Application of NK cells amplified in vitro in treatment of autoimmune diseases

Abstract

The invention provides an application of in-vitro amplified NK cells in treatment of autoimmune diseases, the NK cells are obtained by performing in-vitro induced amplification on peripheral blood mononuclear cells, and the in-vitro induced amplification is performed in a culture medium containing interleukin and / or an antibody. Therefore, the NK cell disclosed by the invention has double advantages that on one hand, the NK cell shows phenotypic characteristics of a CD56light tissue resident type NK cell, has the capability of penetrating pathological tissues, and can be used for directly removing abnormal B cells and T cells in tissues of patients with autoimmune diseases; on the other hand, activated receptors such as NKG2D and CD16 and chemokine receptors such as CXCR3 can be stably and highly expressed, and the removal efficiency is further improved by enhancing the recognition and killing capacity on target cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular, the present application relates to the use of in vitro expanded NK cells in the treatment of autoimmune diseases. BACKGROUND

[0002] Systemic Lupus Erythematosus (SLE) is a severe chronic autoimmune disease that involves multiple organs and tissues. Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that can cause damage to multiple organs and tissues throughout the body. Its main pathogenesis is related to the abnormal activation of T and B lymphocytes, characterized by abnormal B cell function and autoantibody production, including the destruction of immune tolerance to double-stranded DNA (dsDNA) and nuclear antigens, leading to a decrease in T lymphocytes, a decrease in the function of suppressor T cells, and an overgrowth of B cells, resulting in the production of a large number of autoantibodies against dsDNA and other nuclear antigens, the formation of a large number of immune complexes, and the deposition of different tissues and organs (including joints, skin, and kidneys, etc.) to trigger inflammatory reactions and severe damage to tissues and organs.

[0003] Current clinical treatment mainly relies on three types of drugs: glucocorticoids (such as prednisone), immunosuppressants (such as cyclophosphamide, mycophenolate mofetil), and biological agents (such as belimumab). However, these existing therapies have significant limitations. First, there are significant long-term safety issues: high-dose hormone therapy often leads to a significant increase in the risk of osteoporosis, diabetes, and infection; immunosuppressants are associated with bone marrow suppression, gonadotoxicity, and severe side effects such as liver and kidney function damage, especially for patients with moderate to severe active systemic lupus erythematosus who require high-dose hormone and immunosuppressant therapy, who are often at high risk of organ failure and even death. Second, the therapeutic effect is not satisfactory, with a high proportion of insufficient response, for example, the effective rate of belimumab is only about 40-50%, and about 30-40% of patients do not respond well to existing biological agents, with a high proportion of refractory cases. Finally, existing treatment methods focus on symptom control and are difficult to achieve fundamental reconstruction of immune homeostasis, leading to frequent disease recurrence, with a recurrence rate of up to 50-60% after drug withdrawal.

[0004] Currently, there is no truly effective treatment method to address this challenge, and there is no reliable strategy to alleviate or even cure the disease without drugs, and patients usually need to take medication for life. Therefore, there is an urgent need to develop new treatment methods to alleviate the suffering of patients. SUMMARY

[0005] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. Based on this, the present application provides a use of NK cells in the treatment of autoimmune diseases, which have unique phenotypic and functional characteristics: they not only highly express activation receptors such as NKG2D, CD16, and chemotactic factor receptors such as CXCR3, but also present CD56 bright The phenotypic characteristics of tissue-resident NK cells. These properties endow them with the ability to penetrate diseased tissues, allowing them to directly eliminate abnormal B cells and T cells in the tissues of patients with autoimmune diseases, thereby effectively eliminating pathogenic lymphocytes associated with such diseases. In addition, when the NK cells are used in combination with anti-CD19 antibodies or anti-CD20 antibodies, not only can they further enhance the elimination effect of abnormal B cells in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases, but they can also simultaneously eliminate abnormally activated pathogenic T cells in the patient's body, thereby exerting a more comprehensive therapeutic effect.

[0006] The present application is based on the following findings of the inventors: Currently, the existing treatment techniques for systemic lupus erythematosus (SLE) and other autoimmune diseases still have many defects that are difficult to overcome, mainly in the following two aspects: On the one hand, the existing antibody therapy targeting B cells (such as rituximab against CD20 and belimumab against soluble human B lymphocyte stimulating factor BAFF) has limited efficacy. Its limitations are: antibody molecules are difficult to penetrate lymphoid organs and inflammatory tissues, and can only eliminate B cells in the blood circulation, but cannot persistently eliminate tissue-resident autoreactive B cells and memory B cells; anti-CD20 monoclonal antibodies cannot act on CD20-negative plasma cells and the autoantibodies they produce; more importantly, such therapies cannot eliminate abnormally activated autoreactive T cells and the inflammation they cause.

[0007] On the other hand, the existing treatment regimens focus excessively on B cells, while ignoring the key role of T lymphocytes in SLE and other autoimmune diseases. T cells not only participate in immune tolerance regulation, promote inflammatory cytokine secretion, assist B cell differentiation and autoantibody production, but also promote disease progression through various mechanisms: for example, CD4 + IL-2, IL-12, and IFN-γ secreted by T cells can promote the activation and proliferation of cytotoxic T cells and macrophages, which damage renal parenchymal cells by directly infiltrating or recruiting macrophages, NKT cells, etc., leading to renal tubular dysfunction and fibrosis, and playing a central role in the pathogenesis of lupus nephritis; CD4 + Cytokines such as IL-17 secreted by T cells can also induce the expression of pro-inflammatory factors, chemotactic factors, and matrix metalloproteinases, exacerbating inflammatory cell infiltration and tissue destruction.

[0008] Since the prior art can only clear abnormal B cells, it cannot target and clear abnormal activated pathological T cells, resulting in that the treatment can achieve short-term effect, but it is difficult to achieve radical cure, the maintenance of curative effect is poor, the recurrence rate is high, and the long-term remission rate is low.

[0009] Natural killer cells (NK) as the core effector cells of the innate immune system, are the first line of immune surveillance in the body. NK cells express a variety of activating receptors and inhibitory receptors, and can accurately distinguish between normal cells and abnormal cells by recognizing the corresponding ligands on the surface of target cells through NK cell receptors (NKR), rapidly recognize and kill virus-infected, malignant transformed, aged, abnormal and damaged cells, and at the same time, can regulate acquired immune response by secreting a variety of cytokines and chemokines such as interferon and TNF-α, and play the role of immune surveillance and maintaining normal operation of the body. These natural characteristics of NK cells make them have the potential for treatment in the field of autoimmune disease treatment.

