Methods of using antibodies that specifically bind cd132 in the treatment and / or prevention of autoimmune diseases

Abstract

The present application belongs to the field of biological medicine, and particularly relates to a method and application of an antibody specifically binding to CD132 in treating and / or preventing an autoimmune disease. The present application proves the prospect of the antibody specifically binding to human CD132 in treating the autoimmune disease by using the antibody specifically binding to human CD132 to treat and evaluate the autoimmune disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of antibodies that specifically bind to CD132 in the preparation of drugs for the treatment and / or prevention of autoimmune diseases. Background Technology

[0002] Autoimmune diseases are disease states caused by an immune response of the body's own immune system against its own components. Currently used immunomodulatory drugs for autoimmune diseases are broad-spectrum, non-disease-specific, and often cause side effects such as infections and malignant diseases. Therefore, there is a lack of drugs with good therapeutic effects and few side effects. Vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, and / or aplastic anemia are all autoimmune diseases, and currently, there are no effective cures available clinically.

[0003] CD132 (IL-2Rγ) is the common subunit (γc) of the receptor complexes of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, playing a crucial role in signal transduction of various immune cells. These γ-chain cytokines play important roles in the survival, proliferation, and differentiation of lymphocytes (such as T cells, B cells, and NK cells), and in autoimmune diseases, these cytokines often exhibit abnormal activation, mistakenly attacking their own cells. Therefore, CD132 has an important regulatory role in autoimmune diseases, and intervening in its functional activity may be an effective therapeutic strategy; that is, targeting CD132 may be a new direction for the treatment of patients with various autoimmune diseases. Summary of the Invention

[0004] To address the challenges of treating autoimmune diseases with existing clinical drugs, which often result in relapse, this invention provides the application of an antibody that specifically binds to CD132 or its antigen-binding fragment in the preparation of drugs for treating and / or preventing autoimmune diseases.

[0005] The first aspect of this invention provides the use of an antibody that specifically binds to CD132 or an antigen-binding fragment thereof in the preparation of a medicament for treating and / or preventing autoimmune diseases.

[0006] A second aspect of the present invention provides the use of an antibody that specifically binds to CD132 or an antigen-binding fragment thereof in the preparation of a medicament for the treatment and / or prevention of autoimmune diseases, said autoimmune diseases being insensitive to ruxolitinib and / or secukinumab, and / or unresponsive to ruxolitinib and / or secukinumab.

[0007] In some implementations, the autoimmune disease is selected from vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, and / or aplastic anemia.

[0008] A third aspect of the present invention provides the use of an antibody that specifically binds to CD132 or an antigen-binding fragment thereof in the preparation of a medicament for improving skin pigmentation.

[0009] In some implementations, the skin is the skin of the extremities and / or the hands and feet.

[0010] The fourth aspect of the present invention provides the use of an antibody that specifically binds to CD132 or an antigen-binding fragment thereof in the preparation of a medicament for reducing the Psoriasis Area and Severity Index (PASI).

[0011] The fifth aspect of this invention provides the use of an antibody that specifically binds to CD132 or an antigen-binding fragment thereof in the preparation of a medicament for reducing inflammatory factors.

[0012] In some embodiments, the inflammatory factors are selected from IFNγ, TNFα, IL-6, IL-17 and / or IL-5.

[0014] The present invention also provides the use of antibodies that specifically bind to CD132 or antigen-binding fragments thereof in the preparation of medicaments for the treatment of graft-versus-host disease (GVHD).

[0015] The present invention also provides the use of antibodies that specifically bind to CD132 or antigen-binding fragments thereof in the preparation of medicaments for reducing inflammatory cell infiltration in tissues.

[0016] In some embodiments, the tissue is selected from the liver, lungs, and / or kidneys.

[0017] The sixth aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD132.

[0018] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment comprising the following complementarity-determining regions:

[0019] The heavy chain variable region shown in SEQ ID NO:7 contains HCDR1, HCDR2, and HCDR3; and

[0020] The light chain variable region shown in SEQ ID NO:9 contains LCDR1, LCDR2 and LCDR3.

