Anti-CD132 antibodies, and use thereof

Abstract

Provided in the present invention are humanized anti-human CD132 monoclonal antibodies and the use thereof. The present invention performs humanized transformation on the basis of monoclonal chimeras 2D4 and 5H10, so as to obtain high-affinity humanized monoclonal antibodies h2D4H4K12 and h5H10H6K4 targeting the human CD132 by means of screening; therefore, without attenuating the antibody activity, the immunogenicity of parental chimeras is reduced, thus reducing the possible risk that medicated patients may generate immune responses on the antibodies. The monoclonal antibodies can be used for prevention, neutralization or treatment of autoimmune diseases related to IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokines, e.g. systemic lupus erythematosus, rheumatoid arthritis, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease and aplastic anemia.

Description

Anti-CD132 antibodies and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-CD132 antibody and an application thereof. Background Art

[0002] CD132 (IL2Rγ), the γ subunit of the interleukin-2 (IL-2) receptor, is also the γ subunit of the receptor complexes for the following six cytokines: IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Therefore, it is also known as γc (common γ chain). Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organs and systems. γ-chain cytokines, including IL-4, IL-7, IL-9, IL-15, and IL-21, are significantly upregulated in immune cells in SLE. These γ-chain cytokines play a key role in regulating the survival, proliferation, and differentiation of various lymphocytes. Given that SLE is a complex disease involving multiple cells and multiple factors, and that γ-chain cytokines play a key regulatory role in various lymphocytes, which is closely related to the occurrence and development of SLE, intervening in the functional activity of CD132, the common subunit of γ-chain cytokine receptors, may become a potential SLE treatment method.

[0003] In addition, the six γ-chain cytokines upstream of CD132 also play a key role in other different inflammatory diseases. Among them, the role of blocking the IL-21 pathway in autoimmune diseases has been widely studied and applied. IL-21 directly acts on T and B cells, promoting T cell activation, B cell proliferation, differentiation and antibody production. Therefore, blocking IL-21 has a therapeutic effect in various autoimmune diseases caused by T and B cell dysfunction. For example, in rheumatoid arthritis (RA), IL-21 is involved in the development of destructive arthritis. Inhibiting the IL21 signaling pathway has shown certain efficacy in the treatment of RA, which can reduce joint inflammation and improve joint function [1]. In addition, inhibiting IL21 and IL7 has shown efficacy in inflammatory bowel disease (IBD) [2,3]. Many literatures suggest that the IL15 signaling pathway plays a key role in the occurrence and development of vitiligo, and blocking the IL15 signaling pathway can relieve vitiligo symptoms in the long term [4]. Abnormal expression of IL-7 receptor (IL-7R) may be observed in the immune cells of some patients with alopecia areata (AA). Blocking IL-7 can prevent the progression of the disease in mice with alopecia areata and even reverse early-stage alopecia areata [5]. In a systemic sclerosis model, IL-21 drives inflammation and fibrosis in the skin and lungs [6]. IL-15-induced bone marrow CD8+Trm cells are involved in the destruction of HSPCs in human severe aplastic anemia, and IL21 is also involved in the progression of aplastic anemia [7,8]. In addition, the above autoimmune diseases are all caused by abnormal activation of T cells, B cells, or NK cells, which mistakenly attack their own cells.

[0004] CD132 is a common subunit of the IL-4, IL-7, IL-9, IL-15 and IL-21 cytokine receptors, and CD132 is a key regulator of T cell, B cell or NK cell function. Therefore, targeting CD132 may be a new direction for the treatment of patients with various autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease or aplastic anemia.

[0005] [1]YoungD.A, et al. Blockade of the interleukin-21 / interleukin-21receptorpathway ameliorates disease in animal models of rheumatoid arthritis[J].

[0006] [2]Fina D,et al.Regulation of gut inflammation and th17 cell response by interleukin-21[J].

[0007] [3]Belarif L,et al.IL-7 receptor influences anti-TNF responsiveness and T cell gut homing in inflammatory bowel disease[J].

[0008] [4]Richmond,J.M,et al.Antibody blockade of IL-15 signaling has the potential to durably reverse vitiligo[J].

[0009] [5]Dai Z,et al.Blockade of IL-7 signaling suppresses inflammatory responses and reverses alopecia areata in C3H / HeJ mice[J].

[0010] [6]Um,I.G,et al.IL-21 drives skin and lung inflammation and fibrosis in a model for systemic sclerosis[J].

[0011] [7]Zhang,J,et al.Involvement of interleukin-21 in the pathophysiology of aplastic anemia[J].

[0012] [8]Long,J,etal.Bone marrow CD8 + Trm cells induced by IL-15 and CD16 + monocytes contribute to HSPC destruction in human severe aplastic anemia[J]. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to provide a humanized anti-human CD132 monoclonal antibody in response to the deficiencies of the existing technology.

[0014] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0015] The first aspect of the present invention provides an antibody:

[0016] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD132.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0018] (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO. 1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO. 22;

[0019] (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO. 1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO. 11;

[0020] (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO. 1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO. 16;

[0021] (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO. 1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO. 19;

[0022] (e) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO. 6; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO. 24;

[0023] (f) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) shown in SEQ ID NO. 6; and CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) shown in SEQ ID NO. 29.

[0024] Wherein, the CDRs are defined according to the Kabat, IMGT, Chothia or AbM numbering systems.

[0025] In some embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof comprises framework regions (FRs) from human or mouse immunoglobulins.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof binds to human CD132.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0028] The following heavy chain variable region (VH) and light chain variable region (VL):

[0029] (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO. 3, CDR-H2 with a sequence of SEQ ID NO. 4, and CDR-H3 with a sequence of SEQ ID NO. 5; and,

[0030] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO. 18, CDR-L2 of SEQ ID NO. 14, and CDR-L3 of SEQ ID NO. 15;

[0031] (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO. 3, CDR-H2 with a sequence of SEQ ID NO. 4, and CDR-H3 with a sequence of SEQ ID NO. 5; and,

[0032] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO. 13, CDR-L2 of SEQ ID NO. 14, and CDR-L3 of SEQ ID NO. 15;

[0033] (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO. 3, CDR-H2 with a sequence of SEQ ID NO. 4, and CDR-H3 with a sequence of SEQ ID NO. 5; and,

[0034] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO. 21, CDR-L2 of SEQ ID NO. 14, and CDR-L3 of SEQ ID NO. 15;

[0035] (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO. 8, CDR-H2 with a sequence of SEQ ID NO. 9, and CDR-H3 with a sequence of SEQ ID NO. 10; and,

[0036] The light chain variable region (VL) comprises the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.26, CDR-L2 with the sequence of SEQ ID NO.27, and CDR-L3 with the sequence of SEQ ID NO.28.