[0010] Therefore, in the first aspect of the present application, the present application proposes the use of NK cells in the preparation of a drug for treating autoimmune diseases, wherein the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is carried out in a culture medium containing interleukin and / or antibodies. Thus, the NK cells of the present application have double advantages: on the one hand, they present CD56 bright The phenotype characteristics of tissue-resident NK cells, have the ability to penetrate diseased tissues, and can directly clear abnormal B cells and T cells in the tissues of patients with autoimmune diseases; on the other hand, they can stably express activating receptors such as NKG2D and CD16, and chemokine receptors such as CXCR3, thereby enhancing the recognition and killing ability of target cells and further improving the clearance efficiency.

[0011] In the second aspect of the present application, the present application proposes a pharmaceutical composition for combined treatment of autoimmune diseases, characterized in that it comprises: an anti-CD19 antibody or an anti-CD20 antibody, and NK cells; wherein the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is carried out in a culture medium containing interleukin and / or antibodies. As mentioned above, the NK cells of the present application present CD56 brightThe tissue-resident NK cells have the phenotypic characteristics and the ability to penetrate the diseased tissue, and can directly eliminate abnormal B cells and T cells in the tissue of the patient with autoimmune diseases. In addition, the tissue-resident NK cells can stably express FcγRIIIa receptor (CD16), and can synergize with the anti-CD19 or anti-CD20 antibody through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby significantly enhancing the elimination effect on the autoreactive B cells. The combination of the two mechanisms can greatly improve the treatment efficiency of the autoimmune diseases such as systemic lupus erythematosus (SLE).

[0012] In a third aspect, the present application provides a drug combination for treating autoimmune diseases, comprising: NK cells as a first active ingredient, and an anti-CD19 antibody or an anti-CD20 antibody as a second active ingredient; wherein the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is performed in a culture medium containing interleukins and / or antibodies. As described above, the NK cells can synergize with the anti-CD19 or anti-CD20 antibody through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby enhancing the elimination effect on the autoreactive B cells, and being used for treating autoimmune diseases such as SLE. Therefore, the drug combination of the present application can effectively treat autoimmune diseases.

[0013] In a fourth aspect, the present application provides a kit for treating autoimmune diseases, comprising: NK cells as a first active ingredient, and an anti-CD19 antibody or an anti-CD20 antibody as a second active ingredient; wherein the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is performed in a culture medium containing interleukins and / or antibodies. As described above, the NK cells can synergize with the anti-CD19 or anti-CD20 antibody through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby enhancing the elimination effect on the autoreactive B cells, and being used for treating autoimmune diseases such as SLE. Therefore, the kit of the present application can effectively treat autoimmune diseases.

[0014] In a fifth aspect, the present application provides the use of the pharmaceutical composition of the first aspect, the drug combination of the second aspect, or the kit of the third aspect in the preparation of a medicament for treating autoimmune diseases. As described above, the pharmaceutical composition, the drug combination and the kit of the present application can effectively eliminate abnormal B cells in the patient with autoimmune diseases, and therefore, the preparation of the medicament can effectively treat autoimmune diseases.

[0015] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. Attached Figure Description

[0016] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This describes the expression of receptors in PB-NK cells before and after expansion according to Example 1 of the present invention. Figure 2 This describes the expression of CD19 and CD20 in RAJI cells and RPMI8226 cells according to Example 2 of the present invention; Figure 3 These are the test results of the killing rate of RAJI cells in each experimental group according to Example 3 of the present invention; Figure 4 These are the test results of the killing rate of RPMI8226 cells in each experimental group according to Example 3 of the present invention; Figure 5 CD19 in the SLE-PBMC according to Embodiment 4 of the present invention + and CD20 + The sorting effect of B cells; Figure 6 The results of the killing rate test on SLE-PBMC-B cells in each experimental group according to Example 5 of the present invention; Figure 7 This describes the in vivo clearance effect of each experimental group on B cells according to Embodiment 6 of the present invention; Figure 8 This describes the in vivo clearance effect of T cells in the NK cell therapy group according to Example 6 of the present invention; Figure 9 This describes the sorting effect of T cells in SLE-PBMCs and healthy donor PBMCs according to Embodiment 7 of the present invention; Figure 10 This describes the expression of NKR-related ligands in SLE-PBMC-T cells and PBMC-T cells according to Example 8 of the present invention. Detailed Implementation

[0017] The embodiments of the present invention are described in detail below, and are intended to explain the present invention, but should not be construed as limiting the present invention.

[0018] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0019] For the sake of brevity, only some numerical ranges are specifically disclosed herein. However, any upper limit can be combined with any lower limit to create a range not explicitly recited; and any lower limit can be combined with any other lower limit to create a range not explicitly recited, as well as any upper limit can be combined with any other upper limit to create a range not explicitly recited. Further, each individual disclosed point or singular number can be combined as a lower limit or upper limit with any other point or singular number or with other lower limits or upper limits to create a range not explicitly recited.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application; the description and drawings of this application and the terms used therein, including the abstract, are not intended to limit the scope of the present application and are provided for illustrative purposes only. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] In this document, the terms "comprise" or "comprising" are open- ended, that is, they mean "including but not limited to," and thus specify the presence of stated features but do not preclude the presence of other features.

[0022] In this document, the terms "optional," "optionally," or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0023] In this document, the term "treatment" of a disease refers to preventing or ameliorating a disease or at least one clinical symptom of a disease, reducing the risk of developing a disease or at least one clinical symptom of a disease, reducing the development of a disease or at least one clinical symptom of a disease, or reducing the risk of a disease or at least one clinical symptom of a disease. "Treatment" also refers to inhibiting a disease, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both, and to abrogation of at least one physical parameter or of a manifestation of the disease, which can or can not be discernible to the patient. In some embodiments, "treatment" refers to delaying the onset of a disease or at least one or more symptoms thereof in an individual who can be exposed to or predisposed to a disease or condition, even though the individual can not yet be experiencing or displaying symptoms of the disease.

[0024] In the present text, the term "therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treatment of a disease or at least one of the clinical symptoms of the disease, is sufficient to treat the disease or the clinical symptoms thereof. The "therapeutically effective amount" can vary depending on, for example, the drug, the disease and / or the symptoms of the disease, the severity of the disease and / or the symptoms of the disease, the age, weight and / or health condition of the subject to be treated, and the judgment of the prescribing physician. The appropriate amount in any given case can be determined by the person skilled in the art or can be determined by routine experimentation.

[0025] In the case of description of the time relationship, for example, in the case of description of the time sequence such as "~ after", "then ~", "~ then", "~ before" and the like, unless "immediately" or "directly" is used, discontinuous cases are also included.

[0026] The present application proposes the use of NK cells in the preparation of a drug for treating autoimmune diseases, a pharmaceutical composition, a drug combination, a kit and the use thereof, which will be described in detail respectively as follows.

