Application of NK cell in treatment of systemic lupus erythematosus

Abstract

The invention relates to application of NK cells in treating systemic lupus erythematosus. Specifically, the invention relates to application of the NK cell in preparation of a medicine for treating systemic lupus erythematosus in an individual, application of the NK cell in preparation of a medicine for treating systemic lupus erythematosus in combination with an anti-CD20 antibody, and application of the NK cell and the anti-CD20 antibody in preparation of a medicine for treating a patient with systemic lupus erythematosus. Wherein the NK cells are expanded in vitro.

Description

Technical Field

[0001] This application relates to the use of NK cells in the treatment of systemic lupus erythematosus. Background Art

[0002] Natural killer cells (NK cells) are derived from bone marrow lymphoid stem cells and are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs, and lymph nodes. NK cells are a type of lymphocyte that has no MHC restriction, does not require prior sensitization, and does not require the participation of antibodies, and can directly kill tumor cells and virus-infected cells non-specifically. NK cells usually contain a large amount of perforin and granzyme B. When activated NK cells encounter target cells, the perforin and granzyme B released by NK cells attack the target cells. NK cells directly kill target cells through antibody-dependent cell-mediated cytotoxicity (ADCC), etc. NK cells can also secrete cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-3, and these cytokines can directly act on target cells or attack target cells by activating other types of immune cells.

[0003] Cell therapy is a hot topic in the current medical field, and NK cells are also a key point in cell therapy due to their own immunological advantages. Developing NK cell-based therapies for different diseases is urgently needed in this field.

[0004] Systemic lupus erythematosus (SLE) is a complex classical autoimmune disease with unknown etiology. At present, the standard therapy for SLE in clinical practice cannot completely cure the disease, and some drugs may have large toxic side effects after long-term use. Summary of the Invention

[0005] In a first aspect, this application provides the use of NK cells in the preparation of a drug for treating systemic lupus erythematosus in an individual.

[0006] In some embodiments, the individual also has Sjogren's syndrome.

[0007] In a second aspect, this application provides the use of NK cells in the preparation of a drug for co-treating a patient with systemic lupus erythematosus in combination with an anti-CD20 antibody.

[0008] In some embodiments, the anti-CD20 antibody is rituximab or obinutuzumab.

[0009] In a third aspect, this application provides the use of NK cells and an anti-CD20 antibody in the preparation of a drug for treating a patient with systemic lupus erythematosus.

[0010] In some embodiments, the anti-CD20 antibody is rituximab or obinutuzumab.

[0011] In some embodiments, the patient also has Sjogren's syndrome.

[0012] In some embodiments of any one of the first to third aspects, NK cells are expanded in vitro.

[0013] In some embodiments of any one of the first to third aspects, the in vitro expansion of NK cells is carried out using a rocking bioreactor in a serum-free culture system, and the culture process parameters for in vitro expansion include: total ventilation volume of 20 - 100 mL / min, culture temperature of 36.5 - 37.5 °C, rocking speed of 8 - 10 rpm, rocking angle of 4 - 6 °, dissolved oxygen (DO) ≥ 20%, and pH of 6.8 - 7.2.

[0014] In some embodiments of any one of the first to third aspects, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0015] In some embodiments of any one of the first to third aspects, more than 3 billion NK cells are infused per cycle, and at least 1 cycle of infusion is carried out.

[0016] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + proportion of NK cells ≥ 95.0%.

[0017] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + CD16 + proportion of NK cells ≥ 90.0%.

[0018] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + NKG2D + proportion of NK cells ≥ 95.0%.

[0019] In some embodiments of any one of the first to third aspects, the CD19 + proportion of B cells ≤ 2.0%.

[0020] In some embodiments of any one of the first to third aspects, CD3 in the NK cells + CD56 - T cell proportion ≤ 3.0%.

[0021] In some embodiments of any one of the first to third aspects, NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in the NK cell suspension is 0.8 - 1.2×10 8 cells / mL.

[0022] In some embodiments of any one of the first to third aspects, NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the total number of viable cells in the NK cell suspension is 2.0 - 3.0×10 9 NK cells. Description of the Drawings

[0023] Figure 1 Shows the killing results of NK cell injection combined with anti-CD20 antibody on SLE-B cells; wherein the effector-to-target ratios of NK cells to SLE-B cells are 10:1, 3:1, 1:1, and 0 respectively; the anti-CD20 antibodies are 10000 ng / mL, 100 ng / mL, 1 ng / mL, and 0 ng / mL respectively.

[0024] Figure 2 Shows the treatment results of an example of using NK cells and rituximab to treat patients with systemic lupus erythematosus.

[0025] Figure 3 Shows the treatment results of an example of using NK cells and rituximab to treat patients with systemic lupus erythematosus complicated with Sjogren's syndrome.

[0026] Figure 4 Shows the treatment results of another example of using NK cells and rituximab to treat patients with systemic lupus erythematosus. Detailed Description of the Invention

[0027] Cell immunotherapy represented by T cells and NK cells is an emerging treatment method for SLE in recent years. The latest research shows that CAR-T therapy has shown good efficacy in the treatment of SLE, but its long preparation cycle, high cost, and the risk of cytokine release syndrome (CRS) seriously limit its clinical application. The specific role of NK cells as important cytotoxic cells in SLE has not been fully understood.

[0028] In a first aspect, the present application provides the use of NK cells in the preparation of a medicament for treating systemic lupus erythematosus in an individual.

[0029] In some embodiments, NK cells are expanded in vitro.

[0030] In some embodiments, the in vitro expansion of the NK cells is carried out in a serum-free culture system using a rocking bioreactor for the in vitro expansion of NK cells, and the culture process parameters for the in vitro expansion include: a total ventilation volume of 20 - 100 mL / min, a culture temperature of 36.5 - 37.5 °C, a rocking rotation speed of 8 - 10 rpm, a rocking angle of 4 - 6 °, a dissolved oxygen (DO) ≥ 20%, and a pH of 6.8 - 7.2.

[0031] In some embodiments, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0032] In some embodiments, the NK cells are obtained from NK cells derived from umbilical cord blood.

[0033] In some embodiments, the NK cells are obtained from in vitro culture of an NK cell line.

[0034] In some embodiments, the NK cells are obtained from in vitro induction and culture of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs). Only NK cells derived from PBMCs are used, and the rest of the sources are deleted.

[0035] In some embodiments, more than 3 billion NK cells are infused per cycle, and at least 1 cycle of infusion is carried out.

[0036] In some embodiments, more than 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion NK cells are infused per cycle, or a range between any of the above values.

[0037] In some embodiments, 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion NK cells are infused per cycle, or a range between any of the above values.

[0038] In some embodiments, the NK cells are infused for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 cycles (or a range between any of the above values).

[0039] In some embodiments, the NK cells are infused for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 cycles (or a range between any of the above values).

[0040] In some embodiments, the CD3 in the NK cells - CD56 + The proportion of NK cells ≥ 95.0%.

[0041] In some embodiments, the CD3 in the NK cells - CD56 + CD16 + The proportion of NK cells ≥ 90.0%.

[0042] In some embodiments, the CD3 in the NK cells - CD56 + NKG2D + The proportion of NK cells ≥ 95.0%.