[0037] (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO. 8, CDR-H2 with a sequence of SEQ ID NO. 9, and CDR-H3 with a sequence of SEQ ID NO. 10; and,

[0038] The light chain variable region (VL) comprises the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.26, CDR-L2 with the sequence of SEQ ID NO.27, and CDR-L3 with the sequence of SEQ ID NO.31.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups:

[0040] (g) VH of SEQ ID NO. 1 and VL of SEQ ID NO. 22;

[0041] (h) VH of SEQ ID NO. 1 and VL of SEQ ID NO. 11;

[0042] (i) VH of the sequence shown in SEQ ID NO. 1 and VL of the sequence shown in SEQ ID NO. 16;

[0043] (j) VH of the sequence shown in SEQ ID NO. 1 and VL of the sequence shown in SEQ ID NO. 19;

[0044] (k) VH of SEQ ID NO. 6 and VL of SEQ ID NO. 24;

[0045] (l) VH of the sequence shown in SEQ ID NO.6 and VL of the sequence shown in SEQ ID NO.29.

[0046] In some embodiments, the monoclonal antibody further comprises a constant region, and the constant region is the kappa chain constant region Ckappa and the human IgG4 constant region CH1-CH3.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof is selected from a chimeric antibody, a humanized antibody, a monoclonal antibody, a scFv, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide-linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

[0048] The second aspect of the present invention further provides a nucleic acid molecule, specifically, a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to the first aspect.

[0049] Wherein, the nucleic acid molecule encoding the antibody heavy chain variable region consists of the nucleotide sequence shown in SEQ ID NO. 2 or 7, and the nucleic acid molecule encoding the antibody light chain variable region consists of the nucleotide sequence shown in SEQ ID NO. 23, 12, 17, 20, 25 or 30.

[0050] The third aspect of the present invention further provides an expression vector, specifically, comprising the nucleic acid molecule described in the second aspect.

[0051] The fourth aspect of the present invention further provides a host cell, specifically comprising the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0052] Wherein, the host cell is HEK293 cell.

[0053] The fifth aspect of the present invention also provides a method for preparing an antibody or antigen-binding fragment, which comprises culturing the host cell according to the fourth aspect of claim 1 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0054] The sixth aspect of the present invention further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the first aspect, or the vector described in the third aspect, or the host cell described in the fourth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0055] The seventh aspect of the present invention further provides a detection reagent or kit, which contains the antibody or antigen-binding fragment thereof described in the first aspect, or the vector described in the third aspect, or the host cell described in the fourth aspect, or the pharmaceutical composition described in the sixth aspect, and optionally instructions for use.

[0056] In some embodiments, the detection reagent or kit comprises the monoclonal antibodies h2D4H4K12 and / or h5H10H6K4.

[0057] The use of the above-mentioned antibodies or antigen-binding fragments thereof, vectors, host cells, and pharmaceutical compositions in the preparation of drugs for inhibiting or reducing human CD132 activity is also within the scope of protection of the present invention.

[0058] The use of the above-mentioned antibodies or antigen-binding fragments thereof, vectors, host cells, and pharmaceutical compositions in the preparation of therapeutic drugs for preventing, neutralizing, or treating autoimmune diseases is also within the scope of protection of the present invention.

[0059] Methods for preventing, neutralizing or treating autoimmune diseases using the above-mentioned antibodies or antigen-binding fragments thereof, vectors, host cells, and pharmaceutical compositions are also within the scope of protection of the present invention.

[0060] Among them, the autoimmune diseases are immune diseases related to IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokine receptors, which are all immune diseases caused by abnormal activation of T cells, B cells or NK cells and mistakenly attacking their own cells, including: systemic lupus erythematosus, rheumatoid arthritis, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis and aplastic anemia.

[0061] Furthermore, the common subunit of the IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokine receptors is CD132. Therefore, the above cytokine receptors can be regulated by inhibiting or reducing human CD132.

[0062] Specifically, in some embodiments of the present invention, in an in vitro activity experiment to detect the inhibitory activity of humanized candidate antibodies on CD132, the humanized anti-human CD132 antibody h2D4H4K12 can inhibit the activity of IL-21 stimulating NK92 cells to release IFN-γ; the humanized anti-human CD132 antibody h5H10H6K4 can inhibit the activity of IL-4 stimulating Ramos cells to express CD23.

[0063] Furthermore, CD132 is also a key regulatory factor in the function of T cells, B cells or NK cells. Therefore, the function of T cells, B cells or NK cells can be regulated by inhibiting or reducing human CD132.

[0064] Specifically, in some embodiments, both h2D4H4K12 and h5H10H6K4 antibodies have inhibitory effects on B cells, T cells, and NK cells, but do not affect the number of neutrophils, monocytes, red blood cells, and platelets.

[0065] Furthermore, antibodies h2D4H4K12 and / or h5H10H6K4 can be used to treat autoimmune diseases such as lupus erythematosus, vitiligo, psoriasis, and graft-versus-host disease (GVHD).

[0066] Specifically, in some embodiments, the two antibodies h2D4H4K12 and h5H10H6K4 can significantly reduce urine protein and plasma anti-dsDNA levels and improve survival rate.

[0067] Specifically, in some embodiments, the h2D4H4K12 antibody can significantly reduce the area of ​​white spots and significantly improve the disease phenotype of vitiligo.

[0068] Specifically, in some embodiments, the h2D4H4K12 antibody can significantly alleviate the symptoms of psoriasis.

[0069] Specifically, in some embodiments, the h2D4H4K12 antibody can reduce the infiltration of inflammatory cells in the liver, lungs, and kidneys in graft-versus-host disease. Beneficial effects:

[0070] The present invention humanizes two antibodies (2D4 and 5H10) previously obtained in the laboratory, and after screening, obtains two high-affinity monoclonal humanized antibodies (h2D4H4K12 and h5H10H6K4) that can target human CD132. The inhibitory activity of these two antibodies on five γc cytokines is consistent with that of the parent chimeras 2D4 and 5H10, while also reducing the immunogenicity of the parent chimeras, further reducing the risk that patients taking the drugs may develop an immune response to the antibodies. Both antibodies can be used in therapeutic drugs for the prevention, neutralization or treatment of autoimmune diseases associated with IL-4, IL-7, IL-9, IL-15 and / or IL-21 cytokines (such as systemic lupus erythematosus, rheumatoid arthritis, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease and aplastic anemia). In addition, the present invention demonstrates the significant inhibitory effects of these two antibodies on T cells, B cells, and NK cells in CD132 humanized mice, demonstrating their potential therapeutic effects on autoimmune diseases caused by overactive T cells, B cells, and NK cells (such as systemic lupus erythematosus, rheumatoid arthritis, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease, and aplastic anemia). The present invention further illustrates the prospects of these two antibodies for the treatment of autoimmune diseases through animal models of systemic lupus erythematosus, vitiligo, psoriasis, and graft-versus-host disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0072] Figure 1 Inhibition of IFN-γ secretion by NK92 cells under constant IL-21 stimulation by the parental chimeric anti-human CD132 antibody 2D4 and multiple humanized 2D4 antibodies.