[0027] Use of NK cells in the preparation of a drug for treating autoimmune diseases In the first aspect of the present application, the present application proposes the use of NK cells in the preparation of a drug for treating autoimmune diseases, wherein the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is carried out in a culture medium containing interleukins and / or antibodies. Thus, the NK cells of the present application have double advantages: on the one hand, they present CD56 bright The NK cells of the present application have the phenotypic characteristics of tissue-resident NK cells, have the ability to penetrate diseased tissues, and can directly eliminate abnormal B cells and T cells in the tissues of patients with autoimmune diseases; on the other hand, they can stably express high levels of activation receptors such as NKG2D and CD16, and chemotactic factor receptors such as CXCR3, thereby enhancing the recognition and killing ability of target cells and further improving the elimination efficiency.

[0028] In some embodiments, the CD3 - CD56 + The proportion of NK cells is ≥ 99.0%. In some embodiments, the CD3 - CD56 + CD16 + The proportion of NK cells is ≥ 96.0%. In some embodiments, the CD3 - CD56 + NKG2D + The proportion of NK cells is ≥ 98.0%. In some embodiments, the CD3 - CD56 Bright The proportion of NK cells is ≥97%.

[0029] In some embodiments, the CD19 + The proportion of B cells is ≤1.0%. In some embodiments, the CD3 + CD56 - The proportion of T cells is ≤1.0%.

[0030] In some embodiments, the interleukins include at least one of IL-21, IL-2. In some embodiments, the final concentration of the interleukins in the culture medium is 1-100 ng / mL. For example, it can be 1 ng / mL, 3.5 ng / mL, 6.5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the above values.

[0031] In some embodiments, the antibodies include at least one of CD16 antibodies, CD3 antibodies. In some embodiments, the final concentration of the antibodies in the culture medium is 0.5 ng / mL-5 μg / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, etc., or can be a range consisting of any of the above values.

[0032] In some embodiments, the in vitro induction and expansion of peripheral blood mononuclear cells includes: separating and purifying first NK cells from the peripheral blood mononuclear cells; placing the first NK cells in a first culture system for first culture treatment; after 3-5 days of culture, adding first cytokines to the first culture system for second culture treatment to obtain the NK cells; wherein during the second culture treatment, the culture system is replaced with a second culture system. Thus, the method of the present application can make the NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0033] In some embodiments, the first culture system includes a serum-free culture medium and at least one of the CD3 antibodies, the CD16 antibodies, the IL-2. Thus, by using the first culture system of the present application to culture cells, the activation and proliferation signaling pathways of NK cells can be pre-activated, the NK cells are in a good pre-differentiation and proliferation state, and good cell activity is maintained.

[0034] In some embodiments, the concentration of the CD3 antibody in the first culture system is 0.5-5 ng / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0035] In some embodiments, the concentration of the CD16 antibody in the first culture system is 1-5 μg / mL. For example, it can be 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, etc., or a range consisting of any of the aforementioned values.

[0036] In some embodiments, the concentration of the IL-2 in the first culture system is 3.5-6.5 ng / mL. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0037] In some embodiments, the first cytokine is selected from the IL-21. In some embodiments of the present application, the concentration of the IL-21 in the first culture system is 20-100 ng / mL. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0038] In some embodiments, the second culture system comprises a serum-free medium and at least one of the IL-2, the IL-21. Thus, by changing to the second culture system, the activation and proliferation of the NK cells can be further maintained, and the cell aging signal during the cell expansion process can be prevented or delayed, so that the NK cells can maintain a persistent proliferation ability and an activated state.

[0039] In some embodiments, the concentration of the IL-2 in the second culture system is 3.5-6.5 ng / mL. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0040] In some embodiments, the IL-21 has a concentration of 20-100 ng / mL in the second culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the above values.

[0041] In some embodiments, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

[0042] Pharmaceutical composition In a second aspect of the present application, a pharmaceutical composition for treating autoimmune diseases is provided. According to embodiments of the present application, the pharmaceutical composition comprises: an anti-CD19 antibody or an anti-CD20 antibody, and NK cells; wherein the NK cells are obtained by in vitro induced expansion of peripheral blood mononuclear cells, and the in vitro induced expansion is performed in a culture medium containing interleukin and / or an antibody. As described above, the NK cells of the present application exhibit the phenotypic characteristics of tissue-resident NK cells, have the ability to penetrate diseased tissues, and can directly eliminate abnormal B cells and T cells in the tissues of patients with autoimmune diseases. In addition, the NK cells can stably express high levels of FcγRIIIa receptors (i.e., CD16), and can synergize with anti-CD19 or anti-CD20 antibodies through antibody-dependent cell-mediated cytotoxicity (ADCC) to significantly enhance the elimination of autoreactive B cells. The combination of these dual mechanisms can significantly improve the treatment efficacy of autoimmune diseases such as systemic lupus erythematosus (SLE). bright Tissue-resident NK cells exhibit the phenotypic characteristics of tissue-resident NK cells, have the ability to penetrate diseased tissues, and can directly eliminate abnormal B cells and T cells in the tissues of patients with autoimmune diseases. In addition, the NK cells can stably express high levels of FcγRIIIa receptors (i.e., CD16), and can synergize with anti-CD19 or anti-CD20 antibodies through antibody-dependent cell-mediated cytotoxicity (ADCC) to significantly enhance the elimination of autoreactive B cells. The combination of these dual mechanisms can significantly improve the treatment efficacy of autoimmune diseases such as systemic lupus erythematosus (SLE).

[0043] In some embodiments, the NK cells have a CD3 - CD56 + The proportion of NK cells is ≥99.0%. In some embodiments, the NK cells have a CD3 - CD56 + CD16 + The proportion of NK cells is ≥96.0%. In some embodiments, the NK cells have a CD3 - CD56 + NKG2D + The proportion of NK cells is ≥98.0%. In some embodiments, the CD3 - CD56 Bright The proportion of NK cells is ≥97%.

[0044] In some embodiments, the CD19 + The proportion of B cells is ≤1.0%. In some embodiments, the CD3 + CD56 - The proportion of T cells is ≤1.0%.

[0045] In some embodiments, the interleukin comprises at least one of IL-21, IL-2. In some embodiments, the final concentration of the interleukin in the culture medium is 1-100 ng / mL. For example, it can be 1 ng / mL, 3.5 ng / mL, 6.5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, etc., or it can be a range consisting of any of the above values.

[0046] In some embodiments, the antibody comprises at least one of CD16 antibody, CD3 antibody. In some embodiments, the final concentration of the antibody in the culture medium is 0.5 ng / mL-5 μg / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, etc., or it can be a range consisting of any of the above values.

[0047] In some embodiments, the in vitro induction and expansion of peripheral blood mononuclear cells comprises: separating and purifying first NK cells from the peripheral blood mononuclear cells; placing the first NK cells in a first culture system for first culture treatment; after 3-5 days of culture, adding a first cytokine to the first culture system for second culture treatment to obtain the NK cells; wherein during the second culture treatment, the culture system is replaced with a second culture system. Thus, the method of the present application can make the NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0048] In some embodiments, the first culture system comprises a serum-free culture medium and at least one of the CD3 antibody, the CD16 antibody, the IL-2. Thus, by using the first culture system of the present application to culture cells, the activation and proliferation signaling pathways of NK cells can be pre-activated, making the NK cells in a good pre-differentiation and proliferation state, and maintaining good cell activity.

