and 23 days, for a total of 4 administrations. On the 26th day after the end of administration, the animals were bled and dissected for analysis of eosinophil and IgE levels. The experimental results are shown in Figure 10. The results show that the MTM molecule has a pharmacodynamic activity comparable to that of the m8 control molecule, indicating that the MTM molecule has a potential for asthma treatment comparable to that of the monoclonal antibody m8 in the in vivo efficacy evaluation targeting the hIL4R target pathway.

[0221] Example 15: In vivo efficacy evaluation of MTM antibody in the B-hTSLP / TSLPR / hIL7R humanized mouse asthma model

[0222] An asthma model was established in B-hTSLP / TSLPR / hIL7R humanized mice using TSLP (ACRO, Catalog No. TSP-H52Hb) + OVA (chicken ovalbumin, Sigma, Catalog No. A5503) as inducers. Animals in group G1 received intranasal instillations of 20 μL of PBS, while animals in groups G2-G4 received 20 μL of a mixture of TSLP + OVA (1 μg + 10 μg) on ​​days 0, 2, 4, 6, 8, 10, and 12, for a total of seven intranasal inductions. Group G1 served as the blank control, group G2 as the isotype control, group G3 as the tezepelumab (10 mg / kg) control, and group G4 as the test article. Administration was intraperitoneally administered twice, on days 1 and 6, respectively. After administration, blood was collected from the animals and the animals were dissected for analysis of eosinophil and IgE levels. The experimental results are shown in Figure 11. The results show that the MTM molecule has a pharmacodynamic activity comparable to that of the Tezepelumab control molecule, indicating that the MTM molecule has a potential for treating asthma comparable to that of the monoclonal antibody Tezepelumab in in vivo efficacy evaluation in terms of its action pathway against the hTSLP target.

[0223] Example 16: Preparation of MTM high concentration samples

[0224] 1. Construction of cells stably expressing MTM molecules

[0225] The heavy and light chain cDNA sequences of the MTM molecule were cloned into the pKS001 vector to generate recombinant expression plasmids expressing the dual antibody. Using MSX pressure screening, the recombinant plasmids were transfected into CHO-K1 cells (Merck). Stable cell pools expressing MTM were obtained by pressure screening. These stable cell pools were then fermented, and the fermentation supernatant expressing MTM was harvested.

[0226] 2.MTM molecular ultrafiltration concentration

[0227] The fermentation supernatant was purified by Protein A affinity chromatography and samples with a purity greater than 95% were collected. The samples were then concentrated by ultrafiltration using a 30 kDa ultrafiltration tube and replaced with 20 mM sodium acetate, pH 5.0, and 100 mM L-proline buffer. The final concentration could be greater than 100 mg / mL.

[0228] Example 17: Stability evaluation experiment

[0229] The concentrated sample was diluted to 100 mg / mL and 10 mg / mL using 20 mM sodium acetate, pH 5.0, plus 100 mM L-proline buffer, and stability studies were performed at 4°C, 25°C, and 40°C. The concentration was 100 mg / mL at the first two temperatures, and 10 mg / mL at 40°C. The purity changes of the samples were detected by SEC-HPLC. The test results are shown in Table 15. After 4 weeks of high-temperature acceleration, the monomer purity changed by less than 5%. At 4°C and 25°C, the monomer content decreased by less than 2%, indicating that the MTM bispecific antibody molecule has good stability.

[0230] Table 15 Stability evaluation results

[0231] It is understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present invention, but the present invention is not limited thereto. Those skilled in the art may make various modifications and improvements without departing from the principles and essence of the present invention, and such modifications and improvements shall also be considered as protections of the present invention.

Claims

1. An antibody to the α-subunit of the interleukin-4 receptor (Latin IL-4Rα) or an antigen-binding fragment thereof, characterized in that it contains a complementarity-determining region of the heavy chain and a complementarity-determining region of the light chain, the heavy chain complementarity determining region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 12; HCDR2 is the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 10 or SEQ ID NO: 13; HCDR3 is the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 14; The light chain complementarity determining region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 is the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 15; LCDR2 is the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 16; LCDR3 is the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 11 or SEQ ID NO:

17.