[0043] In some embodiments, the CD19 in the NK cells + The proportion of B cells ≤ 2.0%.

[0044] In some embodiments, the CD3 in the NK cells + CD56 - The proportion of T cells ≤ 3.0%.

[0045] In some embodiments, the individual also has Sjogren's syndrome.

[0046] In a second aspect, the present application provides the use of NK cells in the preparation of a medicament for treating a patient with systemic lupus erythematosus in combination with an anti-CD20 antibody.

[0047] In some embodiments, the NK cells are expanded in vitro.

[0048] In some embodiments, the in vitro expansion of the NK cells is carried out using a rocking bioreactor in a feeder cell-free and serum-free culture system, and the culture process parameters for in vitro expansion include: total ventilation volume of 20 - 100 mL / min, culture temperature of 36.5 - 37.5 °C, rocking rotation speed of 8 - 10 rpm, rocking angle of 4 - 6 °, dissolved oxygen (DO) ≥ 20%, and pH of 6.8 - 7.2.

[0049] In some embodiments, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0050] In some embodiments, more than 3 billion NK cells are infused per cycle, and the infusion is performed for at least 1 cycle.

[0051] In some embodiments, more than 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion NK cells are infused per cycle, or a range between any of the above values.

[0052] In some embodiments, 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion NK cells are infused per cycle, or a range between any of the above values.

[0053] In some embodiments, the NK cells are infused for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 cycles (or a range between any of the above values).

[0054] In some embodiments, the NK cells are infused for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 cycles (or a range between any of the above values).

[0055] In some embodiments, the CD3 - CD56 + proportion of NK cells in the NK cells ≥ 95.0%.

[0056] In some embodiments, the CD3 - CD56 + CD16 + proportion of NK cells in the NK cells ≥ 90.0%.

[0057] In some embodiments, the CD3 - CD56 + NKG2D + proportion of NK cells in the NK cells ≥ 95.0%.

[0058] In some embodiments, the proportion of CD19 in the NK cells + in B cells ≤ 2.0%.

[0059] In some embodiments, the proportion of CD3 + CD56 - in T cells ≤ 3.0%.

[0060] In some embodiments, the anti-CD20 antibody is rituximab or obinutuzumab.

[0061] In a third aspect, the present application provides the use of NK cells and anti-CD20 antibodies in the preparation of a medicament for treating patients with systemic lupus erythematosus.

[0062] In some embodiments, the NK cells are expanded in vitro.

[0063] In some embodiments, the in vitro expansion of the NK cells is carried out using a rocking bioreactor in a culture system without feeder cells and serum, and the culture process parameters for the in vitro expansion include: the total ventilation volume is 20 - 100 mL / min, the culture temperature is 36.5 - 37.5 °C, the rocking rotation speed is 8 - 10 rpm, the rocking angle is 4 - 6 °, the dissolved oxygen (DO) ≥ 20%, and the pH is 6.8 - 7.2.

[0064] In some embodiments, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMC).

[0065] In some embodiments, more than 3 billion NK cells are infused per cycle, and at least 1 cycle of infusion is carried out.

[0066] In some embodiments, more than 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion or 15 billion NK cells are infused per cycle, or the range between any of the above values.

[0067] In some embodiments, 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion or 15 billion NK cells are infused per cycle, or the range between any of the above values.

[0068] In some embodiments, the NK cells are infused for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 cycles (or a range between any of the above values).

[0069] In some embodiments, the NK cells are infused for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 cycles (or a range between any of the above values).

[0070] In some embodiments, CD3 - CD56 + in the NK cells accounts for ≥ 95.0%.

[0071] In some embodiments, CD3 - CD56 + CD16 + in the NK cells accounts for ≥ 90.0%.

[0072] In some embodiments, CD3 - CD56 + NKG2D + in the NK cells accounts for ≥ 95.0%.

[0073] In some embodiments, CD19 + in the B cells accounts for ≤ 2.0%.

[0074] In some embodiments, CD3 + CD56 - in the T cells accounts for ≤ 3.0%.

[0075] In some embodiments, the anti-CD20 antibody is rituximab or ofatumumab.

[0076] In some embodiments, the patient also has Sjogren's syndrome.

[0077] In some embodiments of any one of the first to third aspects, the total ventilation volume is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mL / min, or a range between any of the above values.

[0078] In some embodiments of any one of the first to third aspects, the culture temperature is 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4 or 37.5 °C, or a range between any of the above values.

[0079] In some embodiments of any one of the first to third aspects, the rocking speed is 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 rpm, or a range between any of the above values.

[0080] In some embodiments of any one of the first to third aspects, the rocking angle is 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6 °, or a range between any of the above values.

[0081] In some embodiments of any one of the first to third aspects, the dissolved oxygen (DO) ≥ 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, or a range between any of the above values.

[0082] In some embodiments of any one of the first to third aspects, the pH is 6.8, 6.9, 7, 7.1 or 7.2, or a range between any of the above values.

[0083] In some embodiments of any one of the first to third aspects, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMC).

[0084] In some embodiments of any one of the first to third aspects, more than 3 billion NK cells are infused per cycle, and the infusion is performed for at least 1 cycle.

[0085] In some embodiments of any one of the first to third aspects, more than 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion or 15 billion NK cells are infused per cycle, or a range between any of the above values.

[0086] In some embodiments of any one of the first to third aspects, 3 billion, 5 billion, 6 billion, 7 billion, 7.5 billion, 8 billion, 8.5 billion, 9 billion, 9.5 billion, 10 billion, 12 billion, 13 billion, 14 billion, or 15 billion NK cells are infused per cycle, or a range between any of the above values.

[0087] In some embodiments of any one of the first to third aspects, NK cells are infused for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 cycles (or a range between any of the above values).

[0088] In some embodiments of any one of the first to third aspects, NK cells are infused for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 cycles (or a range between any of the above values).

[0089] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + proportion of NK cells ≥ 95.0%.

[0090] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + proportion of NK cells ≥ 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, or 99.5%.

[0091] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + CD16 + proportion of NK cells ≥ 90.0%.

[0092] In some embodiments of any one of the first to third aspects, the CD3 - CD56 + CD16 +The proportion of NK cells is ≥90.0%, 90.5%, 91.0%, 91.5%, 92.0%, 92.5%, 93.0%, 93.5%, 94.0%, 94.5%, 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5%.

[0093] In some embodiments of any one of the first to third aspects, CD3 in the NK cells - CD56 + NKG2D + The proportion of NK cells is ≥95.0%.

[0094] In some embodiments of any one of the first to third aspects, CD3 in the NK cells - CD56 + NKG2D + The proportion of NK cells is ≥95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5%.

[0095] In some embodiments of any one of the first to third aspects, CD19 in the NK cells + The proportion of B cells is ≤2.0%.

[0096] In some embodiments of any one of the first to third aspects, CD19 in the NK cells + The proportion of B cells is ≤2.0%, 1.5%, 1.0% or 0.5%.

[0097] In some embodiments of any one of the first to third aspects, CD3 in the NK cells + CD56 - The proportion of T cells is ≤3.0%.

[0098] In some embodiments of any one of the first to third aspects, CD3 in the NK cells + CD56 - The proportion of T cells is ≤3.0%, 2.5%, 2.0%, 1.5%, 1.0% or 0.5%.