[0073] Figure 2. The parental chimeric anti-human CD132 antibody 5H10 and multiple humanized 5H10 antibodies inhibited CD23 expression by Ramos cells under constant IL-4 stimulation.

[0074] Figure 3 h5H10H6K4 inhibits IL21-stimulated IFN-γ secretion from NK92 cells.

[0075] Figure 4 h2D4H4K12 inhibits the expression of CD23 in IL4-stimulated Ramos cells.

[0076] Figure 5 h5H10H6K4 (Figure 5-1) and h2D4H4K12 (Figure 5-2) inhibit IL15-stimulated NK92 cells from secreting IFN-γ.

[0077] Figure 6 h5H10H6K4 (Figure 6-1) and h2D4H4K12 (Figure 6-2) inhibit IL7-stimulated CD4 + Phosphorylation of STAT5 downstream of T cells.

[0078] Figure 7 h5H10H6K4 (Figure 7-1) and h2D4H4K12 (Figure 7-2) inhibit IL9-stimulated M07E cell proliferation.

[0079] Fig. 8 Immunogenicity of h2D4H4K12 and h5H10H6K4.

[0080] Figure 9: Effects of h2D4H4K12 and h5H10H6K4 antibodies on CD132 hu / hu Effects of background mouse T cells (9A), B cells (9B), NK cells (9C), neutrophils (9D), red blood cells (9E), monocytes (9F), platelets (9G) and Treg cells (9H).

[0081] Figure 10 Effects of h2D4H4K12 and h5H10H6K4 antibodies on pristane-induced CD132 hu / hu Anti-dsDNA levels (10A) and urine protein / creatinine ratio (10B) in a mouse lupus erythematosus model.

[0082] Figure 11 Effect of h2D4H4K12 on the treatment of vitiligo.

[0083] Figure 12 Effect of h2D4H4K12 on the treatment of psoriasis.

[0084] FIG13 Effect of h2D4H4K12 on the treatment of graft-versus-host disease (GVHD). DETAILED DESCRIPTION

[0085] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0086] In previous research by our research group, we have obtained monoclonal chimeras 2D4 and 5H10 that can target human CD132 through phage screening (detailed screening, construction, and sequence information for the monoclonal chimeras 2D4 and 5H10 of CD132 have been disclosed in patent CN116970081A). However, antibodies with chimera configurations are highly immunogenic and can easily lead to the production of anti-antibodies in the body, thereby weakening the antibody's efficacy. Therefore, this patent further humanizes the target antibodies 2D4 and 5H10 to reduce the chimera's immunogenicity without weakening the antibody's activity. At the same time, the target antibodies (h2D4H4K12 and h5H10H6K4) are validated by in vivo pharmacodynamics to further demonstrate the effectiveness of the two antibodies.

[0087] All experiments were approved and performed in accordance with the guidelines of the Ethics Committee of the Dermatology Hospital, Chinese Academy of Medical Sciences (Institute of Dermatology, Chinese Academy of Medical Sciences).

[0088] Example 1: Humanization of monoclonal antibodies 2D4 and 5H10

[0089] The variable region sequences of monoclonal antibodies 2D4 and 5H10 were compared with human germline antibody sequences to identify sequences with high homology for CDR transplantation. Simultaneously, computer modeling was performed to analyze the CDR regions and surrounding framework amino acid sequences to examine their spatial binding patterns. By calculating electrostatic forces, van der Waals forces, hydrophilicity, and entropy, key amino acids within the gene sequences of each positive monoclonal antibody were identified for potential interaction with CD132 and maintenance of the spatial framework. Back-mutation sites were then designed based on these analyses. HLA-DR affinity was analyzed to select human germline framework sequences with low immunogenicity. All of these humanization modifications were designed and completed by Conoya Biotechnology Co., Ltd.

[0090] For 2D4, one heavy chain variable region derivative (h2D4-VH4) and four light chain variable region derivatives (h2D4-VL3, h2D4-VL9, h2D4-VL11, and h2D4-VL12) were designed. For 5H10, one heavy chain variable region derivative (h5H10-VH6) and two light chain variable region derivatives (h5H10-VL3 and h5H10-VL4) were designed. The sequences of all the above light and heavy chain variable region derivatives are shown in Tables 1 and 2. The light chain variable region derivatives were cloned into the pHCT2 vector (purchased from Addgene), which contains the human IgGκ light chain constant region and regulatory elements and is developed by Conoya, to express the complete IgGκ light chain in mammalian cells. The heavy chain variable region derivatives were cloned into the pHCT1s vector (purchased from Addgene), a Conoya-developed vector containing the human IgG4 subtype heavy chain constant region and regulatory elements, to express the complete IgG4 subtype heavy chain in mammalian cells. After plasmid pairing, the plasmids were transfected into HEK293 cells (purchased from ATCC) and expressed for 5-6 days. The supernatant was collected, filtered, and purified using a protein A column to construct candidate humanized antibodies h2D4H4K3, h2D4H4K9, h2D4H4K11, h2D4H4K12, h5H10H6K4, and h5H10H6K5.

[0091] Table 1 Heavy chain variable region derivative sequences of candidate CD132 humanized antibodies (SEQ ID NO.** omitted before the number)

[0092] Table 2: Light chain variable region derivative sequences of candidate CD132 humanized antibodies (SEQ ID NO.** omitted before the numbers)

[0093] Specifically, the humanized antibody heavy chain variable region derivative sequence is:

[0094] 1) The amino acid sequence of h2D4-VH4 is shown in SEQ ID NO. 1, the encoding nucleotide sequence is shown in SEQ ID NO. 2, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 3, 4, and 5, respectively.

[0095] Nucleotide sequence

[0096] 2) The amino acid sequence of h5H10-VH6 is shown in SEQ ID NO. 6, the encoding nucleotide sequence is shown in SEQ ID NO. 7, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 8, 9, and 10, respectively.

[0097] Nucleotide sequence

[0098] Specifically, the sequence of the humanized antibody light chain variable region derivative is:

[0099] 3) The amino acid sequence of h2D4-VL3 is shown in SEQ ID NO. 11, the encoding nucleotide sequence is shown in SEQ ID NO. 12, and the CDR1, CDR2, and CDR3 sequence are shown in SEQ ID NOs. 13, 14, and 15, respectively.

[0100] Nucleotide sequence

[0101] 4) The amino acid sequence of h2D4-VL9 is shown in SEQ ID NO. 16, its encoding nucleotide sequence is shown in SEQ ID NO. 17, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 18, 14 and 15, respectively.