[0021] In some embodiments, the antibody or its antigen-binding fragment comprises the following heavy chain variable regions and light chain variable regions:

[0022] The heavy chain variable region contains the following three CDRs: HCDR1 with sequence SEQ ID NO.1, HCDR2 with sequence SEQ ID NO.2, and HCDR3 with sequence SEQ ID NO.3; and,

[0023] The light chain variable region contains the following three CDRs: LCDR1 with sequence SEQ ID NO.4, LCDR2 with sequence SEQ ID NO.5, and LCDR3 with sequence SEQ ID NO.6.

[0024] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0025] The heavy chain variable region (VH) shown in SEQ ID NO.7 and the light chain variable region (VL) shown in SEQ ID NO.9.

[0026] In some embodiments, the nucleotides encoding the heavy chain variable region (VH) of the antibody or antigen-binding fragment are shown in SEQ ID NO. 8, and the nucleotides encoding the light chain variable region (VL) are shown in SEQ ID NO. 10.

[0027] In some implementations, the antibody or antigen-binding fragment further includes a constant region, which is the kappa chain constant region Ckappa and the human IgG4 constant region CH1-CH3.

[0028] Sequence information

[0029] The sequence information involved in this invention is provided in Table 1 below.

[0030] Table 1. Sequence information involved in this invention.

[0031]

[0032]

[0033] The seventh aspect of the present invention provides a method for treating an autoimmune disease, wherein a therapeutically effective amount of an antibody or antigen-binding fragment of the sixth aspect of the present invention is provided to the patient.

[0034] In some embodiments, the autoimmune disease is an autoimmune disease that is insensitive to ruxolitinib and / or secukinumab treatment, and / or relapses after ruxolitinib and / or secukinumab treatment.

[0035] In some implementations, the autoimmune disease is vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis, or aplastic anemia.

[0036] The eighth aspect of the present invention provides a method for improving skin pigmentation, wherein a therapeutically effective amount of an antibody or antigen-binding fragment of the sixth aspect of the present invention is provided to a patient.

[0037] In some implementations, the skin is the skin of the extremities and / or the hands and feet.

[0038] The ninth aspect of the present invention provides a method for reducing the Psoriasis Area and Severity Index (PASI), wherein the patient is provided with a therapeutically effective amount of an antibody or antigen-binding fragment of the sixth aspect of the present invention.

[0039] The tenth aspect of the present invention provides a method for reducing inflammatory factors, wherein a therapeutically effective amount of an antibody or antigen-binding fragment of the sixth aspect of the present invention is provided to a patient.

[0040] The eleventh aspect of the present invention provides a method for treating graft-versus-host disease (GVHD), wherein a therapeutically effective amount of an antibody or antigen-binding fragment of the sixth aspect of the present invention is provided to the patient.

[0041] The twelfth aspect of the present invention provides a method for reducing inflammatory cell infiltration in tissues, wherein a therapeutically effective amount of an antibody or antigen-binding fragment of the sixth aspect of the present invention is provided to the patient.

[0042] In some embodiments, the tissue is selected from the liver, lungs, and / or kidneys.

[0043] Beneficial effects:

[0044] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0045] The antibody of this invention can effectively treat autoimmune diseases that are insensitive to ruxolitinib and / or secukinumab, and / or unresponsive to ruxolitinib and / or secukinumab, especially vitiligo that is insensitive to ruxolitinib or relapses after discontinuation of ruxolitinib, and severe psoriasis that is insensitive to secukinumab or relapses after discontinuation of secukinumab. Treatment with the antibody of this invention can significantly increase skin pigmentation, reduce the area of ​​white patches, decrease the probability of vitiligo recurrence, and effectively reduce the Psoriasis Area and Severity Index (PASI), decrease inflammatory factors in the body, reduce inflammatory cell infiltration and tissue damage, showing broad promise in the treatment of autoimmune diseases. Attached Figure Description

[0046] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0047] Figure 1 h2D4H4K12 is used to reduce the area of ​​vitiligo.

[0048] Figure 2 h2D4H4K12 is effective in relieving psoriasis symptoms.

[0049] Figure 3 h2D4H4K12 is used to reduce inflammatory factors.