[0099] Fourth aspect, the present application provides a method for treating systemic lupus erythematosus in an individual, the method comprising administering to the individual a therapeutically effective amount of NK cells.

[0100] Fifth aspect, the present application provides a method for treating systemic lupus erythematosus in an individual, the method comprising administering to the individual a therapeutically effective amount of NK cells and an anti-CD20 antibody.

[0101] In some embodiments, the anti-CD20 antibody is rituximab or obinutuzumab.

[0102] In some embodiments of any one of the fourth to fifth aspects, the NK cells are expanded in vitro.

[0103] In some embodiments of any one of the fourth to fifth aspects, the in vitro expansion of NK cells is carried out using a rocking bioreactor in a serum-free culture system, and the culture process parameters for in vitro expansion include: total ventilation volume of 20 - 100 mL / min, culture temperature of 36.5 - 37.5 °C, rocking rotation speed of 8 - 10 rpm, rocking angle of 4 - 6 °, dissolved oxygen (DO) ≥ 20%, and pH of 6.8 - 7.2.

[0104] In some embodiments of any one of the fourth to fifth aspects, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMCs).

[0105] In some embodiments of any one of the fourth to fifth aspects, more than 3 billion NK cells are infused per cycle, and at least 1 cycle of infusion is performed.

[0106] In some embodiments of any one of the fourth to fifth aspects, the proportion of CD3 - CD56 + in the NK cells is ≥ 95.0%.

[0107] In some embodiments of any one of the fourth to fifth aspects, the proportion of CD3 - CD56 + CD16 + in the NK cells is ≥ 90.0%.

[0108] In some embodiments of any one of the fourth to fifth aspects, the proportion of CD3 - CD56 + NKG2D + in the NK cells is ≥ 95.0%.

[0109] In some embodiments of any one of the fourth to fifth aspects, the proportion of CD19 + in the B cells in the NK cells is ≤ 2.0%.

[0110] In some embodiments of any one of the fourth to fifth aspects, the proportion of CD3 + CD56 - in the T cells in the NK cells is ≤ 3.0%.

[0111] In some embodiments of any one of the fourth to fifth aspects, NK cells are stored in a bag, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in the NK cell suspension is 0.8 - 1.2×10 8 cells / mL.

[0112] In some embodiments of any one of the fourth to fifth aspects, NK cells are stored in a bag, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the total number of viable cells in the NK cell suspension is 2.0 - 3.0×10 9 NK cells.

[0113] "Therapeutically effective amount" means an amount of a compound sufficient to treat a disease or at least one clinical symptom of a disease when administered to an individual to treat the disease or the clinical symptom. The "therapeutically effective amount" can vary depending on, for example, the drug, the disease and / or the clinical symptom of the disease, the severity of the disease and / or the symptom of the disease or disorder, the age, weight and / or health condition of the individual to be treated, and the judgment of the prescribing physician. The appropriate amount in any given case can be determined by those skilled in the art or can be determined by routine experimentation.

[0114] "Treatment" of a disease means preventing or ameliorating the disease or at least one clinical symptom of the disease, reducing the risk of having the disease or at least one clinical symptom of the disease, reducing the development of the disease or at least one clinical symptom of the disease or reducing the risk of developing the disease or at least one clinical symptom of the disease. "Treatment" also means inhibiting the disease physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters) or both, and means inhibiting at least one physical parameter or manifestation that may or may not be discernible to the patient. In some embodiments, "treatment" means delaying the onset of a disease or at least one or more of its symptoms in an individual who may be exposed to or is susceptible to the disease or disorder, even if the individual has not yet experienced or manifested symptoms of the disease.

[0115] In a sixth aspect, the present application provides a pharmaceutical composition, the pharmaceutical composition comprising any one of the following:

[0116] (i) NK cells and a pharmaceutically acceptable carrier;

[0117] (ii) NK cells, an anti-CD20 antibody and a pharmaceutically acceptable carrier.

[0118] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, etc. that are compatible with the administration of a drug. The use of these dispersion media and solvents for the active ingredients of a pharmaceutical composition is well known in the art, unless it is incompatible with the active ingredients of the pharmaceutical composition.

[0119] In some embodiments, the anti-CD20 antibody is rituximab or obinutuzumab.

[0120] In some embodiments, the pharmaceutical composition is used for treating systemic lupus erythematosus in an individual.

[0121] In some embodiments, the individual also has Sjögren's syndrome.

[0122] In a seventh aspect, the present application provides a kit for treating systemic lupus erythematosus, which comprises any one of the following:

[0123] (i) a first container containing NK cells;

[0124] (ii) a first container containing NK cells and a second container containing an anti-CD20 antibody.

[0125] In some embodiments, the anti-CD20 antibody is rituximab or obinutuzumab.

[0126] In some embodiments, the kit further comprises an instruction manual.

[0127] In some embodiments, the kit is used for treating systemic lupus erythematosus in an individual.

[0128] In some embodiments, the individual also has Sjögren's syndrome.

[0129] In some embodiments of any one of the sixth aspect to the seventh aspect, the NK cells are expanded in vitro.

[0130] In some embodiments of any one of the sixth aspect to the seventh aspect, the in vitro expansion of NK cells is carried out in a serum-free culture system using a rocking bioreactor, and the culture process parameters for in vitro expansion include: the total ventilation volume is 20 - 100 mL / min, the culture temperature is 36.5 - 37.5 °C, the rocking rotation speed is 8 - 10 rpm, the rocking angle is 4 - 6 °, the dissolved oxygen (DO) ≥ 20%, and the pH is 6.8 - 7.2.

[0131] In some embodiments of any one of the sixth aspect to the seventh aspect, the NK cells are obtained from NK cells derived from peripheral blood mononuclear cells (PBMC).

[0132] In some embodiments of any one of the sixth aspect to the seventh aspect, more than 3 billion NK cells are infused per cycle, and at least 1 cycle of infusion is carried out.

[0133] In some embodiments of any one of the sixth aspect to the seventh aspect, the CD3 in the NK cells -CD56 + The proportion of NK cells ≥ 95.0%.

[0134] In some embodiments of any one of the sixth to seventh aspects, CD3 in the NK cells - CD56 + CD16 + The proportion of NK cells ≥ 90.0%.

[0135] In some embodiments of any one of the sixth to seventh aspects, CD3 in the NK cells - CD56 + NKG2D + The proportion of NK cells ≥ 95.0%.

[0136] In some embodiments of any one of the sixth to seventh aspects, CD19 in the NK cells + The proportion of B cells ≤ 2.0%.

[0137] In some embodiments of any one of the sixth to seventh aspects, CD3 in the NK cells + CD56 - The proportion of T cells ≤ 3.0%.

[0138] In some embodiments of any one of the sixth to seventh aspects, NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in the NK cell suspension is 0.8 - 1.2×10 8 cells / mL.

[0139] In some embodiments of any one of the sixth to seventh aspects, NK cells are stored in bags, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the total number of viable cells in the NK cell suspension is 2.0 - 3.0×10 9 NK cells.