[0102] Nucleotide sequence

[0103] 5) The amino acid sequence of h2D4-VL11 is shown in SEQ ID NO. 19, the encoding nucleotide sequence is shown in SEQ ID NO. 20, and the CDR1, CDR2, and CDR3 thereof are shown in SEQ ID NOs. 21, 14, and 15, respectively.

[0104] Nucleotide sequence

[0105] 6) The amino acid sequence of h2D4-VL12 is shown in SEQ ID NO. 22, the encoding nucleic acid thereof is shown in SEQ ID NO. 23, and its CDR1, CDR2 and CDR3 are shown in SEQ ID NOs. 18, 14 and 15, respectively.

[0106] Nucleotide sequence

[0107] 7) The amino acid sequence of h5H10-VL3 is shown in SEQ ID NO. 24, the encoding nucleotide sequence is shown in SEQ ID NO. 25, and the CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs. 26, 27 and 28, respectively.

[0108] Nucleotide sequence

[0109] 8) The amino acid sequence of h5H10-VL4 is shown in SEQ ID NO. 29, the encoding nucleotide sequence is shown in SEQ ID NO. 30, and the CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NOs. 26, 27, and 31, respectively.

[0110] Nucleotide sequence

[0111] Example 2: In vitro activity assay to detect the inhibitory activity of candidate humanized antibodies against CD132

[0112] To further evaluate the activity gap between the candidate humanized antibodies and the parent chimera, the experimental method in patent CN116970081A was referred to. Because the parent chimera 2D4 has strong inhibitory activity against IL21, the method of IL21 stimulating the NK92 cell line to secrete IFN-γ was used to evaluate the 2D4 candidate humanized antibody. Because the activity gap between the 5H10 candidate humanized antibodies was more significant in the IL4 inhibition experimental system, the method of IL4 stimulating the Ramos cell line to upregulate CD23 was selected to further evaluate the 5H10 candidate humanized antibody.

[0113] 2.1 Verification of the 2D4 humanized anti-human CD132 candidate antibody's ability to inhibit NK92 cell secretion of IFN-γ

[0114] 2 × 10 cells were seeded in a 96-well plate using growth medium (prepared according to the instructions of Pronose, but without IL-2). 4 NK-92 cells (Punosai CL-0530) were incubated at 37°C in 5% CO2 and starved overnight. The next day, each 2D4 humanized anti-human CD132 candidate antibody was serially diluted in a 3-fold gradient from 400nM to 1.6nM in growth medium (without IL-2), added to the NK-92 cells and incubated for 30 minutes. After incubation, 250pM IL-21 was added to the NK-92 cells containing different 2D4 humanized anti-human CD132 candidate antibodies. After incubation for 72h at 37°C in 5% CO2, IFN-γ secretion was measured by ELISA.

[0115] Using a human IFN-γ ELISA kit (BD Catalog No. 555142), the capture antibody was coated at a 1:250 ratio on a 96-well microtiter plate and incubated overnight at 4°C. The next day, the plates were washed three times with PBS buffer (pH 7.2), blocked with 10% FBS for 1 hour at room temperature, and then washed three times with PBS buffer (pH 7.2). Then, 100 μl of sample (cell supernatant) and standard were added, and the plates were incubated for 2 hours at room temperature. The plates were then washed five times with PBS buffer (pH 7.2). 5AV-HRP was added at a 1:250 ratio in a dilution containing 0.4% detection antibody. Finally, the plates were incubated for 1 hour at room temperature, washed seven times with PBS buffer (pH 7.2), and 100 μl of color development solution (TMB solution, Sigma Catalog No. T2885) was added. The plates were incubated at 37°C for 10 minutes, and the reaction was terminated by adding 50 μl of 2M concentrated sulfuric acid solution. The plates were immediately placed in a microtiter plate and the OD was read. 450 Figure 1 shows that the activity of humanized anti-human CD132 antibody h2D4H4K12 in inhibiting IL-21-stimulated IFN-γ release from NK92 cells is comparable to that of the parental 2D4 chimera.

[0116] 2. Validation of 25H10 humanized anti-CD132 candidate antibody inhibiting IL-4-stimulated CD23 expression in Ramos cells

[0117] IL-4 can stimulate Ramos cells to express CD23, so this property can be used to evaluate the inhibitory effect of the 5H10 humanized anti-CD132 candidate antibody on IL-4 signaling. Ramos cell line (ATCC) was cultured in RPMI 1640 complete medium containing 10% FBS at 37°C and 5% CO2, with a cell concentration of 2×10 cells per ml. 5 -2×10 6 Cells were plated in 96-well flat-bottom cell culture plates, with 100 μl per well. Serial dilutions of each 5H10 humanized anti-CD132 candidate antibody were prepared in RPMI 1640 complete medium, with a four-fold serial dilution from 200 nM to 0.003 nM. After incubation with the cells for 30 minutes, 50 μl of IL-4 was added for a final concentration of 2.67 nM, and the cells were cultured at 37°C, 5% CO2 for 48 hours.

[0118] The cultured cells were transferred to a U-shaped 96-well cell culture plate, centrifuged at 300 g for 3 minutes at 4°C, discarded the supernatant, and washed twice with flow cytometry buffer (PBS buffer containing 4% calf serum, pH 7.2), 200 μl per well. 50 μl of blocking buffer (buffer containing 100 μg / ml hIgG) was added to the cells, blocked on ice for 10 minutes, centrifuged at 300 g for 3 minutes, and the supernatant discarded. Then, 50 μl of anti-human CD23 FITC antibody dilution (BD EBVCS-5) was added to each well and blocked on ice for 20 minutes. After centrifugation at 300 g for 3 minutes and the supernatant discarded, the cells were washed twice with flow cytometry buffer, 200 μl per well. 100 μl of PI staining solution was added to the cells, ice-bathed in the dark for 5 minutes, centrifuged at 300 g for 3 minutes and the supernatant discarded. The cells were washed twice with 200 μl of flow cytometry buffer per well, and then resuspended in 100 μl of PBS buffer (pH 7.2) per well. The mean fluorescence intensity was measured by flow cytometry and the results were recorded. As shown in Figure 2, the humanized anti-human CD132 antibody h5H10H6K4 inhibited IL-4-stimulated CD23 expression in Ramos cells at a level comparable to that of the parental 5H10 chimera.

[0119] Example 3: Revalidation of humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12

[0120] In Example 2, the inhibitory activity of candidate humanized antibodies against CD132 was tested by in vitro activity experiments, and two humanized anti-human CD132 antibodies, h5H10H6K4 and h2D4H4K12, were screened. This example further verified whether the inhibitory activity of the two antibodies against five γc cytokines was consistent with that of the parent chimeras 2D4 and 5H10.