[0050] Figure 4 h2D4H4K12 is used to alleviate graft-versus-host disease (GVHD). Detailed Implementation

[0051] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0052] Example 1:

[0053] Experimental Methods: On day 0, C57BL / 6 background mice (genetically modified C57BL / 6 background mice from the Biocytogen breeding cluster to replace the endogenous full-length CD132 domain with the corresponding human sequence) were injected intradermally with 2×10⁻⁶ oz. 5B16F10 cells (melanoma cells, purchased from Wuhan Pronosei Life Science Technology Co., Ltd.) were administered subcutaneously twice on days 4 and 10 at a dose of 10 mg / kg to deplete Treg cells in mice. Melanoma was surgically removed on day 12. On day 30, the disease progression in mice was observed. Mice with successfully induced vitiligo were selected and divided into two groups. Group A received subcutaneous PBS as a non-treatment control group, while Group B received subcutaneous h2D4H4K12 at a dose of 10 mg / kg twice weekly for 4 weeks (Table 2). The area of ​​vitiligo patches in mice was photographed before and after treatment.

[0054] Table 2. Experimental drug administration and treatment protocols for each group of mice.

[0055] Grouping Recipient lineage Number of animals in each group Modeling situation mAb processing Group A <![CDATA[CD132 hu / hu ]]> 3 Subcutaneous injection of B16F10 No mAb (PBS) Group B <![CDATA[CD132 hu / hu ]]> 3 Subcutaneous injection of B16F10 h2D4H4K12

[0056] The results are as follows Figure 1 As shown, in vitiligo model mice, after the onset of the disease, mice treated with PBS (Group A) for one month showed progressive vitiligo with a significant increase in the area of ​​white patches; while mice treated with h2D4H4K12 (Group B) showed a significant reduction in the area of ​​white patches. This indicates that h2D4H4K12 can effectively alleviate the symptoms of vitiligo.

[0057] We also compared the efficacy of h2D4H4K12 with that of the existing vitiligo treatment drug ruxolitinib.

[0058] Ruxolitinib topical formulations have been approved by the FDA for the treatment of localized vitiligo lesions, demonstrating good efficacy and tolerability, particularly in sensitive areas such as the face. However, ruxolitinib is less effective in areas such as the extremities and hands and feet, and patients still face the risk of relapse after discontinuation of the medication. IL-15 and IL-21 signaling are crucial to the source of vitiligo recurrence (T cells resident at the lesion site). Because h2D4H4K12 can effectively block these two factors, it has a significant advantage in reducing vitiligo recurrence.

[0059] We validated this by intradermal injection of 2 × 10⁻⁶ C57BL / 6 background mice (genetically modified C57BL / 6 background mice from the Biocytogen breeding cluster to replace the endogenous full-length CD132 domain with the corresponding human sequence) 5A vitiligo model was induced using B16F10 cells (melanoma cells). The disease progression in mice was observed from day 30 to 60. Mice with successfully induced vitiligo were selected and divided into three groups. One group received no treatment, one group received subcutaneous h2D4H4K12 treatment (once a week), and one group received ruxolitinib cream (applied daily). Treatment lasted for one month before discontinuation. Skin pigmentation was assessed weekly, and pigment recovery was recorded. The speed, extent, and duration of skin color recovery after h2D4H4K12 and ruxolitinib treatments were compared. The results showed that:

[0060] h2D4H4K12 is significantly more effective than ruxolitinib in treating vitiligo, especially in areas such as the extremities and hands and feet, where it is highly effective. Furthermore, h2D4H4K12 can effectively reduce the recurrence of vitiligo.

[0061] Example 2:

[0062] Experimental Methods: On day 0, C57BL / 6 background mice (genetically modified C57BL / 6 background mice from the Biocytogen breeding cluster with the corresponding human sequence replacing the full-length domain of endogenous CD132) underwent dorsal hair removal (2×3cm) and were subcutaneously administered 300μg h2D4H4K12; from day 1 to day 5, 5% imiquimod was applied to the hair removal area of ​​the mice's back once daily at 62.5mg / time. The mice were weighed, and the dorsal skin lesions, rashes, and inflammation were observed; on day 7, PASI scores were performed, the mice were sacrificed, and the dorsal skin was harvested. After HE staining, the skin was observed for abnormal keratinization, inflammatory infiltration, etc. The experimental mice were grouped as shown in Table 3 below.

[0063] Table 3. Experimental drug administration and treatment protocols for each group of mice.