[0140] Examples

[0141] Example 1: Preparation of NK cell injection

[0142] The preparation of NK cell injection is divided into three parts: preparation of peripheral blood mononuclear cells (PBMC), preparation of the stock solution of NK cell injection, and preparation of the preparation.

[0143] Preparation of peripheral blood mononuclear cells (PBMC)

[0144] PBMC preparation includes hPB Leukopak pretreatment, CTS TM Rotea TMFive major steps: processing, PBMC aliquoting, labeling, and PBMC cryopreservation.

[0145] hPB Leukopak pretreatment

[0146] Add PPC3 diluent (source of purchase or composition: RosetteSep 9 to hPB Leukopak (source of purchase or composition: peripheral blood mononuclear cells) at an addition amount of 0.7 - 5.0 mL / 1×10 TM cells. The addition amount of Human CD3 Depletion Cocktail is 1.5 - 5.0 mL / 1×10 9 cells) or PPC3S diluent (source of purchase or composition: IgG4 antibody conjugated with red blood cells and T cells, addition amount is 1.5 - 5.0 mL / 1×10 9 cells). Incubate at room temperature for 10 - 40 min. Add erythrocyte sedimentation diluent (3% m / v hydroxyethyl starch) to hPB Leukopak at a final concentration range of 1.2% - 2.0% for hydroxyethyl starch. Hang vertically and let stand at room temperature for (120 ± 45) min. After the erythrocytes sediment and form layers, use a blood plasma separator clamp to press the upper cell suspension into the sample bag.

[0147] CTS TM RoteaTM treatment

[0148] Dilute the cell suspension with an equal volume of sodium chloride injection solution. Circulate the cell suspension in the sample bag to form a stable fluidized bed, then continue to load the cell suspension in the sample bag to concentrate the PBMC cells in the fluidized bed. After washing with sodium chloride injection solution and replacing with cryopreservation solution, the cryopreservation solution collects the PBMC cells into the harvest bag. Count the PBMC cells in the harvest bag. According to the counting result, adjust the viable cell density of the PBMC cell suspension to (2.7 - 6.0)×10 7 cells / mL. The cell viability should be ≥80.0%.

[0149] PBMC separation

[0150] Calculate the aliquot volume per bag according to 6.8×10 8 viable cells / bag. Sterilely connect the harvest bag and the cryopreservation bag. Mix the PBMC cells to be aliquoted on a shaker and aliquot them into the cryopreservation bag through a peristaltic pump. After aliquoting, keep a sample for inspection (such as sterility inspection, mycoplasma inspection, etc.).

[0151] Labeling

[0152] Attach a PBMC label to the body of the above - aliquoted cryopreservation bag.

[0153] PBMC cryopreservation

[0154] Place the blood bag box flat in the cryopreservation chamber of the programmable freezer and cool it down to -70 to -90 °C. After the cooling is completed, transfer the cryopreserved PBMC cells to a vapor-phase liquid nitrogen tank and store them at ≤ -150 °C.

[0155] Preparation of the stock solution of NK cell injection

[0156] The preparation of the stock solution of NK cell injection includes three major processes: initial culture of NK cells, amplified culture of NK cells, and concentration, washing, and formulation of the stock solution of NK cells.

[0157] Initial culture of NK cells

[0158] PBMC resuscitation

[0159] Coat three T225 cm 2 culture flasks, add 1 mL of coating factor mixture (composition: CD33-CLL1 (0 - 50 μg / mL), CD52 (0 - 50 μg / mL), CD38 (0 - 50 μg / mL), CD3 antibody (0 - 50 μg / mL)) to each flask, and then add 29 mL of sodium chloride injection to each flask and mix well. Place the T225 cm 2 culture flasks in a biosafety cabinet and incubate at room temperature for 1 - 4 h or in a carbon dioxide incubator at 37.0 ± 0.5 °C for 1 - 4 h.

[0160] Take out 1 bag of cryopreserved PBMC cells from the liquid nitrogen tank, quickly thaw them in an anhydrous thawing instrument (thawing temperature 37 ± 0.5 °C), with a thawing time of 2 - 5 min. Transfer the thawed PBMC cells to a 50 mL centrifuge tube containing 15 mL of compound electrolyte injection or sodium chloride injection, and then add compound electrolyte injection or sodium chloride injection to 45 mL and mix the cells well.

[0161] Centrifuge at 600 g at room temperature for 8 ± 2 min. Discard the supernatant, add 45 mL of NK cell activation medium (composition: mainly 1640 medium, heparin sodium (0 - 10 IU / mL), platelet lysate (volume ratio 0 - 10%), human albumin (volume ratio 1.0% - 5.0%), IL2 (0 - 2000 IU / mL), IL-15 (0 - 50 ng / mL), etc.) to resuspend, and mix well for counting. According to the cell counting results, if the total number of viable cells ≥ 3.6 × 10 8 cells, and the cell viability ≥ 80.0%, then proceed with inoculation and culture. If the total number of viable cells < 3.6 × 10 8 cells or the cell viability < 80.0%, then terminate the resuscitation of PBMC cells this time.

[0162] Remove the T225 cm 2Coating solution in the culture flask. According to the counting results, the above PBMC cell suspension was evenly transferred to three coated T225 cm 2 culture flasks (coating agent composition: CD33 - CLL1 (0 - 50 μg / mL), CD52 (0 - 50 μg / mL), CD38 (0 - 50 μg / mL), CD3 antibody (0 - 50 μg / mL)). In each T225 cm 2 culture flask, the total inoculation volume was 120 mL, and the inoculated viable cell density was ≥1.00×10 6 cells / mL. The three flasks were respectively labeled as Flask A, Flask B, and Flask C. After making marks on the flask bodies, the three T225 cm 2 culture flasks were placed in a CO₂ incubator at 37.0 ± 0.5 °C and 5.0 ± 0.5% CO₂ for static culture for 3 days.

[0163] T225 cm 2 Flask replenishment culture

[0164] On the 3rd day of culture, 60 mL of NK cell activation medium was added to each of the above three T225 cm 2 culture flasks, and the static culture was continued.

[0165] T225 cm 2 Flask transfer bag culture

[0166] On the 5th - 7th day of culture, the cell suspensions in the three T225 cm 2 culture flasks were mixed. After sampling and counting respectively, if the average viable cell density of the three flasks was ≥6.7×10 5 cells / mL, then it was transferred to an 825 cm 2 cell culture bag for culture. NK cell activation medium was added for replenishment, and the replenishment volume was adjusted according to the average viable cell density of the three flasks. After replenishment, the viable cell density was ≥4.00×10 5 cells / mL, and the volume after replenishment was 900 - 1800 mL. If the average viable cell density was lower than 6.70×10 5 cells / mL, then the culture was continued until the next day for sampling and counting to make the above judgment on whether to transfer to a bag. If the transfer to a bag was not carried out on the second day either, then the sampling and counting were carried out on the third day to make the above judgment on whether to transfer to a bag. If the average viable cell density in the counting result on the third day was lower than 6.70×10 5 cells / mL, then this culture was terminated. After the replenishment was completed, it was continued to be placed in a CO₂ incubator at 37.0 ± 0.5 °C and a CO₂ concentration of 5.0 ± 0.5% for static culture for two days.