[0049] In some embodiments, the concentration of the CD3 antibody in the first culture system is 0.5-5 ng / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0050] In some embodiments, the concentration of the CD16 antibody in the first culture system is 1-5 μg / mL. For example, it can be 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, etc., or a range consisting of any of the aforementioned values.

[0051] In some embodiments, the concentration of the IL-2 in the first culture system is 3.5-6.5 ng / mL. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0052] In some embodiments, the first cytokine is selected from the IL-21. In some embodiments of the present application, the concentration of the IL-21 in the first culture system is 20-100 ng / mL. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0053] In some embodiments, the second culture system comprises a serum-free medium and at least one of the IL-2, the IL-21. Thus, by changing to the second culture system, the activation and proliferation of the NK cells can be further maintained, and the cell aging signal during the cell expansion process can be prevented or delayed, so that the NK cells can maintain a persistent proliferation ability and an activated state.

[0054] In some embodiments, the concentration of the IL-2 in the second culture system is 3.5-6.5 ng / mL. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or a range consisting of any of the aforementioned values.

[0055] In some embodiments, the IL-21 has a concentration of 20-100 ng / mL in the second culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the above values.

[0056] In some embodiments, the CD19 antibody includes, but is not limited to, at least one of inebilizumab, tanibotumab. In some embodiments, the CD20 antibody includes, but is not limited to, at least one of rituximab, obinutuzumab, ofatumumab, ocrelizumab.

[0057] In some embodiments, the autoimmune disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma.

[0058] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable excipient or carrier.

[0059] In this context, the term "pharmaceutically acceptable" indicates that the pharmaceutical composition is capable of administration to a subject without producing adverse physiologic effects that would outweigh the benefits of the administration of the pharmaceutical composition. For example, a "pharmaceutically acceptable excipient" refers to an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable.

[0060] In some embodiments, the pharmaceutically acceptable excipient includes one or more of a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, a clathrate agent, a humectant, an absorbent, a diluent, a flocculating agent, a deflocculating agent, a filter aid, and a release retardant.

[0061] In some embodiments, the pharmaceutically acceptable carrier includes water, saline, a liposome, a lipid, a protein, a protein-antibody conjugate, a peptide substance, cellulose, a nanogel, or a combination thereof. The selection of the carrier should be matched with the administration method, which is well known to those of ordinary skill in the art.

[0062] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space as long as it can act together to achieve the purpose of the present application. For example, the anti-CD19 antibody or anti-CD20 antibody in the pharmaceutical composition, and the NK cell can be administered to the subject as a whole, or administered to the subject separately, for example, the anti-CD19 antibody or anti-CD20 antibody (or the NK cell) is administered first, and then the NK cell (or the anti-CD19 antibody or anti-CD20 antibody) is administered. When the components contained in the pharmaceutical composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0063] Drug combination and kit In a third aspect of the present application, a drug combination for treating autoimmune diseases is provided. According to an embodiment of the present application, the drug combination comprises: NK cells as a first active ingredient, and an anti-CD19 antibody or an anti-CD20 antibody as a second active ingredient; wherein the NK cells are obtained by in vitro induced expansion of peripheral blood mononuclear cells, and the in vitro induced expansion is performed in a culture medium containing interleukin and / or antibody. As described above, the NK cells can synergize with the anti-CD19 or anti-CD20 antibody through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby enhancing the clearance effect on autoreactive B cells, and being used for the treatment of autoimmune diseases such as SLE. Therefore, the drug combination of the present application can effectively treat autoimmune diseases.

[0064] In some embodiments, the CD3 - CD56 + The proportion of NK cells is ≥99.0%. In some embodiments, the CD3 - CD56 + CD16 + The proportion of NK cells is ≥96.0%. In some embodiments, the CD3 - CD56 + NKG2D + The proportion of NK cells is ≥98.0%. In some embodiments, the CD3 - CD56 Bright The proportion of NK cells is ≥97%.

[0065] In some embodiments, the CD19 + The proportion of B cells is ≤1.0%. In some embodiments, the CD3 + CD56 -T cell percentage ≤ 1.0%.

[0066] In some embodiments, the interleukin comprises at least one of IL-21, IL-2. In some embodiments, the final concentration of the interleukin in the culture medium is 1-100 ng / mL. For example, it can be 1 ng / mL, 3.5 ng / mL, 6.5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the above values.

[0067] In some embodiments, the antibody comprises at least one of CD16 antibody, CD3 antibody. In some embodiments, the final concentration of the antibody in the culture medium is 0.5 ng / mL-5 μg / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, etc., or can be a range consisting of any of the above values.

[0068] In some embodiments, the in vitro induction and expansion of peripheral blood mononuclear cells comprises: separating and purifying first NK cells from the peripheral blood mononuclear cells; placing the first NK cells in a first culture system for first culture treatment; after 3-5 days of culture, adding a first cytokine to the first culture system for second culture treatment to obtain the NK cells; wherein during the second culture treatment, the culture system is replaced with a second culture system. Thus, the method of the present application can make the NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0069] In some embodiments, the first culture system comprises a serum-free culture medium and at least one of the CD3 antibody, the CD16 antibody, and the IL-2. Thus, by using the first culture system of the present application to culture cells, the activation and proliferation signaling pathways of NK cells can be pre-activated, making the NK cells in a good pre-differentiation and proliferation state, and maintaining good cell activity.

[0070] In some embodiments, the concentration of the CD3 antibody in the first culture system is 0.5-5 ng / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, etc., or can be a range consisting of any of the above values.

[0071] In some embodiments, the CD16 antibody is at a concentration of 1-5 pg / mL in the first culture system. For example, it can be 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, etc., or can be a range consisting of any of the aforementioned values.

[0072] In some embodiments, the IL-2 is at a concentration of 3.5-6.5 ng / mL in the first culture system. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0073] In some embodiments, the first cytokine is selected from IL-21. In some embodiments of the present application, the IL-21 is at a concentration of 20-100 ng / mL in the first culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0074] In some embodiments, the second culture system comprises a serum-free medium and at least one of the IL-2, the IL-21. Thereby, by changing to the second culture system, the activation and proliferation of the NK cells can be further maintained and the cell senescence signal during the cell expansion process can be prevented or delayed, so that the NK cells can maintain a persistent proliferative capacity and activated state.

[0075] In some embodiments, the IL-2 is at a concentration of 3.5-6.5 ng / mL in the second culture system. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0076] In some embodiments, the IL-21 is at a concentration of 20-100 ng / mL in the second culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0077] In some embodiments, the CD19 antibody includes, but is not limited to, at least one of inebilizumab, tanibemcept.