[0064] Grouping Recipient lineage Number of animals in each group Modeling situation mAb processing Blank group <![CDATA[CD132 hu / hu ]]> 6 Unmade No mAb (PBS) NC Group <![CDATA[CD132 hu / hu ]]> 6 Imiquimod modeling No mAb (PBS) h2D4H4K12 group <![CDATA[CD132 hu / hu ]]> 6 Imiquimod modeling h2D4H4K12

[0065] Table 4 PASI scores of mouse dorsal skin

[0066]

[0067] Table 5. Weight statistics of mice in each group

[0068]

[0069] The results are as follows Figure 2As shown in Table 4, after the onset of psoriasis in mice, the skin on the backs of mice in the untreated group (Blank group) was rosy and without scales, indicating normal skin. In the imiquimod-treated but untreated group (NC group), obvious erythematous scaling appeared on the backs, and the PASI score was significantly higher than that of the Blank group (p<0.01). However, after treatment with h2D4H4K12, the erythematous scaling was significantly reduced compared to the NC group (p<0.01), the PASI score was significantly lower (p<0.01), and the body weight was significantly increased (p<0.01) (Table 5). This indicates that h2D4H4K12 can significantly alleviate the symptoms of psoriasis.

[0070] We also compared the efficacy of h2D4H4K12 with that of the existing psoriasis treatment secukinumab.

[0071] Secukinumab is a humanized antibody targeting IL-17A and is currently widely used to treat psoriasis. We compared the therapeutic effects of h2D4H4K12 and secukinumab on psoriasis, and the experimental mice were grouped as shown in Table 6 below.

[0072] Table 6. Experimental drug administration and treatment protocols for each group of mice.

[0073] Modeling situation quantity Dosage time Dosage Administration method Blank group Unmade 6 / / / NC Group Imiquimod modeling 6 / / / h2D4H4K12-A Group Imiquimod modeling 6 Day 7, -3, 0 100μg / time Tail vein injection h2D4H4K12-B Group Imiquimod modeling 6 Days 0, 2, and 5 100μg / time Tail vein injection secukinumab - Group A Imiquimod modeling 6 Day 7, -3, 0 100μg / time Tail vein injection secukinumab-Group B Imiquimod modeling 6 Days 0, 2, and 5 100μg / time Tail vein injection

[0074] Mice were monitored daily by body weight and skin photography scores. On Day 7, mice were sacrificed, and skin was subjected to HE staining for pathological examination. Flow cytometry was used to detect differences in immune cells in the skin of different groups. qPCR was used to detect differences in the expression of various inflammatory factors in the skin, and CBA flow cytometry was used to detect differences in various inflammatory factors in peripheral blood. The results showed that h2D4H4K12 was significantly more effective than secukinumab in treating psoriasis.

[0075] Example 3:

[0076] Experimental Methods: Four cynomolgus macaques (two males and two females) were selected and divided into two groups (as shown in Table 7). H2D4H4K12 was administered subcutaneously weekly at a concentration of 200 mg / kg in the high-concentration group and 50 mg / kg in the low-concentration group for five consecutive weeks. Peripheral blood inflammatory factor levels were measured 3 days before administration, on the day of administration, and on days 15 and 30 after administration.

[0077] Table 7. Experimental drug administration and treatment protocols for each group of mice.

[0078] Grouping Recipient lineage Number of animals in each group Dosage serial number Group A Crab-eating macaques 2(1 male and 1 female) 200mg / kg 161、162 Group B Crab-eating macaques 2(1 male and 1 female) 50mg / kg 164、165

[0079] The results are as follows Figure 3As shown, after administration of h2D4H4K12, the levels of TNFα, IFNγ, and IL-6 in the peripheral blood of four monkeys gradually decreased over time.

[0080] Example 4:

[0081] Experimental procedure: On day 0, 1×10⁻⁶ mice with B-NDG background (Biocytogen, catalog number 110586) were injected via the tail vein. 7 Personally sourced PBMCs. Peripheral blood was collected from model mice on day 14 for human T / B cell staining and flow cytometry analysis to observe cell colonization. Mice with stable human T / B cell colonization were considered to have successfully established graft-versus-host disease (GVHD). Based on cell colonization and body weight, successfully modeled mice were evenly divided into two groups: an NC group (no treatment) and an h2D4H4K12 group (h2D4H4K12 administered subcutaneously at a dose of 10 mg / kg), administered twice weekly until the endpoint. A group of unmodeled mice was retained as the Blank group (negative control). Mouse body weight and survival rate were recorded during treatment. At the endpoint, liver, spleen, and kidney tissues were collected for HE staining to observe tissue damage and inflammatory infiltration. The experimental drug administration and treatment regimens for each group are shown in Table 8.