[0167] 825 cm 2 Cell culture bag replenishment culture 1

[0168] On the 7th - 10th day of culture: Transfer the 825 cm2 Mix the cell suspension in the cell culture bag and take a sample for counting. The supplementary fluid is NK cell expansion medium (composition: mainly RPMI 1640 medium, sodium heparin (0 - 10 IU / mL), platelet lysate (volume ratio 0 - 10%), human albumin (volume ratio 1.0% - 5.0%), IL2 (0 - 2000 IU / mL), IL-15 (0 - 50 ng / mL), etc.). Adjust the volume of the supplementary fluid according to the viable cell density. After supplementation, the viable cell density ≥ 4.00×10 5 cells / mL. Add 5 - 10 mL of serum substitute (II) (composition: platelet lysate and human albumin, volume ratio 1:1) before supplementation. The volume after supplementation is 1800 mL - 2700 mL, and continue static culture. If it still does not meet the 825 cm 2 cell culture bag culture 1 supplementation standard on the 10th day, terminate the culture.

[0169] 825 cm 2 Cell culture bag supplementary fluid culture 2

[0170] On the 9th - 12th day, mix the cell suspension in the 825 cm 2 cell culture bag and take a sample for cell counting. The total number of viable cells ≥ 2.0×10 9 cells, and the cell viability ≥ 80.0%. If the above requirements are met, send the sample for flow cytometry. If the above requirements are not met, continue the culture. If the culture system is less than 2700 mL, supplementary fluid can be added, and NK cell expansion medium is added. After supplementation, the viable cell density ≥ 6.00×10 5 cells / mL, and the total volume after supplementation does not exceed 2700 mL. If the flow cytometry result does not meet the flow cytometry standard for sample submission, terminate this culture.

[0171] Take the total number of viable cells, cell viability, NK purity, total T cell impurities, and B cell impurities before supplementation on the 9th - 12th day as in - process control indicators. If the total number of viable cells and cell viability meet the in - process control standards, take a sample and send it for flow cytometry detection; if the total number of viable cells and cell viability do not meet the in - process control standards, continue the culture until the 12th day. Until the in - process control standards are met, take a sample and send it for flow cytometry detection. If it still does not meet the standards, terminate the culture. If the flow cytometry result meets the in - process control standards in the above situations, inoculate and culture in the reactor; if the flow cytometry result does not meet the in - process control standards, terminate the culture. The specific in - process control standards are shown in Table 1 below.

[0172] Table 1: Reactor in - process control standards (9th - 12th day)

[0173]

[0174] Note 1: *The total number of viable cells is calculated according to the viable cell density, and the total number of viable cells = viable cell density × volume.

[0175] Note 2: The above process is for a 50L reactor (working volume 25L). For a 100L reactor, the total number of inoculated cells (and the number of inoculated culture flasks) during the PBMC resuscitation operation in this process is twice that of the 50L reactor. Other culture and fluid addition parameters are the same. During the bag transfer culture from D5 to D7, every three culture flasks are transferred to one bag for culture, and two bags of cells are transferred to the reactor for culture.

[0176] NK cell expansion culture

[0177] Reactor inoculation culture

[0178] On the 9th - 12th day, after the in - process control results are confirmed, when the total number of viable cells per bag ≥ 2.0×10 9 cells, the cell viability ≥ 80.0%, the NK purity (CD3 - CD56 + ) ≥ 70.0%, the total T - cell impurity (CD3 + ) ≤ 9.0%, and the B - cell impurity (CD19 + ) ≤ 5.0%, then it can be transferred to the reactor for continuous expansion culture.

[0179] Transfer all of the above cell suspension to the reactor cell culture bag. Adjust the fluid addition volume according to the viable cell density. The fluid addition volume is the NK cell expansion medium, and add 25 - 100 mL of serum replacement (II) (composition: platelet lysate and human albumin, volume ratio 1:1). After fluid addition, the viable cell density ≥ 4.00×10 5 cells / mL, and the glucose concentration ≥ 1.00 g / L. If the glucose concentration after fluid addition is lower than 1.00 g / L, then add glucose solution to make the glucose concentration of the cell suspension 1.00 - 2.00 g / L.

[0180] The reactor process parameters are controlled as follows:

[0181] a. Culture parameters: The total ventilation volume is 20 - 100 mL / min, the culture temperature is 37.0 ± 0.5 °C, the rotation speed is 8 - 10 rpm, the angle is 4 - 6 °, pH: 7.0 ± 0.2, DO: ≥ 20%.

[0182] b. pH control is related to carbon dioxide and 1 mol / L sodium bicarbonate, and DO control is related to oxygen and nitrogen.

[0183] Reactor expansion culture

[0184] The reactor process parameters are controlled as follows:

[0185] Culture parameters: The total ventilation volume is 20 - 40 mL / min, the culture temperature is 37.0 ± 0.5 °C, the rotation speed is 8 - 10 rpm, the angle is 4 - 6 °, pH: 7.0 ± 0.2, DO: ≥ 20%.

[0186] pH / DO control: pH control is associated with carbon dioxide and 1 mol / L sodium bicarbonate, and DO control is associated with oxygen and nitrogen.

[0187] Sampling: After adjusting the rotation speed and angle to mix the cell suspension evenly, sample for detecting pH value, viable cell density, cell viability, glucose, lactate, ammonia, and glutamine. If the absolute value of the deviation between the detected pH value and the on-line pH value is ≥ 0.10, pH calibration is carried out, and flow cytometry detection is carried out on the 15th to 18th days.

[0188] Fluid replacement: Adjust the fluid replacement volume according to the viable cell density. After fluid replacement, the viable cell density ≥ 5.00×10 5 cells / mL. If fluid replacement has not been carried out for three consecutive days, the cultivation of this batch is terminated. After the second fluid replacement, add 25 - 50 mL of serum substitute (Ⅱ). If no fluid replacement is carried out and the glucose concentration in the cell suspension is lower than 1.00 g / L, add glucose solution to make the glucose concentration in the cell suspension 1.00 - 2.00 g / L until the total volume is made up to 25 L.

[0189] Perfusion: When the culture system reaches the maximum volume of the culture bag and the lactate concentration ≥ 1.00 g / L, start to adopt the intermittent perfusion culture mode to replace the fresh NK cell expansion medium (using a hollow fiber column). Add 25 - 100 mL of serum substitute (Ⅱ) after the first perfusion, and add 25 - 50 mL of serum substitute (Ⅱ) after intermittent perfusions (such as the third, fifth, and seventh perfusions). Requirements after perfusion: The theoretical glucose concentration ≥ 1.00 g / L. If the glucose concentration after perfusion < 1.00 g / L, add glucose solution to make the theoretical glucose concentration in the cell suspension 1.00 - 2.00 g / L.

[0190] Take the cell viability, NK purity, total T cell impurities, and double-negative cell impurities before fluid replacement on the 15th to 18th days as the in-process control indicators. If they meet the in-process control standards, the next step of the amplification culture operation can be carried out; otherwise, the culture is terminated. The specific in-process control standards are shown in Table 2 below.

[0191] Table 2: In-process control standards for reactor amplification culture (15th - 18th days)

[0192]

[0193] Note: If on the 15th to 18th days, the cell viability, NK purity (CD3 - CD56 + ), total T cell impurities (CD3 + ), and double-negative cell impurities (CD3 - CD56 - ) have met the in-process control standards in Table 2, the NK cell concentration washing and stock solution preparation procedures can be carried out on D18 - D23.