[0078] In some embodiments, the autoimmune disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, and asthma.

[0079] In a fourth aspect of the present application, a kit for treating an autoimmune disease is provided. According to embodiments of the present application, the kit comprises: NK cells as a first active ingredient, and an anti-CD19 antibody or an anti-CD20 antibody as a second active ingredient; wherein the NK cells are obtained by in vitro induced expansion of peripheral blood mononuclear cells, and the in vitro induced expansion is performed in a culture medium containing interleukin and / or an antibody. As described above, the NK cells can synergize with the anti-CD19 or anti-CD20 antibody through antibody-dependent cell-mediated cytotoxicity (ADCC), thereby enhancing the clearance effect on autoreactive B cells, for the treatment of autoimmune diseases such as SLE. Therefore, the kit of the present application can effectively treat autoimmune diseases.

[0080] In some embodiments, the CD3 - CD56 + The proportion of NK cells is ≥ 99.0%. In some embodiments, the CD3 - CD56 + CD16 + The proportion of NK cells is ≥ 96.0%. In some embodiments, the CD3 - CD56 + NKG2D + The proportion of NK cells is ≥ 98.0%. In some embodiments, the CD3 - CD56 Bright The proportion of NK cells is ≥ 97%.

[0081] In some embodiments, the CD19 + The proportion of B cells is ≤ 1.0%. In some embodiments, the CD3 + CD56 - T cells account for ≤ 1.0%.

[0082] In some embodiments, the interleukin comprises at least one of IL-21, IL-2. In some embodiments, the final concentration of the interleukin in the culture medium is 1-100 ng / mL. For example, it can be 1 ng / mL, 3.5 ng / mL, 6.5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 70 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the above values.

[0083] In some embodiments, the antibody comprises at least one of CD16 antibody, CD3 antibody. In some embodiments, the final concentration of the antibody in the culture medium is 0.5 ng / mL-5 μg / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 100 ng / mL, 500 ng / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, etc., or can be a range consisting of any of the above values.

[0084] In some embodiments, the in vitro induction and expansion of peripheral blood mononuclear cells comprises: separating and purifying first NK cells from the peripheral blood mononuclear cells; placing the first NK cells in a first culture system for first culture treatment; after 3-5 days of culture, adding a first cytokine to the first culture system for second culture treatment to obtain the NK cells; wherein during the second culture treatment, the culture system is replaced with a second culture system. Thus, the method of the present application can make the NK cells in a good pre-differentiation and proliferation state, and maintain good cell activity.

[0085] In some embodiments, the first culture system comprises a serum-free culture medium and at least one of the CD3 antibody, the CD16 antibody, the IL-2. Thus, by using the first culture system of the present application to culture cells, the activation and proliferation signaling pathways of NK cells can be pre-activated, making the NK cells in a good pre-differentiation and proliferation state, and maintaining good cell activity.

[0086] In some embodiments, the concentration of the CD3 antibody in the first culture system is 0.5-5 ng / mL. For example, it can be 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, etc., or can be a range consisting of any of the above values.

[0087] In some embodiments, the CD16 antibody is at a concentration of 1-5 pg / mL in the first culture system. For example, it can be 1 pg / mL, 2 pg / mL, 3 pg / mL, 4 pg / mL, 5 pg / mL, etc., or can be a range consisting of any of the aforementioned values.

[0088] In some embodiments, the IL-2 is at a concentration of 3.5-6.5 ng / mL in the first culture system. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0089] In some embodiments, the first cytokine is selected from the IL-21. In some embodiments of the present application, the IL-21 is at a concentration of 20-100 ng / mL in the first culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0090] In some embodiments, the second culture system comprises a serum-free medium and at least one of the IL-2, the IL-21. Thereby, by changing to the second culture system, the activation and proliferation of the NK cells can be further maintained and the cell senescence signal during the cell expansion process can be prevented or delayed, so that the NK cells can maintain a persistent proliferative capacity and activated state.

[0091] In some embodiments, the IL-2 is at a concentration of 3.5-6.5 ng / mL in the second culture system. For example, it can be 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0092] In some embodiments, the IL-21 is at a concentration of 20-100 ng / mL in the second culture system. For example, it can be 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, etc., or can be a range consisting of any of the aforementioned values.

[0093] In some embodiments, the CD19 antibody includes, but is not limited to, at least one of inebilizumab, tanibirumab. In some embodiments, the CD20 antibody includes, but is not limited to, at least one of rituximab, obinutuzumab, ofatumumab, ocrelizumab.

[0094] In some embodiments, the autoimmune disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma.

[0095] Uses and methods In a fifth aspect, the present application provides use of the pharmaceutical composition of the second aspect, the pharmaceutical combination of the third aspect, or the kit of the fourth aspect in the preparation of a medicament for treating an autoimmune disease. As described above, the pharmaceutical composition, the pharmaceutical combination, and the kit of the present application can effectively eliminate abnormal B cells in a patient with an autoimmune disease, and thus, the preparation of a medicament can effectively treat an autoimmune disease.

[0096] In some embodiments, the autoimmune disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma.

[0097] In a sixth aspect, the present application provides a method for treating an autoimmune disease. According to embodiments of the present application, the method includes administering to a subject a therapeutically effective amount of the pharmaceutical composition of the second aspect, the pharmaceutical combination of the third aspect, or the kit of the fourth aspect. As described above, the pharmaceutical composition, the pharmaceutical combination, and the kit of the present application can effectively eliminate abnormal B cells in a patient with an autoimmune disease, and thus, the method of the present application can effectively treat an autoimmune disease.

[0098] In some embodiments, the autoimmune disease includes at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma.

[0099] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not intended to be limiting of the present application. Unless otherwise indicated, technical or scientific terms used in the embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains. The materials, methods, and examples provided herein are illustrative only and in no way limiting. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein.

[0100] Example 1: Expression profile of important functional proteins of allogeneic peripheral blood-derived in vitro expanded NK cells The allogeneic peripheral blood NK cells were expanded by factor expansion method. Peripheral blood was collected from healthy donors to obtain peripheral blood mononuclear cells (PBMCs). Then, the NK cells were isolated and purified using NK cell purification kit (purchased from Miltenyi). The isolated NK cells were initially cultured in KBM581 serum-free medium containing 1 ng / mL CD3 antibody, 2 μg / mL CD16 antibody and 5 ng / mL IL-2. After 3-5 days of culture, 50 ng / mL IL-21 was supplemented to the culture system for culture. During the culture process, the culture medium (KBM581 serum-free medium) was replaced regularly according to the cell growth state and fresh cytokines (5 ng / mL IL-2 and 50 ng / mL IL-21) were supplemented. The culture was continued for 20-40 days. After the culture was completed, the cell population was harvested. Finally, allogeneic NK cells with high purity, high activity and enhanced cytotoxic activity were obtained. This method can achieve efficient expansion of allogeneic NK cells without feeder cells, providing a stable and reliable cell source for the preparation of "off-the-shelf" NK cell immunotherapy products.