2. An antibody to IL-4Rα or an antigen-binding fragment thereof according to claim 1, characterized in that it contains (1) a heavy chain complementarity determining region comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; a light chain complementarity determining region comprising LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 6; or (2) a heavy chain complementarity determining region comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 7, and HCDR3 as shown in SEQ ID NO: 3; a light chain complementarity determining region comprising LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 8; or (3) a heavy chain complementarity determining region comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 7, and HCDR3 as shown in SEQ ID NO: 9; a light chain complementarity determining region comprising LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 8; or (4) a heavy chain complementarity determining region comprising HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 10, and HCDR3 as shown in SEQ ID NO: 3; a light chain complementarity determining region comprising LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO: 11; or (5) a heavy chain complementarity determining region comprising HCDR1 as shown in SEQ ID NO: 12, HCDR2 as shown in SEQ ID NO: 13, and HCDR3 as shown in SEQ ID NO: 14; a light chain complementarity determining region comprising LCDR1 as shown in SEQ ID NO: 15, LCDR2 as shown in SEQ ID NO: 16, and LCDR3 as shown in SEQ ID NO:

17.

3. An antibody to IL-4Rα or an antigen-binding fragment thereof according to claim 1, characterized in that it additionally contains framework region sequences derived from the human germline heavy chain IGHV1-46*01+IGHJ1*01, or reverse-mutated sequences at least 90% identical thereto; or framework region sequences derived from the human germline light chain IGKV1-39*01+IGKJ2*01, or reverse-mutated sequences at least 90% identical thereto.

4. An antibody to IL-4Rα or an antigen-binding fragment thereof according to claim 1, characterized in that the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, a bispecific antibody, or an antigen-binding fragment thereof.

5. An antibody to IL-4Rα or an antigen-binding fragment thereof according to claim 1, characterized in that it contains (1) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29; (2) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 31; (3) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 33; (4) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19; or (5) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

21.

6. An antibody to IL-4Rα or an antigen-binding fragment thereof according to claim 5, characterized in that it contains an amino acid sequence with at least 90% identity.

7. A fusion protein comprising an antibody to IL-4Rα or an antigen-binding fragment thereof according to any one of claims 1-6.

8. A polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-6.

9. An expression vector containing the polynucleotide according to claim 8.

10. A host cell comprising the vector of claim 8, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

11. A pharmaceutical composition comprising an antibody to IL-4Rα or an antigen-binding fragment thereof according to any one of paragraphs 1-6 or a fusion protein according to paragraph 7, and pharmaceutically acceptable excipients.

12. The use of an antibody to IL-4Rα or an antigen-binding fragment thereof according to any of paragraphs 1-6, a fusion protein according to paragraph 7, or a pharmaceutical composition according to paragraph 11 in the preparation of a medicament for the treatment of an allergic disease.

13. The use according to claim 12, wherein the allergic disease is atopic dermatitis, asthma, allergic rhinitis, certain cases of chronic nasosinusitis with nasal polyps or chronic obstructive pneumonia.

14. A bispecific antibody to IL-4Rα and thymic stromal lymphopoietin (TSLP), characterized in that the bispecific antibody includes an antibody to IL-4Rα or an antigen-binding fragment thereof, selected from any of claims 1-6.

15. The bispecific antibody according to claim 14, characterized in that the bispecific antibody contains immunoglobulin IgG and a single-chain antibody of the scFv type, wherein the bispecific antibody is selected from one of the following groups: (1) the immunoglobulin IgG comprises an antibody to TSLP or an antigen-binding fragment thereof, and the single-chain antibody of the scFv type comprises an antibody to IL-4Rα or an antigen-binding fragment thereof, selected from any of claims 1-6; or (2) the immunoglobulin IgG comprises an antibody to IL-4Rα or an antigen-binding fragment thereof, selected from any of paragraphs 1-6, and the single-chain antibody of the scFv type comprises an antibody to TSLP or an antigen-binding fragment thereof.