[0194] 48 hours before the preparation of the stock solution, samples are taken for sterility inspection. The sterility inspection is used as an in-process control index. If it meets the in-process control standards, the next operation of Pre-stock solution preparation can be carried out.

[0195] NK Cell Concentration, Washing and Stock Solution Preparation

[0196] Centrifugal Washing of NK Cell Suspension:

[0197] Concentration: The NK cell suspension obtained above is processed using a fully enclosed automated cell processing system. The temperature is set at 4°C (temperature control range: 2 - 8°C). The sample is continuously flow-concentrated with an injection flow rate of 250 mL / min and a centrifugal speed of 2200 rpm.

[0198] Washing: After the continuous flow concentration is completed, it enters the washing step with Wash Solution - 1. The number of washing cycles is 6 - 8 times, the single washing volume is set at 140 mL, the flow rate is 150 mL / min, and the centrifugal speed is 1500 rpm. After the washing step with Wash Solution - 1 is completed, it enters the washing step with Cryopreservation Solution - 2. The number of washing times is 1 - 2 times, the single washing volume is set at 140 mL, the flow rate is 150 mL / min, and the centrifugal speed is 1500 rpm.

[0199] Pre-stock solution Preparation:

[0200] According to the NK cell count result, calculate the resuspension volume of Cryopreservation Solution - 2. The resuspension volume range of the Pre-stock solution is ≤600 mL, the flow rate is 150 mL / min, the number of cycles is 1 time, the high mixing speed is 800 rpm, the mixing acceleration is 200 rpm, and the mixing time is 180 - 300 s. After resuspension, the resuspended solution is transferred to the stock solution bag at a flow rate of 200 mL / min to obtain the Pre-stock solution.

[0201] Calculation formula for resuspension volume:

[0202]

[0203] If the calculated resuspension volume > 600 mL, resuspend at a volume of 600 mL.

[0204] Take about 1 mL for cell counting. Use the cell viability and viable cell density as in-process control indicators. If they meet the in-process control standards, the next operation can be carried out. The specific in-process control standards for the Pre-stock solution are shown in Table 3 below.

[0205] Table 3: In-process Control Standards for Pre-stock Solution (Days 18 - 23)

[0206]

[0207] Dilute Preparation of Stock Solution:

[0208] Based on the Pre-stock solution cell count result and the viable cell density of the stock solution ((0.8 - 1.2)×10 8 cells / mL), calculate the volume of the stock solution and the volume of cryopreservation solution - 2 to be supplemented.

[0209] Calculation formula: Volume of stock solution = (Pre-stock solution viable cell density × Pre-stock solution volume) / Set stock solution viable cell density;

[0210] Volume of cryopreservation solution - 2 = Volume of stock solution - Pre-stock solution volume.

[0211] According to the calculation result of the volume of cryopreservation solution - 2, directly add the supplemented cryopreservation solution - 2 into the centrifuge cup, and the injection flow rate of cryopreservation solution - 2 is 250 mL / min. The cryopreservation solution - 2 after washing the centrifuge cup will enter the stock solution bag.

[0212] After mixing the stock solution bag, take a sample for counting. If the counting result is (0.8 - 1.2)×10 8 cells / mL, then the stock solution is obtained. If the counting result exceeds the range of the stock solution viable cell density, then continue to add cryopreservation solution - 2 to adjust the viable cell density to (0.8 - 1.2)×10 8 cells / mL, and the NK cell stock solution is obtained.

[0213] Preparation of preparation

[0214] Filling: Use a closed filling system (temperature controlled at 2 - 8°C) to fill the stock solution. The peristaltic pump filling flow rate is 50 - 200 mL / min, and fill 20 mL (viable cell density (0.8 - 1.2)×10 8 cells / mL) or 25 mL (viable cell density (0.8 - 1.2)×10 8 cells / mL) or 30 mL (viable cell density (0.8 - 1.2)×10 8 cells / mL) or 50 mL (viable cell density (0.8 - 1.2)×10 8 cells / mL) into a single-use cell cryopreservation bag.

[0215] Sealing: Use a handheld heat sealer to seal the long catheter nozzle of the single-use cell cryopreservation bag. After heat sealing is completed, check that the preparation packaging is intact and there is no liquid leakage.

[0216] Labeling: Affix a label and transfer the product with the label affixed to a cryopreservation box (labels need to be affixed to both the cryopreservation box and the cryopreservation bag).

[0217] Programmed cooling:

[0218] a. Turn on the programmed cooler, and quickly transfer the labeled sample to the chamber of the programmed cooler for programmed cooling (temperature drops to -80°C).

[0219] b. For the relevant parameters of the programmed procedure, refer to Table 4.

[0220] Table 4: Relevant Parameters of the Programmed Procedure

[0221] Wait, chamber: 4.0 ± 1.0 °C. Temperature control, rate: 1.0 °C / min, sample: -8.0 to -10.0 °C. Temperature control, rate: 20.0 °C / min, chamber: -45.0 to -60.0 °C. Temperature control, rate: 10.0 °C / min, chamber: -25.0 to -30.0 °C. Temperature control, rate: 1.0 °C / min, sample: -40.0 °C. Temperature control, rate: 5.0 °C / min, sample: -80.0 °C. Hold, time: 5 min, chamber: -80.0 °C.

[0222] Cryopreservation: After the programmed cooling is completed, transfer the NK cell injection to a vapor-phase liquid nitrogen tank for cryopreservation (storage condition: ≤ -150°C).

[0223] The exemplary simplified preparation steps of the NK cell injection are as follows:

[0224] NK cells are derived from peripheral blood, prepared into PBMC cells in vitro, and stored in liquid nitrogen. The resuscitated PBMC cells are subjected to initial culture and amplification culture in vitro to obtain high-quality NK cells. After the NK cell suspension is concentrated and washed, the NK cells are adjusted to a viable cell density of (0.8 - 1.2) × 10 8 cells / mL, which is the NK cell stock solution. Use a closed filling system to fill the NK cell stock solution into a single-use cell cryopreservation bag. Heat-seal and label the single-use cell cryopreservation bag, place it in a cryopreservation box, and then transfer it into the programmed cooling instrument cavity for programmed cooling to -80°C. After the programmed cooling is completed, transfer it to a liquid nitrogen tank for storage (storage condition: ≤ -150°C), and the NK cell injection is obtained. The final cell purity quality control of the NK cell injection is shown in Table 5:

[0225] Table 5: Final Cell Purity Quality Control of NK Cell Injection

[0226]

[0227] Example 2: In vitro killing experiment of NK cells combined with anti-CD20 antibody on SLE-B cells

[0228] CFSE / DRAQ7 fluorescence labeling method

[0229] The experimental method of the CFSE / DRAQ7 fluorescence labeling method includes seven steps: experimental grouping, preparation before the experiment, cell sorting, preparation of SLE-B cells, antibody preparation, preparation of NK cell injection, and co-incubation and detection.

[0230] Experimental grouping

[0231] Experimental conditions:

[0232] Target cells: SLE-B cells.

[0233] Antibody: CD20 monoclonal antibody injection.