[0101] To verify the expression of important functional proteins such as receptors of allogeneic peripheral blood-derived in vitro expanded NK cells, the expression levels of representative molecules such as NCR receptor family (NKp30, NKp44), C lectin receptor family (CD69, NKG2D, NKG2C), activation-related receptor (CD25, DNAM-1), chemotaxis-related (CXCR3, LFA-1), proliferation-related (Ki67), killing-related (CD107a, Granzyme B, Perforin, IFN-γ, 4-1BB, TRAIL, CD16) were detected by flow cytometry. Specifically, the allogeneic peripheral blood-derived in vitro expanded NK cells and their corresponding PB-NK cells before expansion (i.e. the NK cells obtained directly after isolation and purification, as a control group) were collected. After washing twice with PBS, they were resuspended in FACS fluid (0.1% sodium azide and 0.4% BSA in PBS), counted, and the cell concentration was adjusted to 1×10 6 / ml; the corresponding antibody and isotype control antibody were added respectively, and incubated at 4℃ for 30 minutes; after washing twice with PBS, resuspended in FACS fluid, and analyze cell fluorescence by flow cytometry, and analyze the results by FlowJo software. As shown in Figure 1 As shown in A-C, compared with PB-NK control, allogeneic peripheral blood-derived in vitro expanded NK cells highly express NKp30, NKp44, CD69, NKG2D, CD25, CXCR3, Ki67, CD107a, Granzyme B, Perforin, IFN-γ, 4-1BB, which indicates that the in vitro expansion culture comprehensively enhances the killing, activation, proliferation and chemotaxis ability of NK cells. For the molecules beneficial to NK cell function, such as NKG2C, TRAIL, CD16, DNAM-1, which are highly expressed in PB-NK, the allogeneic peripheral blood-derived in vitro expanded NK cells also maintain high expression levels, which proves that the allogeneic peripheral blood-derived in vitro expanded NK cells related to the application have a unique functional related protein expression profile, which means a significant improvement in NK cell function. bright CD56 bright NK cells are considered as a subset of tissue-resident NK cells with the advantage of tissue infiltration and residence. The present application unexpectedly found that the NK cell expansion method of the present application can make CD56 dim NK cells originally low in CD56 bright NK cells in peripheral blood into CD56 bright NK cells, and after activation by factors in the expansion system, the CD56 Figure 1 NK cells reach 97.65% (as shown in Table 1 and

[0102] Table 1 NK cell subset ratio table

[0103] Example 2: Target cell line screening for in vitro function verification To verify the killing effect of allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD19 or CD20 antibodies on target cells positive for CD19 and CD20, the expression of CD19 and CD20 in two tumor cell lines, human Burkitt's lymphoma cells RAJI and human multiple myeloma RPMI8226, was detected and analyzed. Specifically, the tested tumor cells were collected, washed twice with PBS, resuspended in FACS fluid (0.1% sodium azide and 0.4% bovine serum albumin BSA in PBS), counted, and adjusted to a cell concentration of 1×10 6 / ml; PE-CD19, FITC-CD20 antibodies and isotype control antibodies were added, respectively, and incubated at 4°C for 30 minutes; after washing twice with PBS, resuspended in FACS fluid, analyzed for cell fluorescence by flow cytometry, and analyzed for results using FlowJo software. As shown in the following table (note: red is the antibody isotype control group, and subsequent experiments are the same), RAJI cells highly express CD19 and CD20; in contrast, RPMI8226 does not express CD19 and CD20. This indicates that RAJI can be used as a target cell for testing the killing effect of allogeneic peripheral blood-derived in vitro expanded NK cells combined with antibodies, and RPMI8226 can be used as a control. Figure 2

[0104] Example 3: In vitro killing function verification of allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD19 or CD20 antibodies The in vitro killing activity of allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD19 or CD20 antibodies was tested by DAPI and CFSE staining. After CFSE fluorescent staining, 1×10 5 cells per well were plated in the culture plate. The groups set were as follows: (1) Blank control group: naturally dead target cells (2) Antibody group: CD19 antibody (Inebilizumab, Selleck) or CD20 antibody (Rituximab, Selleck) killing target cells (3) Allogeneic peripheral blood-derived in vitro expanded NK cell group: allogeneic peripheral blood-derived in vitro expanded NK cells (4) Combined group: allogeneic peripheral blood-derived in vitro expanded NK cells + CD19 antibody (Inebilizumab, Selleck); allogeneic peripheral blood-derived in vitro expanded NK cells + CD20 antibody (Rituximab, Selleck) killing target cells ​The antibody group and the combination group were 50 μg / mL, and the effector-target ratio of allogeneic peripheral blood-derived in vitro expanded NK cells and target cells was 1:1. After 4 hours of mixed culture, the supernatant was removed by centrifugation, and the cell pellet was washed and stained with DAPI. The stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. Figure 3 For the killing rate test results of RAJI cells as target cells, it can be seen that for RAJI cells with high expression of CD19 and CD20, NK cells alone have a certain killing effect, and the killing effect of NK cells combined with antibodies is significantly improved compared with NK cells alone. Figure 4 For the killing rate test results of RPMI8226 cells as target cells, it can be seen that for RPMI8226 cells that do not express CD19 and CD20, NK cells alone have a certain killing effect, but the killing effect of NK cells combined with antibodies is not improved compared with NK cells alone. It is proved that NK cells can enhance the killing effect on CD19 and CD20 positive target cells by combining with CD19 or CD20 antibodies, and have no effect on CD19 and CD20 negative target cells.

[0105] Example 4: SLE patient peripheral blood B cell (SLE-PBMC-B) sorting for in vitro functional verification In order to verify whether allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD19 or CD20 antibodies have a killing effect on SLE-PBMC-B cells, we sorted CD19 + and CD20 + B cells from the PBMC of SLE patients to detect the effect of combined killing. Specifically: take the frozen SLE-PBMC (1×10 8 from liquid nitrogen, let the liquid nitrogen volatilize, and then quickly place it in a 37-degree water bath for thawing; immediately after thawing, add it to Buffer and transfer it to a 15-mL centrifuge tube, centrifuge at 4°C, 300 g for 5 minutes; after removing the supernatant, resuspend with 800 μL of Buffer (Buffer is commercial PBS + 0.5% human serum albumin HSA).