16. The bispecific antibody of claim 15, wherein the antibody to TSLP or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 is the amino acid sequence of SEQ ID NO: 48; HCDR2 is the amino acid sequence of SEQ ID NO: 49; HCDR3 is the amino acid sequence of SEQ ID NO: 50; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 is the amino acid sequence of SEQ ID NO: 51; the amino acid sequence of LCDR2 is DDS; LCDR3 is the amino acid sequence of SEQ ID NO:

52.

17. The bispecific antibody according to claim 15, characterized in that the antibody to IL-4Rα or its antigen-binding fragment contains a variable region of a heavy chain and a variable region of a light chain, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 is the amino acid sequence of SEQ ID NO: 1; HCDR2 is the amino acid sequence of SEQ ID NO: 2; HCDR3 is the amino acid sequence of SEQ ID NO: 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 is the amino acid sequence of SEQ ID NO: 4; LCDR2 is the amino acid sequence of SEQ ID NO: 5; LCDR3 is the amino acid sequence of SEQ ID NO:

6.

18. The bispecific antibody of claim 15, wherein the antibody to TSLP or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the variable region of the heavy chain has the amino acid sequence of SEQ ID NO: 42, and the variable region of the light chain has the amino acid sequence of SEQ ID NO: 43; or the variable region of the heavy chain has the amino acid sequence of SEQ ID NO: 34, and the variable region of the light chain has the amino acid sequence of SEQ ID NO:

36.

19. The bispecific antibody according to claim 15, characterized in that the antibody to IL-4Rα or its antigen-binding fragment contains a variable region of the heavy chain and a variable region of the light chain, and the variable region of the heavy chain has the amino acid sequence of SEQ ID NO: 44, and the variable region of the light chain has the amino acid sequence of SEQ ID NO: 45; or the variable region of the heavy chain has the amino acid sequence of SEQ ID NO: 28, and the variable region of the light chain has the amino acid sequence of SEQ ID NO:

29.

20. The bispecific antibody according to claim 15, characterized in that the immunoglobulin IgG and the single-chain antibody of the scFv type are connected via a linker, wherein the linker is (GGGGS)n, where n is a positive integer from 1 to 5, preferably the amino acid sequence presented under SEQ ID NO:

46.

21. The bispecific antibody according to claim 15, characterized in that the single-chain antibody of the scFv type further comprises a linker connecting the variable region of the heavy chain to the variable region of the light chain of the scFv, wherein the linker is (GGGGS)n, where n is a positive integer from 1 to 5, preferably the amino acid sequence presented under SEQ ID NO:

47.

22. A bispecific antibody according to claim 15, characterized in that the bispecific antibody is selected from one of the following groups: (1) the IgG immunoglobulin comprises a heavy chain variable region as set forth in SEQ ID NO: 34, a heavy chain constant region as set forth in SEQ ID NO: 35, a light chain variable region as set forth in SEQ ID NO: 36, and a light chain constant region as set forth in SEQ ID NO: 37, wherein the scFv-type single-chain antibody portion comprises a heavy chain variable region as set forth in SEQ ID NO: 44 and a light chain variable region as set forth in SEQ ID NO: 45; or (2) the IgG immunoglobulin comprises a heavy chain variable region as set forth in SEQ ID NO: 28, a heavy chain constant region as set forth in SEQ ID NO: 40, a light chain variable region as set forth in SEQ ID NO: 29, and a light chain constant region as set forth in SEQ ID NO: 41, wherein the scFv-type single-chain antibody portion comprises a heavy chain variable region as set forth in SEQ ID NO: 42 and a light chain variable region as set forth in SEQ ID NO:

43.

23. The bispecific antibody according to claim 22, characterized in that the bispecific antibody is selected from one of the following groups: (1) the bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 57 and a light chain as shown in SEQ ID NO: 58; or (2) the bispecific antibody comprises a heavy chain as shown in SEQ ID NO: 59 and a light chain as shown in SEQ ID NO:

60.

24. A pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 14-23 and pharmaceutically acceptable excipients.

25. The use of a bispecific antibody according to any one of claims 14 to 23 or a pharmaceutical composition according to claim 24 in the preparation of a medicament for the treatment of moderate to severe asthma or moderate to severe atopic dermatitis, allergic rhinitis, certain cases of chronic nasosinusitis with nasal polyps or chronic obstructive pulmonary diseases.