[0082] Table 8. Experimental drug administration and treatment protocols for each group of mice.

[0083] Grouping Recipient lineage Number of animals in each group Modeling situation mAb processing Blank group NSG 6 Unprocessed No mAb (PBS) NC Group NSG 6 Tail vein injection of PBMCs No mAb (PBS) h2D4H4K12 group NSG 6 Tail vein injection of PBMCs h2D4H4K12

[0084] The results are as follows Figure 4 As shown: Mice in the negative control group (Blank group) maintained continuous weight gain and good survival status, with no inflammatory cell infiltration or tissue damage observed in any tissue at the endpoint. In contrast, mice in the NC group (after GVHD modeling) experienced weight loss over time, poor condition, and eventual death. At the endpoint, the liver, lungs, and kidneys of NC group mice showed extensive inflammatory infiltration and significant tissue damage. Administration of h2D4H4K12 effectively blocked the occurrence of GVHD in mice. The infiltration of inflammatory cells in the liver, lungs, and kidneys of h2D4H4K12-treated mice was significantly less than that of the NC group, and most mice treated with h2D4H4K12 were spared from weight loss and death.

[0085] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. All components not explicitly stated in this embodiment can be implemented using existing technology.

Claims

1. The use of an antibody that specifically binds to CD132 or an antigen-binding fragment thereof in the preparation of a medicament for the treatment and / or prevention of an autoimmune disease, wherein the autoimmune disease is an autoimmune disease that is insensitive to ruxolitinib and / or secukinumab, and / or unresponsive to ruxolitinib and / or secukinumab.

2. The application according to claim 1, wherein the autoimmune disease is selected from vitiligo, psoriasis, alopecia areata, inflammatory bowel disease, systemic sclerosis and / or aplastic anemia.

3. Application of antibodies that specifically bind to CD132 or their antigen-binding fragments in the preparation of drugs for improving skin pigmentation.

4. The application according to claim 3, wherein, The skin in question is the skin of the extremities and / or the hands and feet.

5. The use of antibodies that specifically bind to CD132 or their antigen-binding fragments in the preparation of drugs for reducing the Psoriasis Area and Severity Index (PASI).

6. The use of antibodies that specifically bind to CD132 or their antigen-binding fragments in the preparation of drugs for reducing inflammatory factors.

7. The application according to claim 6, wherein, The inflammatory factors are selected from IFNγ, TNFα, IL-6, IL-17 and / or IL-5.

8. The use of antibodies that specifically bind to CD132 or their antigen-binding fragments in the preparation of drugs for the treatment of graft-versus-host disease (GVHD).

9. The use of antibodies that specifically bind to CD132 or their antigen-binding fragments in the preparation of drugs for reducing inflammatory cell infiltration in tissues.

10. The application according to claim 9, wherein, The tissues are selected from the liver, lungs, and / or kidneys.

11. The application according to any one of claims 1-10, wherein, The antibody or its antigen-binding fragment comprises: HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO:7; and / or The light chain variable region shown in SEQ ID NO:9 contains LCDR1, LCDR2 and LCDR3.

12. The application according to claim 11, wherein, The antibody or its antigen-binding fragment comprises heavy chain variable regions of the following three CDRs: HCDR1 with the sequence of SEQ ID NO.1, HCDR2 with the sequence of SEQ ID NO.2, and HCDR3 with the sequence of SEQ ID NO.3; and / or, The light chain variable region contains the following three CDRs: LCDR1 with sequence SEQ ID NO.4, LCDR2 with sequence SEQ ID NO.5, and LCDR3 with sequence SEQ ID NO.

6.

13. The application according to claim 12, wherein, The antibody or antigen-binding fragment comprises the heavy chain variable region shown in sequence SEQ ID NO.7 and the light chain variable region shown in sequence SEQ ID NO.9.

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