[0106] Mix 100 μL of CD19 + MACS microbeads and CD20 + MACS microbeads at a ratio of 1:1, add to 800 μL of cell resuspension, mix well, and incubate at 4°C for 15 minutes; then add 10 mL of Buffer for resuspension, centrifuge at 4°C, 300 g for 5 minutes, remove the supernatant, and finally resuspend the cells with 500 μL of Buffer; wash the LS column with 3 mL of Buffer, and add the resuspended cells to the LS column. Collect the eluate, which is CD19 - and CD20- cells; then the LS column was washed with 3 mL of Buffer three times; finally, 2.5 mL of Buffer was added to wash out the CD19 + and CD20 + cells, and this step was repeated once. The collected CD19 + and CD20 + cells were counted and adjusted to a concentration of 1 x 10 6 cells / mL; PE-CD19, FITC-CD20 antibodies and isotype control antibodies were added, respectively, and incubated at 4°C for 30 minutes; after washing twice with PBS, the cells were resuspended in FACS solution, and the fluorescence of the cells was analyzed by flow cytometry, and the results were analyzed by FlowJo software. The results are shown in Figure 5 , and the cells collected in the enrichment solution highly expressed CD19 and CD20, proving that the B cells expressing CD19 and CD20 were successfully sorted from SLE-PBMC.

[0107] Example 5: Verification of the in vitro killing function of allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD19 antibodies or CD20 antibodies on SLE-PBMC-B According to the steps and experimental grouping of the killing detection experiment in Example 3, the amount of antibodies was 50 μg / mL, the effector-target ratio of allogeneic peripheral blood-derived in vitro expanded NK cells to target cells was 1:1, and after 4 hours and 24 hours of mixed culture, the supernatant was removed by centrifugation, the cell pellet was washed, and then stained with DAPI. The stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. Figure 6 The results of the killing rate test for 4 hours when SLE-PBMC-B was the target cell are shown in

[0108] Example 6: Verification of the in vivo killing function of allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD20 antibodies on B cells BRGSF mice are a severe immunodeficient model constructed by combining the knockout of Rag2, Il2rg and Flk2 genes and the introduction of SirpaNOD alleles. Its characteristics are the simultaneous lack of T cells, B cells, NK cells, and a significant reduction in myeloid cells. This strain is superior to traditional models due to its extremely high degree of immunodeficiency and excellent compatibility with human cells / tissues, and it also has stronger radiation tolerance due to the lack of PrkdcSCID mutation, making it easier to construct humanized models.

[0109] To verify whether the allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD20 antibody have a killing effect on B cells in vivo of SLE patients, the inventors constructed a hPBMC humanized mouse model using SLE patient-derived PBMC on the basis of BRGSF female mice to verify the killing effect of allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD20 antibody on human SLE-B cells in vivo. The tail vein was administered 5 days after the mouse was humanized and reconstructed. The groups were set as follows: (1) Blank control group: injection of PBS (2) Antibody group: injection of CD20 antibody (rituximab, Selleck) (3) Allogeneic peripheral blood-derived in vitro expanded NK cell group: injection of allogeneic peripheral blood-derived in vitro expanded NK cells (4) Allogeneic peripheral blood-derived in vitro expanded NK cell combined with antibody group: injection of allogeneic peripheral blood-derived in vitro expanded NK cells + CD20 antibody (rituximab, Selleck) Among them, the antibody dosage in the antibody group and the combined antibody group was 0.25 mg per mouse, and the dose of allogeneic peripheral blood-derived in vitro expanded NK cells was 5 x 10 6 / mouse. The percentage of human immune cells in vivo was detected one day after administration. The mouse peripheral blood mononuclear cell suspension was prepared as follows: the mouse peripheral blood was collected and anticoagulated with sodium heparin; centrifuged at 500 g for 5 minutes at 4°C, and after removing the supernatant, 100 μL of whole blood was added to 1 ml of red blood cell lysis solution to lyse for 5 minutes in the dark; centrifuged at 500 g for 5 minutes at 4°C to remove the supernatant; resuspended in PBS twice, counted, and adjusted to a cell concentration of 1 x 10 6 / ml, blocked with mouse serum for 15 minutes; then added with anti-human AC7-CD45, PC5.5-CD3, BV785-CD56, BV510-CD19, FITC-CD4, BV605-CD8, BV660-CD14 antibodies, and incubated at 4°C for 30 minutes; the cell precipitate was washed and stained with DAPI, and the stained cells were obtained by flow cytometry, and the results were analyzed by FlowJo software. The results are shown in Figure 7 It can be seen that allogeneic peripheral blood-derived in vitro expanded NK cells and CD20 antibody alone have a certain degree of B cell clearance effect, but allogeneic peripheral blood-derived in vitro expanded NK cells combined with CD20 antibody have a more significant effect on the clearance of human SLE-B cells in mice, and have a significant effect enhancement. Figure 8 For the proportion of human SLE-T cells in white blood cells in mice one day after administration, it can be seen that allogeneic peripheral blood-derived in vitro expanded NK cells have a certain effect on the clearance of human SLE-T cells (including CD4 + T cells and CD8+ T cells) also have certain killing effects on CD4 + T cells, and the killing effect of CD8

[0110] Example 7: SLE-PBMC-T sorting for in vitro functional verification To further verify the reason why the allogeneic peripheral blood-derived in vitro expanded NK cells have killing effect on SLE-PBMC-T cells, the inventors sorted CD4 + and CD8 + T cells from the PBMC of SLE patients for subsequent detection of NKR-related ligands. The specific steps are as follows: Example 4, CD4 + MACS microbeads and CD8 + MACS microbeads were used to sort SLE-PBMC-T cells, and the collected CD4 + and CD8 + T cells were counted, and the cell concentration was adjusted to 1x10 6 FITC-CD4, BV605-CD8 antibodies and isotype control antibodies were added, and incubated at 4°C for 30 minutes; after washing twice with PBS, resuspended in FACS fluid, analyzed the cell fluorescence by flow cytometry, and analyzed the results by FlowJo software. The results are shown in Figure 9 , the cells collected in the enrichment solution highly expressed CD4 and CD8, proving that the T cells expressing CD4 and CD8 were successfully sorted from SLE-PBMC, and the T cells expressing CD4 + and CD8 + in the PBMC of healthy donors were also sorted as controls.

[0111] Example 8: SLE-PBMC-T related ligand detection To explore the reason why the T cells of SLE patients are killed by allogeneic peripheral blood-derived in vitro expanded NK cells, the inventors directly compared whether there were differences in the expression of NKR-related ligands between SLE patient T cells and healthy donor T cells (PBMC-T) by flow cytometry, and then detected the expression of NKG2D-related ligands ULBP1 / 2 / 3 / 4 / 5 / 6 and MICA / B in SLE-PBMC-T cells and PBMC-T cells. The specific steps are as follows: the cells were washed twice with PBS and resuspended in FACS fluid, counted, and the cell concentration was adjusted to 1x10 6 APC-ULBP1, Alexa Fluor 700-ULBP2 / 5 / 6, APC-ULBP3, PE-ULBP4, APC-MICA / B antibodies and isotype control antibodies were added, and incubated at 4°C for 30 minutes; after washing twice with PBS, resuspended in FACS fluid, analyzed the cell fluorescence by flow cytometry, and analyzed the results by FlowJo software. The results are shown inFigure 10 As shown, it can be seen that SLE-PBMC-T highly express NKG2D ligands ULBP1, ULBP4, MICA / B compared with PBMC-T. Therefore, the pathogenic T cells in SLE should be killed and eliminated by the allogeneic peripheral blood-derived in vitro expanded NK cells after being recognized by the above ligands.