[0121] 3.1 Humanized anti-human CD132 antibody h5H10H6K4 inhibits IL21-stimulated NK92 cell secretion of IFN-γ

[0122] To further evaluate the ability of h5H10H6K4 to block IL-21-stimulated IFN-γ secretion from NK92 cells, the specific experimental steps were as detailed in Example 2.1, except that the antibody was replaced with h5H10H6K4. The results are shown in Figure 3, which shows that the anti-human CD132 antibody h5H10H6K4 can inhibit IL-21-stimulated IFN-γ release from NK92 cells, but its inhibitory activity is weaker than that of h2D4H4K12 compared to the results of h2D4H4K12 in Figure 1.

[0123] 3.2 Humanized anti-human CD132 antibody h2D4H4K12 inhibits IL4-stimulated CD23 expression in Ramos cells

[0124] To further evaluate the ability of h2D4H4K12 to block CD23 secretion and expression by IL-4-stimulated Ramos cells, the specific experimental steps were as detailed in Example 2.2, except that the antibody was replaced with h2D4H4K12. The results are shown in Figure 4, which demonstrates that the anti-human CD132 antibody h2D4H4K12 can inhibit IL-4-stimulated CD23 expression by Ramos cells.

[0125] 3.3 Humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 inhibit IL15-stimulated NK92 cell secretion of IFN-γ

[0126] To further evaluate the ability of h2D4H4K12 and h5H10H6K4 to block IL-15-stimulated IFN-γ secretion from NK92 cells, the specific experimental procedures are detailed in Example 2.1. Antibodies h5H10H6K4 and h2D4H4K12 were used, respectively, and 250 pM IL-21 was replaced with 2.5 nM IL-15. The experimental results are shown in Figures 5-1 and 5-2. Figures 5-1 and 5-2 respectively demonstrate that both humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 can inhibit IL-15-stimulated IFN-γ release from NK92 cells, but the inhibitory activity of h5H10H6K4 is weaker than that of h2D4H4K12.

[0127] 3.4 people CD4 + Flow cytometric analysis of STAT phosphorylation in T cells (human PBMC)

[0128] To further evaluate the in vitro properties of humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12, their ability to block IL-7-induced CD4 + The experimental process is the same as that of Example 8 in patent CN116970081A, except that the chimeric anti-human CD132 antibodies 5H10 and 2D4 are replaced by humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12. The experimental results are shown in Figures 6-1 and 6-2. Figures 6-1 and 6-2 respectively show that anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 can inhibit IL-7 stimulation of human peripheral blood CD4 + Downstream cellular STAT5 phosphorylation.

[0129] 3.5 Humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 inhibit IL-9-stimulated M07E cell proliferation

[0130] The inhibitory activity of humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 against IL-9 was further evaluated. The experimental procedure was the same as that of Example 10 in patent CN116970081A, except that the chimeric anti-human CD132 antibodies 5H10 and 2D4 were replaced with humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12. The results are shown in Figure 7. Figure 7-1 and Figure 7-2 respectively show that humanized anti-human CD132 antibodies h5H10H6K4 and h2D4H4K12 can inhibit IL-9-stimulated M07E cell proliferation.

[0131] Example 4: Evaluation of the immunogenicity of humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4

[0132] Sample preparation:

[0133] Under sterile conditions, h2D4H4K12, h5H10H6K4, and KLH (independently expressed by Conoya) were diluted to 200 μg / mL using X-vivo15 medium (Shanghai Peiyuan) and sterilized for use.

[0134] Determination method:

[0135] Peripheral venous blood was collected from 25 healthy volunteers and PBMCs were collected by Ficoll centrifugation (Healthcare). PBMCs were washed once with PBS, counted, and the cell density was adjusted to 2.5×10 5 / mL, 100 μL was added to a 96-well U-shaped plate (corning). 100 μL of the diluted sample was added to the cells and incubated at 37°C, 5% CO2 for 48 hours. KLH was used as a positive control, and wells without sample were used as negative controls. The assay was performed in the same manner. The 96-well plate was discarded, 200 μL / well of the supernatant was washed once with flow cytometry buffer (PBS + 10% FBS). 100 μg / mL hIgG (Jackson) was added, 50 μL per well, and incubated on ice for 30 minutes. 1 μL / well of FITC anti-human CD4, APC anti-human CD137 (4-1BB), and PE anti-human CD134 (OX40) fluorescent antibodies (all purchased from Biolegend) were added and incubated on ice for 45 minutes. 50 μL of 5 μg / mL PI was added for staining for 5 minutes. The cells were washed twice with flow cytometry buffer, resuspended in 100 μL of PBS, and analyzed by flow cytometry.

[0136] Data Analysis:

[0137] (1) Lymphocytes were gated based on cell size (FSC-A) and granularity (SSC-A);

[0138] (2) In the lymphocyte population, adherent cells were excluded based on FSC-A and FSC-H, and single cell populations were circled;

[0139] (3) Circle the living cell population in the single cell population based on FSC-A and PI light signals;

[0140] (4) Circle the CD4-positive T cells among the living cells and analyze the proportion of CD137 (4-1BB) and CD134 (OX40) double-positive cells in this group of cells.

[0141] The final results are shown in FIG8 . The double-positive cells of humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 were comparable to those of the negative control, indicating that the above two antibodies have extremely low immunogenicity.

[0142] Example 5: In vivo immunosuppressive experiment evaluating the effects of anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 on immune cell populations in the blood

[0143] Experimental Procedure: C57BL / 6 background mice (C57BL / 6 background mice from the Biocytogen breeding colony that have been genetically modified to replace the endogenous full-length CD132 domain with the corresponding human sequence) were subcutaneously administered with antibodies h2D4H4K12 or h5H10H6K4 at a dose of 8 mg / kg or 16 mg / kg once every four days for three doses. No administration of antibodies h2D4H4K12 or h5H10H6K4 served as a control.