[0234] Effector-to-target ratio: 10:1, 3:1, 1:1, 0:1.

[0235] Antibody concentrations: 10000 ng / mL, 1000 ng / mL, 1 ng / mL, 0 ng / mL. Number of replicates: 2.

[0236] Layout distribution:

[0237] Table 6: Experimental design layout table

[0238]

[0239] Preparation before the experiment

[0240] Prepare sorting wash solution:

[0241] Weigh 3722.8 mg of EDTA-Na2-2H2O and dissolve it in 50 mL of ultrapure water. Filter it with a Syringe Filter Unit to prepare a 0.2 M EDTA solution.

[0242] Take 49 mL of PBS, add 1 mL of FBS, and add 250 μL of 0.2 M EDTA solution to prepare PBS containing 2% FBS and 1 mM EDTA, which is the sorting wash solution.

[0243] CFSE preparation:

[0244] Take a tube of CFSE dry powder, centrifuge it at 7000 RPM for 30 s in a compound rotor centrifuge, add 36 μL of DMSO, shake it for 30 s, and then centrifuge it at 7000 RPM for 30 s in a compound rotor centrifuge to obtain a 5 mM CFSE solution.

[0245] Complete medium preparation:

[0246] Take 450 mL of RPMI 1640 and add 50 mL of FBS, mix well, which is the complete medium.

[0247] Cell sorting:

[0248] Preheat the water bath to 37 °C.

[0249] Take out the cryopreserved PBMC and thaw it in a 37 °C water bath. Transfer it to a 15 mL centrifuge tube, add an appropriate amount of complete medium to wash. Centrifuge at 400 g for 10 min, discard the supernatant, add an appropriate amount of sorting wash solution to resuspend, take a sample and dilute it 20 times for AO / PI counting, and adjust the cell density to be between 5×10 7 to 1×10 8 cells / mL.

[0250] Take an appropriate amount of resuspended PBMC into a 5 mL polystyrene round-bottom tube, add CD19 Selection Cocktail at 10%-20% of the PBMC volume, blow it evenly, and incubate at room temperature for 3-5 min.

[0251] Resuspend by shaking RapidSpheres for 30 s. Add RapidSpheres at 10%-20% of the PBMC volume to the above PBMC, mix well, and incubate at room temperature for 3-5 min.

[0252] Supplement the sorting wash buffer to the PBMC to a total volume of 2.5 mL, and mix well.

[0253] Place a 5 mL polystyrene round-bottom tube into the EasySep TM Magnet and incubate at room temperature for 3-5 min. Pick up the EasySep TM Magnet and slowly and steadily pour the supernatant in the tube into a 15 mL centrifuge tube labeled as depleted B PBMC. Remove the 5 mL polystyrene round-bottom tube from the EasySep TM Magnet, add 2.5 mL of sorting wash buffer, mix well, and blow down the precipitate on the tube wall. Repeat this step 3 times.

[0254] After resuspending with an appropriate amount of RPMI 1640 for the last time, transfer the cell suspension to a 15 mL centrifuge tube and label it as SLE-B. Count using AO / PI.

[0255] Preparation of SLE-B cells

[0256] According to the experimental design, take an appropriate amount of SLE-B cells (5×10 5 to 3×10 6 ) into a 15 mL centrifuge tube, supplement RPMI1640 to 4 mL, add 2-3 μL of CFSE solution to each tube, mix well, place in an incubator at 37 °C for 30 min, and mix once in the middle.

[0257] After the culture, add 4 mL of complete medium to each tube, shake well, centrifuge at 400 g for 10 min, and discard the supernatant. Add 4 mL of complete medium to each tube, shake well, centrifuge at 400 g for 10 min, and discard the supernatant. Resuspend with an appropriate amount of complete medium and count using AO / PI.

[0258] Adjust the density of the stained target cells to 2×10 5 cells / mL with complete medium according to the counting results, and prepare the corresponding target cell working solution.

[0259] Antibody preparation

[0260] The original concentration of the antibody is 10 mg / mL, and the planned final concentrations for use are 10000 ng / mL, 100 ng / mL, and 1 ng / mL.

[0261] Take an appropriate amount of the antibody stock solution and add it to an appropriate amount of complete medium to prepare antibody working solution I with a concentration of 50000 ng / mL.

[0262] Take an appropriate amount of antibody working solution I and add it to an appropriate amount of complete medium to prepare antibody working solution II with a concentration of 500 ng / mL.

[0263] Take an appropriate amount of antibody working solution II and add it to an appropriate amount of complete medium to prepare antibody working solution III with a concentration of 500 ng / mL.

[0264] Preparation of NK cell injection

[0265] Take out the pre-frozen NK cell injection from the liquid nitrogen tank and quickly place it in a water bath at 37 °C for resuscitation.

[0266] After resuscitation, according to the labeled value of 1×10 8 cells / mL, take an appropriate amount of NK cell injection and add an appropriate amount of complete medium to dilute it to the working concentration of 2×10 6 cells / mL to prepare NK working solution I.

[0267] Take an appropriate amount of NK working solution I and add an appropriate amount of complete medium to dilute it to the working concentration of 6.6×10 5 cells / mL to prepare NK working solution II.

[0268] Take an appropriate amount of NK working solution II and add an appropriate amount of complete medium to dilute it to the working concentration of 2.2×10 5 cells / mL to prepare NK working solution III.

[0269] Co-incubation and detection

[0270] According to the experimental design layout table in Table 6, add NK working solution (100 μL / well), target cell working solution (100 μL / well), and antibody working solution (50 μL / well) to the corresponding wells of a 96-well plate in sequence. Then, use complete medium to fill up all wells to 250 μL, and place the 96-well plate on a microplate oscillator and shake it at 400 rpm for 3 min. Then, place it in an incubator at 37 °C and 5% CO2 for 6 h.

[0271] Prepare the live / dead dye working solution within 30 min before the end of incubation. Add 0.4% volume of DRAQ7 to an appropriate amount of PBS. Mix well and store in the dark.

[0272] After incubation, centrifuge the 96-well plate at 400 g for 10 min, discard the supernatant, add 100 μL of the live / dead dye working solution to each well to resuspend the cells, incubate in the dark at room temperature for 20 min, and then add 140 μL of PBS to each well. Detect by flow cytometry.

[0273] Killing detection results

[0274] Using the CFSE / DRAQ7 fluorescence labeling method, the killing activity of NK cell injection combined with anti-CD20 antibody against SLE-B cells was detected. The results showed that NK cell injection combined with anti-CD20 antibody had significant killing activity against SLE-B cells, and the killing effect increased with the increase of the effector-to-target ratio and antibody concentration. The killing of SLE-B cells by NK cell injection combined with anti-CD20 antibody was significantly higher than that of anti-CD20 antibody against SLE-B cells; the killing of SLE-B cells by NK cell injection combined with anti-CD20 antibody was significantly higher than that of NK cell injection against SLE-B cells. The detection results are shown in Table 7 and Figure 1 as follows.

[0275] Table 7: Killing results of NK cell injection combined with anti-CD20 antibody against SLE-B cells

[0276]

[0277] Example 3: Application of NK cells in the treatment of systemic lupus erythematosus

[0278] Patient's past medical history

[0279] Male patient, 50 years old, diagnosed with systemic lupus erythematosus for five years, with multiple organ involvement throughout the body. He had previously received hormone, hydroxychloroquine, cyclophosphamide pulse therapy, etc. to control the condition.