[0112] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0113] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. Use of NK cells in the manufacture of a medicament for the treatment of an autoimmune disease, wherein, The NK cells are obtained by in vitro induced expansion of peripheral blood mononuclear cells, which is performed in a culture medium containing interleukins and / or antibodies.

2. A pharmaceutical composition for combination therapy of autoimmune diseases, characterized by, Comprise: an anti-CD19 antibody or an anti-CD20 antibody, and NK cells; wherein the NK cells are obtained by in vitro induced expansion of peripheral blood mononuclear cells, which is performed in a culture medium containing interleukins and / or antibodies.

3. The use according to claim 1 or the pharmaceutical composition according to claim 2, characterized in that, The final concentration of the interleukins in the culture medium is 1-100 ng / mL; Optionally, the final concentration of the antibodies in the culture medium is 0.5 ng / mL-5 μg / mL.

4. The use or pharmaceutical composition according to claim 3, characterized in that, The interleukins comprise at least one of IL-21, IL-2; Optionally, the antibodies comprise at least one of CD16 antibodies, CD3 antibodies.

5. The use or pharmaceutical composition according to claim 4, characterized in that, The in vitro induced expansion of peripheral blood mononuclear cells comprises: isolating and purifying first NK cells from the peripheral blood mononuclear cells; subjecting the first NK cells to a first culture treatment in a first culture system; after 3-5 days of culture, adding a first cytokine to the first culture system for a second culture treatment, to obtain the NK cells; wherein during the second culture treatment, the culture system is replaced by a second culture system when the culture system is replaced.

6. The use or pharmaceutical composition according to claim 5, characterized in that, The first culture system comprises a serum-free culture medium and at least one of the CD3 antibodies, the CD16 antibodies, the IL-2; Optionally, the concentration of the CD3 antibodies in the first culture system is 0.5-5 ng / mL, preferably 1 ng / mL; Optionally, the concentration of the CD16 antibodies in the first culture system is 1-5 μg / mL, preferably 2 μg / mL; Optionally, the concentration of the IL-2 in the first culture system is 3.5-6.5 ng / mL, preferably 5 ng / mL; Optionally, the first cytokine is selected from the IL-21; Optionally, the concentration of the IL-21 in the first culture system is 20-100 ng / mL, preferably 50 ng / mL; Optionally, the second culture system comprises a serum-free culture medium and at least one of the IL-2, the IL-21; Optionally, the concentration of the IL-2 in the second culture system is 3.5-6.5 ng / mL, preferably 5 ng / mL; Optionally, the concentration of the IL-21 in the second culture system is 20-100 ng / mL, preferably 50 ng / mL.

7. The pharmaceutical composition of claim 2, wherein, The CD19 antibodies are selected from at least one of inebilizumab, tanibirumab; Optionally, the CD20 antibodies are selected from at least one of rituximab, obinutuzumab, ofatumumab, ocrelizumab. Optionally, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma.

8. A combination of drugs for use in the treatment of an autoimmune disease, characterized in that, Comprise: NK cells as a first active ingredient, an anti-CD19 antibody or an anti-CD20 antibody as a second active ingredient; Wherein, the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is carried out in a culture medium containing interleukins and / or antibodies.

9. A kit for treating an autoimmune disease, characterized in that, Comprise: NK cells as a first active ingredient, an anti-CD19 antibody or an anti-CD20 antibody as a second active ingredient; Wherein, the NK cells are obtained by in vitro induction and expansion of peripheral blood mononuclear cells, and the in vitro induction and expansion is carried out in a culture medium containing interleukins and / or antibodies.

10. The pharmaceutical combination according to claim 8 or the kit according to claim 9, wherein, The final concentration of the interleukins in the culture medium is 1-100 ng / mL; Optionally, the final concentration of the antibodies in the culture medium is 0.5 ng / mL-5 μg / mL; Optionally, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, anti-phospholipid antibody syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma. Optionally, the interleukins comprise at least one of IL-21, IL-2; Optionally, the antibodies comprise at least one of CD16 antibodies, CD3 antibodies.

11. The pharmaceutical association or kit according to claim 10, characterized in that it comprises, The in vitro induction and expansion of peripheral blood mononuclear cells comprises: The in vitro induction and expansion of peripheral blood mononuclear cells comprises: Separating and purifying first NK cells from the peripheral blood mononuclear cells; Placing the first NK cells in a first culture system for first culture treatment; After 3-5 days of culture, adding a first cytokine in the first culture system for second culture treatment to obtain the NK cells; Wherein, during the second culture treatment, the culture system is replaced with a second culture system when the culture system is replaced.

12. The pharmaceutical association or kit according to claim 11, characterized in that it comprises, The first culture system comprises a serum-free culture medium and at least one of the CD3 antibodies, the CD16 antibodies, the IL-2; Optionally, the concentration of the CD3 antibodies in the first culture system is 0.5-5 ng / mL, preferably 1 ng / mL; Optionally, the concentration of the CD16 antibodies in the first culture system is 1-5 μg / mL, preferably 2 μg / mL; Optionally, the concentration of the IL-2 in the first culture system is 3.5-6.5 ng / mL, preferably 5 ng / mL; Optionally, the first cytokine is selected from the IL-21; Optionally, the concentration of IL-21 in the first culture system is 20-100 ng / mL, preferably 50 ng / mL; Optionally, the second culture system comprises a serum-free culture medium and at least one of the IL-2, the IL-21; Optionally, the concentration of IL-2 in the second culture system is 3.5-6.5 ng / mL, preferably 5 ng / mL; Optionally, the concentration of IL-21 in the second culture system is 20-100 ng / mL, preferably 50 ng / mL.

13. The pharmaceutical association or kit according to claim 12, characterized in that, The CD19 antibody is selected from at least one of inebilizumab, tanibotumab; Optionally, the CD20 antibody is selected from at least one of rituximab, obinutuzumab, ofatumumab, ocrelizumab.

14. Use of the pharmaceutical composition of any one of claims 2-7, the pharmaceutical association of any one of claims 8, 10-13, or the kit of any one of claims 9-13 for the manufacture of a medicament for the treatment of an autoimmune disease; Optionally, the autoimmune disease comprises at least one of systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pernicious anemia, antiphospholipid syndrome, autoimmune liver disease, ankylosing spondylitis, psoriasis, vitiligo, vasculitis, inflammatory bowel disease, type I diabetes, multiple sclerosis, asthma.

Images