[0144] Table 3 Experimental drug administration and treatment schemes for mice in each group

[0145] Analysis of immune cell populations in the blood over a period of time by flow cytometry. The specific process is as follows: The number of total immune cells, B cells, T cells, NK cells, and neutrophils in peripheral blood at different time points (once every 10 days) was analyzed by flow cytometry to evaluate the effect of h2D4H4K12 and h5H10H6K4 antibodies on the absolute number of these cell types. Briefly, at each time point, blood samples were collected from each mouse, and 50-100 μL of each blood sample was incubated in red blood cell lysis buffer (Biyuntian) at room temperature for 10 minutes to lyse red blood cells. If lysis was not complete, a second round of lysis was performed. After washing twice in PBS buffer (Shanghai Peiyuan, pH 7.2), mouse IgG (Jackson Immunoresearch) was diluted to 200 μg / ml in flow buffer (4% FBS (ExCell Bio)) to prepare FC receptor blocking solution. 50 μL of blocking solution was added to each lysed blood sample and blocked at room temperature for 10 minutes. The cells were then stained for cell surface markers by adding a cocktail of fluorescently labeled antibodies (described in Table 4) diluted in flow buffer to identify CD45 + Cells, T cells, B cells, and NK cells (the absolute number of neutrophils, monocytes, red blood cells, and platelets were all determined using a mouse blood routine analyzer). Finally, the samples were washed twice in flow cytometry buffer, resuspended in PBS buffer (pH 7.2), and sample data were acquired on a BD Celesta flow cytometer. Data analysis was performed using FlowJov10 software. + Immune cells were defined as singlet, viable cells, and within this group, T cells were further defined as CD3 + , Treg cells were further defined as CD4 + 、CD25 + , B cells were further defined as CD3 - 、CD19 + NK cells are further defined as CD3 - 、CD19 - NK1.1 + .

[0146] Table 4 Antibodies used in flow cytometry analysis

[0147] The results are shown in Figure 9: Treatment of background mice with h2D4H4K12 or h5H10H6K4 (8 mg / kg and 16 mg / kg) resulted in a significant decrease in the number of T cells (Figure 9A), B cells (Figure 9B), and NK cells (Figure 9C) in their blood, while neutrophil (Figure 9D), red blood cell (Figure 9E), monocyte (Figure 9F), and platelet counts (Figure 9G) were unaffected. h2D4H4K12 had no significant effect on the proportion of Treg cells (Figure 9H). After the completion of the three doses, serum concentrations of h2D4H4K12 and h5H10H6K4 decreased over time. By the end of the study, all of these levels had returned to similar levels observed before treatment. These results indicate that both h2D4H4K12 and h5H10H6K4 antibodies have varying degrees of inhibitory effects on B cells, T cells, and NK cells, but do not affect the number of neutrophils, monocytes, red blood cells, and platelets. In addition, the inhibitory effect of h2D4H4K12 on Treg cells is weaker than that of h5H10H6K4.

[0148] Example 6: Evaluating the efficacy of anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 in treating pristane-induced mouse lupus erythematosus model.

[0149] Experimental procedure: C57BL / 6 background mice (C57BL / 6 background mice genetically modified from the Biocytogen breeding colony to replace the endogenous CD132 full-length domain with the corresponding human sequence) were injected with 0.5 mL of pristane (MCE) into the peritoneal cavity, and the control group was injected with an equal volume of PBS buffer (pH 7.2). h2D4H4K12 or h5H10H6K4 or anti-KLH isotype control (all antibodies are produced by Conoya Biotechnology Co., Ltd.) was administered subcutaneously at a dose of 20 mg / kg 3 days before modeling, with a frequency of 2 times per week for 6 weeks, or no administration (Table 5). The levels of urine protein, urine creatinine, and plasma anti-dsDNA in the mice were monitored. The mice were killed by cervical dislocation at week 12.

[0150] Specific monitoring experimental steps:

[0151] 1. Use the Mouse Urine Microalbumin ELISA Detection Kit (Elabscience) to detect mouse urine microalbumin: Collect 20μl of urine from each mouse and perform the test according to the operating procedures. Briefly, dilute the urine of each mouse 1000 times with the standard / sample diluent, add 100μl to each well of the 96-well ELISA plate that has been coated in the kit, incubate at 37°C for 90 minutes, dry, add 100μl of biotinylated antibody working solution to each well, and incubate at 37°C for 1 hour. Wash 4 times, add 100μl of HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 minutes, wash 4 times, add 90μl of color development solution, place at 37°C for 10 minutes, add 50μl of stop solution, and immediately place in the microplate reader to read the OD 450 The value of .

[0152] 2. ELISA test for anti-dsDNA level in mouse plasma: Collect 50-100 μl of blood from each mouse, centrifuge at 4500 rpm, take the supernatant and freeze it at -80°C for subsequent testing. TM Salmon sperm DNA solution (Invitrogen) was incubated at 4°C overnight. The next day, the cells were washed three times with PBS buffer (pH 7.2) and blocked with 10% BSA (Sangon) at 37°C for 1 hour. The cells were then washed three times with PBS buffer (pH 7.2). A 50-fold diluted plasma sample was added to each well, 100 μl, and the cells were incubated at 37°C for 90 minutes. The cells were then washed five times with PBS buffer (pH 7.2) and HRP-labeled Fc-specific anti-mouse IgG1 (abclonal) was added at a ratio of 1:4000. The cells were incubated at room temperature for 1 hour, washed seven times with PBS buffer (pH 7.2), and 100 μl of color development solution (TMB solution, Sigma catalog number T2885) was added. The cells were incubated at 37°C for 10 minutes, and then 50 μl of 2M concentrated sulfuric acid solution was added to terminate the reaction. The cells were immediately placed in a microplate reader and the OD was read. 450 The value of .

[0153] Table 5 Experimental drug administration and treatment scheme for each group of mice

[0154] The results are shown in Figure 10: Treatment with h2D4H4K12 or h5H10H6K4 antibodies reduced the amount of anti-dsDNA autoantibodies in mouse plasma (Figure 10A) and lowered the urine protein / creatinine ratio (Figure 10B). This indicates that h2D4H4K12 and h5H10H6K4 antibodies significantly reduced urine protein and plasma anti-dsDNA levels in pristane-induced lupus mice and improved their survival rates, further demonstrating that h2D4H4K12 and h5H10H6K4 antibodies are effective in treating lupus.

[0155] Example 7: Evaluating the efficacy of anti-human CD132 antibody h2D4H4K12 in treating vitiligo mouse models.

[0156] Experimental procedure: On day 0, 2×10 5 B16F10 cells (melanoma cells, purchased from Wuhan Punosai Life Science Technology Co., Ltd.). Anti-CD4 (purchased from Bio X cell) was administered subcutaneously twice on days 4 and 10, respectively, at a dose of 10 mg / kg to deplete Treg cells in the mice. The melanoma was surgically removed on day 12. The mice were observed for onset of disease on day 30, and mice with successful vitiligo modeling were selected and divided into two groups. Group A was subcutaneously administered PBS as an untreated control group, while Group B was administered h2D4H4K12 subcutaneously at a dose of 10 mg / kg twice a week for 4 weeks (Table 6). Photos were taken before and after treatment to record the area of ​​white spots in the mice.

[0157] Table 6 Experimental drug administration and treatment scheme for each group of mice

[0158] The results are shown in Figure 11. After one month of treatment, vitiligo in the PBS-treated group (Group A) progressed, with a significant increase in the area of ​​white spots. However, in the h2D4H4K12-treated group (Group B), the area of ​​white spots significantly decreased. This demonstrates that h2D4H4K12 can effectively alleviate vitiligo symptoms.