[0280] Treatment situation

[0281] In April 2024, the patient presented with manifestations of acute exacerbation such as elevated dsDNA antibody, CRP, and erythrocyte sedimentation rate. The complement C3 and C4 had been low. Starting from May 15, 2024, the patient was given the NK cell injection prepared in Example 1 (cycle and dose: 30 days as a cycle, administered on the first day of each cycle, 7.5 billion per time, a total of 7.5 billion were infused in each cycle), and rituximab injection (trade name: 30 days as a cycle, administered on the first day of each cycle, and the administered dose was 100 mg) for treatment. As of September 2024, a total of 3 cycles of NK cell infusion were performed.

[0282] Treatment results

[0283] The efficacy of the NK cell injection prepared in Example 1 in the treatment of systemic lupus erythematosus is shown in Table 8 and Figure 2 as follows.

[0284] Table 8:

[0285]

[0286] As shown in Table 8, before the NK cell treatment was given on May 15, 2024, the patient's anti-dsDNA antibody, high-sensitivity C-reactive protein, erythrocyte sedimentation rate, etc. were higher than the normal range. After the NK cell treatment, the patient's anti-dsDNA antibody turned negative. The high-sensitivity C-reactive protein increased after the first treatment and returned to the normal level after continuous treatment. The active indexes such as erythrocyte sedimentation rate, total B lymphocytes, and percentage of total B lymphocytes decreased compared with those before medication. Combining the blood routine results in Table 8 and Figure 2 the rash status shown, it is indicated that the symptoms of the patient's systemic lupus erythematosus have been relieved.

[0287] Example 4: Application of NK cells in systemic lupus erythematosus complicated with Sjogren's syndrome

[0288] Patient's past medical history

[0289] Female patient, 67 years old, suffering from systemic lupus erythematosus for more than 4 years. Past medical history: Sjogren's syndrome for more than 10 years, and the condition was controlled with hormones, hydroxychloroquine, etc.

[0290] Treatment situation

[0291] Starting from June 15, 2024, the patient was given the NK cell injection solution prepared in Example 1 (cycle and dose: 30 days as a cycle, administered on the first day of each cycle, 7.5 billion per time, and a total of 7.5 billion were infused in each cycle), and rituximab injection solution (trade name: 30 days as a cycle, administered on the first day of each cycle, and the administered dose was 100 mg) was used in combination for treatment. As of September 2024, a total of 3 cycles of NK cell infusion were carried out.

[0292] Treatment results

[0293] The curative effect of the NK cell injection solution prepared in Example 1 in the treatment of systemic lupus erythematosus is shown in Table 9 and Figure 3 shown.

[0294] Table 9

[0295]

[0296] As shown in Table 9, before the NK cell treatment was given on June 15, 2024, the erythrocyte sedimentation rate was higher than the normal range, indicating disease activity. After the NK cell treatment, the erythrocyte sedimentation rate returned to the normal range and the platelet count increased. Combining the blood routine results in Table 9 and Figure 3 the rash status shown, it is indicated that the symptoms of the patient's systemic lupus erythematosus have been relieved.

[0297] Example 5: Application of NK cells in the treatment of systemic lupus erythematosus

[0298] Patient's past medical history

[0299] A male patient, 62 years old, has suffered from systemic lupus erythematosus for many years. He has previously taken prednisone, tacrolimus, etc. to control the condition. Due to elevated blood sugar, tacrolimus was replaced with cyclosporine; combined with decreased platelets, eltrombopag was taken.

[0300] Treatment situation

[0301] Starting from July 16, 2024, the NK cell injection prepared in Example 1 was administered (cycle and dose: 30 days as a cycle, administered on the first day of each cycle, 7.5 billion per time, a total of 7.5 billion were infused in each cycle), and rituximab injection (trade name: 30 days as a cycle, administered on the first day of each cycle, and the administered dose was 100 mg) was used for treatment. As of September 2024, a total of 2 cycles of NK cell infusion were carried out.

[0302] Treatment results

[0303] The efficacy of the NK cell injection prepared in Example 1 in the treatment of systemic lupus erythematosus is shown in Table 10 and Figure 4 as shown below.

[0304] Table 10

[0305]

[0306] As shown in Table 10, before the NK cell treatment was given on July 16, 2024, the platelet count was lower than the normal value. After the NK cell treatment, the skin symptoms of the patient improved, the platelet count returned to the normal range, the erythrocyte sedimentation rate decreased after the second cycle of NK cell treatment, and the percentage of total B lymphocytes decreased. Combining the blood routine results in Table 10 and the rash status shown in Figure 4 as shown below, it is indicated that the symptoms of systemic lupus erythematosus in the patient have been relieved.

Claims

1. Use of NK cells in the preparation of a medicament for treating systemic lupus erythematosus in an individual; optionally, wherein the individual also suffers from Sjögren's syndrome.

2. Use of NK cells in the preparation of a medicament for treating systemic lupus erythematosus in combination with an anti-CD20 antibody; optionally, the anti-CD20 antibody is rituximab or obinutuzumab.

3. Use of NK cells and anti-CD20 antibodies in the preparation of a medicament for treating a patient with systemic lupus erythematosus; optionally, wherein the anti-CD20 antibody is rituximab or obinutuzumab; optionally, wherein the patient also suffers from Sjögren's syndrome.

4. The use according to any one of claims 1 to 3, wherein the NK cells are expanded in vitro.

5. The use according to claim 4, wherein the NK cells are expanded in vitro in a serum-free culture system using a rocking bioreactor, and the culture process parameters of the in vitro expansion include: The total aeration volume was 20-100 mL / min, the culture temperature was 36.5-37.5°C, the rocking speed was 8-10 rpm, the rocking angle was 4-6°, the dissolved oxygen (DO) was ≥20%, and the pH was 6.8-7.

2.

6. The use according to any one of claims 1 to 3, wherein The NK cells are obtained from the in vitro induction and culture of NK cells derived from peripheral blood mononuclear cells (PBMC).

7. The use according to any one of claims 1 to 3, wherein the number of NK cells infused per cycle is greater than 3 billion, and at least one cycle of infusion is performed.

8. The use according to any one of claims 1 to 3, wherein CD3 in NK cells - CD56 + NK cell percentage ≥ 95.0%; and / or CD3 in NK cells - CD56 + CD16 + NK cell percentage ≥ 90.0%; and / or CD3 in NK cells - CD56 + NKG2D + NK cell percentage ≥ 95.0%; and / or CD19 in NK cells + B cells ≤ 2.0%; and / or CD3 in NK cells + CD56 - T cells account for ≤3.0%.

9. The use according to any one of claims 1 to 3, wherein the NK cells are stored in a bag, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the viable cell density in the NK cell suspension is 0.8-1.2×10 8 Pieces / mL.

10. The use according to any one of claims 1 to 3, wherein the NK cells are stored in a bag, each bag containing 20 mL to 50 mL of NK cell suspension, wherein the total number of viable cells in the NK cell suspension is 2.0-3.0×10 9 NK cells.

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