[0159] Example 8: Evaluating the efficacy of anti-human CD132 antibody h2D4H4K12 in treating psoriasis mouse models.

[0160] Experimental Procedure: On day 0, C57BL / 6 mice (genetically modified from the Biocytogen breeding colony to replace the full-length endogenous CD132 domain with the corresponding human sequence) underwent dorsal hair removal (2×3 cm) and were subcutaneously administered 300 μg of h2D4H4K12. From day 1 to day 5, 5% imiquimod was applied to the depilated area on the back of the mice at a dose of 62.5 mg once daily. The mice were weighed and observed for skin lesions, rash, and inflammation on the back. On day 7, PASI scores were assessed, and the mice were sacrificed. The dorsal skin was removed and stained with HE to observe abnormal keratinization and inflammatory infiltration. The experimental mice were grouped as shown in Table 7.

[0161] Table 7 Experimental drug administration and treatment scheme for each group of mice

[0162] Table 8 PASI scores of mouse back skin

[0163] Table 9 Statistics of weight of mice in each group

[0164] The results are shown in Figure 12. After the onset of psoriasis in mice, the back skin of the untreated (i.e., Blank) mice was ruddy and scale-free, indicating normal skin. However, the imiquimod-treated (NC) group developed significant erythema and scale on the back, with a significantly higher PASI score than the Blank group (p<0.01). However, after treatment with h2D4H4K12 (i.e., h2D4H4K12), the erythema and scale were significantly reduced compared to the NC group (p<0.01), the PASI score was significantly lower (p<0.01) (Table 8), and the body weight was significantly increased (p<0.01) (Table 9). This indicates that h2D4H4K12 can significantly alleviate psoriasis symptoms.

[0165] Example 9: Evaluating the efficacy of the anti-human CD132 antibody h2D4H4K12 in treating graft-versus-host disease (GVHD).

[0166] Experimental procedure: On day 0, 1×10 7 PBMCs of human origin. On the 14th day, peripheral blood of modeling mice was collected, and human T / B cell staining and flow cytometry analysis were performed to observe the cell colonization. Mice with stable colonization of human T / B cells were judged to have successfully modeled graft-versus-host disease (GVHD). The mice with successful modeling were evenly divided into two groups according to cell colonization and body weight, one group was the NC group (no treatment), and the other group was the h2D4H4K12 group (h2D4H4K12 was administered subcutaneously at a dose of 10 mg / kg), with a frequency of 2 times a week, which continued until the end of the mouse. A group of unmodeled mice was retained as the Blank group (negative control group). The weight and survival rate of the mice were recorded during the treatment. At the end of the treatment, the liver, spleen, kidney and other tissues of the mice were collected, and HE staining was used to observe the damage and inflammatory infiltration of each tissue. The experimental dosing and treatment schemes of each group of mice are shown in Table 10.

[0167] Table 10 Experimental drug administration and treatment scheme for each group of mice

[0168] The results are shown in Figure 13: The weight of mice in the negative control group (Blank group) continued to increase, and their survival was good. No inflammatory cell infiltration or tissue damage was observed in the tissues at the endpoint. However, the weight of mice after GVHD modeling (i.e., NC group) decreased over time, the mice were in poor condition, and they died one after another. At the endpoint, a large amount of inflammatory infiltration was observed in the liver, lungs, kidneys and other tissues of the mice in the NC group, and tissue damage was obvious. The administration of h2D4H4K12 can effectively block the occurrence of GVHD in mice. The infiltration of inflammatory cells in the liver, lungs and kidneys of the h2D4H4K12 group mice was less than that of the NC group, and the vast majority of mice treated with h2D4H4K12 were free from weight loss and death.

[0169] In summary, the above in vitro and in vivo experiments have shown that the humanized anti-human CD132 antibodies h2D4H4K12 and h5H10H6K4 can inhibit the activity of IL4, IL-7, IL9, IL-15 and IL-21, thereby significantly inhibiting the functional activity of T cells, B cells and NK cells in vivo. Abnormal activation of T cells, B cells and NK cells is a key mechanism for the pathogenesis of lupus erythematosus, vitiligo and psoriasis. In addition, further experiments on lupus erythematosus, vitiligo model mice, psoriasis model mice and GVHD model mice found that both antibodies can significantly improve the disease phenotype of lupus erythematosus mice, and h2D4H4K12 significantly improved the disease phenotype of vitiligo mice, psoriasis mice and GVHD mice. Other autoimmune diseases such as rheumatoid arthritis, alopecia areata, inflammatory bowel disease, systemic sclerosis and aplastic anemia are also closely related to abnormal activation of T cells, B cells or NK cells. CD132 is a key regulator of T cell, B cell or NK cell function. At the same time, CD132 is a common subunit of the above-mentioned inflammatory factors. Therefore, targeting CD132 is a new direction for the treatment of patients with various autoimmune diseases.

[0170] The foregoing is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications should be considered within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD132, wherein, The antibody or its antigen-binding fragment comprises the following complementarity-determining regions (CDRs): (a) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO.1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light-chain variable region (VL) shown in SEQ ID NO.22; (b) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO.1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light-chain variable region (VL) shown in SEQ ID NO.11; (c) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO.1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light-chain variable region (VL) shown in SEQ ID NO.16; (d) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO.1; and CDR-L1, CDR-L2, and CDR-L3 contained in the light-chain variable region (VL) shown in SEQ ID NO.19; (e) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO.6; and CDR-L1, CDR-L2, and CDR-L3 contained in the light-chain variable region (VL) shown in SEQ ID NO.24; (f) CDR-H1, CDR-H2, and CDR-H3 contained in the heavy-chain variable region (VH) shown in SEQ ID NO.6; and CDR-L1, CDR-L2, and CDR-L3 contained in the light-chain variable region (VL) shown in SEQ ID NO.

29.

2. The antibody or its antigen-binding fragment according to claim 1, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, or AbM numbering system.

3. The antibody or its antigen-binding fragment according to claim 1, wherein the VH and / or VL of the antibody or its antigen-binding fragment comprises framework regions (FRs) from human or murine immunoglobulins.

4. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment binds to human CD132.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: The following heavy-chain variable region (VH) and light-chain variable region (VL): (a) A heavy-chain variable region (VH) comprising the following 3 CDRs: CDR-H1 with the sequence of SEQ ID NO.3, CDR-H2 with the sequence of SEQ ID NO.4, CDR-H3 with the sequence of SEQ ID NO.5; and, The light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.18, CDR-L2 with the sequence of SEQ ID NO.14, and CDR-L3 with the sequence of SEQ ID NO.15; (b) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO.3, CDR-H2 with the sequence of SEQ ID NO.4, and CDR-H3 with the sequence of SEQ ID NO.5; and, The light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.13, CDR-L2 with the sequence of SEQ ID NO.14, and CDR-L3 with the sequence of SEQ ID NO.15; (c) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO.3, CDR-H2 with the sequence of SEQ ID NO.4, and CDR-H3 with the sequence of SEQ ID NO.5; and, The light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.21, CDR-L2 with the sequence of SEQ ID NO.14, and CDR-L3 with the sequence of SEQ ID NO.15; (d) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO.8, CDR-H2 with the sequence of SEQ ID NO.9, and CDR-H3 with the sequence of SEQ ID NO.10; and, The light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.26, CDR-L2 with the sequence of SEQ ID NO.27, and CDR-L3 with the sequence of SEQ ID NO.

28. (e) The heavy chain variable region (VH) contains the following three CDRs: CDR-H1 with the sequence of SEQ ID NO.8, CDR-H2 with the sequence of SEQ ID NO.9, and CDR-H3 with the sequence of SEQ ID NO.10; and, The light chain variable region (VL) contains the following three CDRs: CDR-L1 with the sequence of SEQ ID NO.26, CDR-L2 with the sequence of SEQ ID NO.27, and CDR-L3 with the sequence of SEQ ID NO.

31.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 5, wherein, The antibody or its antigen-binding fragment contains a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups: (a) VH with the sequence of SEQ ID NO.1 and VL with the sequence of SEQ ID NO.22; (b) VH with the sequence of SEQ ID NO.1 and VL with the sequence of SEQ ID NO.11; (c) VH with the sequence of SEQ ID NO.1 and VL with the sequence of SEQ ID NO.16; (d)VH of the sequence shown in SEQ ID NO.1 and VL of the sequence shown in SEQ ID NO.19; (e)VH of the sequence shown in SEQ ID NO.6 and VL of the sequence shown in SEQ ID NO.24; (f)VH of the sequence shown in SEQ ID NO.6 and VL of the sequence shown in SEQ ID NO.

29.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin; and (b) a light chain constant region (CL) of a human immunoglobulin; wherein the heavy chain constant region is an IgG heavy chain constant region.

8. The antibody or its antigen-binding fragment according to claim 7, wherein the antibody or its antigen-binding fragment comprises a human IgG4 heavy chain constant region.

9. The antibody or its antigen-binding fragment according to claim 7, wherein the light chain constant region is the kappa chain constant region Ckappa.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment is selected from chimeric antibodies, humanized antibodies, monoclonal antibodies, scFv, Fab, Fab’, F(ab’)2, Fv fragments, disulfide-linked Fv (dsFv), diabodies, bispecific antibodies, and multispecific antibodies.

11. An isolated nucleic acid molecule encoding the antibody or its antigen-binding fragment according to any one of claims 1-10.

12. The isolated nucleic acid molecule according to claim 11, which comprises a nucleic acid molecule encoding the variable region of the antibody heavy chain and a nucleic acid molecule encoding the variable region of the antibody light chain, wherein, the nucleic acid molecule encoding the variable region of the antibody heavy chain consists of the nucleotide sequence shown in SEQ ID NO.2 or 7, and the nucleic acid molecule encoding the variable region of the antibody light chain consists of the nucleotide sequence shown in SEQ ID NO.23, 12, 17, 20, 25 or 30.

13. A vector comprising the nucleic acid molecule according to any one of claims 11-12.

14. A host cell comprising the nucleic acid molecule according to any one of claims 11-12 or the vector according to claim 13.

15. A method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-10, which comprises culturing the host cell according to claim 14 under conditions allowing the expression of the antibody or its antigen-binding fragment, and recovering the antibody or its antigen-binding fragment from the cultured host cell culture.

16. A pharmaceutical composition comprising the antibody or its antigen-binding fragment according to claims 1-10, or the vector according to claim 13, or the host cell according to claim 14, and a pharmaceutically acceptable carrier and / or excipient.

17. A detection reagent or kit comprising the antibody or its antigen-binding fragment according to claims 1-10, or the vector according to claim 13, or the host cell according to claim 14, or the pharmaceutical composition according to claim 16, and optionally an instruction manual.

18. Use of the antibody or antigen-binding fragment thereof according to claims 1-10, or the vector according to claim 13, or the host cell according to claim 14, or the pharmaceutical composition according to claim 16, in the preparation of a medicament for treating and / or preventing an autoimmune disease, wherein, The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease (GVHD), or aplastic anemia.

19. A method for preventing and / or treating an autoimmune disease, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1-10, or the vector according to claim 13, or the host cell according to claim 14, or the pharmaceutical composition according to claim 16.

20. The method according to claim 19, wherein The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease (GVHD), or aplastic anemia.

21. Use of the antibody or antigen-binding fragment thereof recited in claims 1-10, or the vector recited in claim 13, or the host cell recited in claim 14, or the pharmaceutical composition recited in claim 16, as a medicament for treating and / or preventing autoimmune diseases, wherein, The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease (GVHD), or aplastic anemia.

22. A humanized anti-human CD132 monoclonal antibody, characterized in that, The monoclonal antibody is a humanized monoclonal antibody. Wherein, the monoclonal antibody comprises a light chain complementary determining region and a heavy chain complementary determining region, the light chain complementary determining region includes LCDR1, LCDR2 and LCDR3, and the heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3. The light chain complementary determining region and the heavy chain complementary determining region are as follows: The amino acid sequences of LCDR1, LCDR2 and LCDR3 are successively as shown in SEQ ID NO.18, 14, 15; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are successively as shown in SEQ ID NO.3, 4, 5.

23. The monoclonal antibody according to claim 22, characterized in that, The monoclonal antibody comprises a light chain variable region and a heavy chain variable region, and the light chain variable region and the heavy chain variable region are as follows: The amino acid sequence of the light chain variable region is as shown in SEQ ID NO.22; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.

1.

24. The monoclonal antibody according to claim 22, characterized in that, The monoclonal antibody further comprises a constant region, and the constant region is the kappa chain constant region Ckappa and the human IgG4 constant regions CH1-CH3.

25. A nucleic acid molecule, characterized in that, Encoding the monoclonal antibody according to any one of claims 22-24.

26. An expression vector, characterized in that, Comprising the nucleic acid molecule according to claim 25.

27. A host cell, characterized in that, Comprising the expression vector according to claim 26.

28. The host cell according to claim 27, wherein The host cell is a HEK293 cell.

29. A detection reagent or kit, characterized in that, It contains the monoclonal antibody according to any one of claims 22-24.

30. Use of the monoclonal antibody according to any one of claims 22-24, or the expression vector according to claim 26, or the host cell according to claims 27-28 in the preparation of a drug for treating an autoimmune disease.

31. The application according to claim 30, wherein, The autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, graft-versus-host disease (GVHD), or aplastic anemia.

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