Methods of administering natural killer cells containing anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR)

Abstract

The present invention provides methods of administering NK cells comprising a polynucleotide comprising a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR).

Description

[Technical Field]

[0001] Priority claim This application claims priority to U.S. Provisional Application No. 63 / 398,834, filed August 17, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Targeted therapies, including adoptive cell therapies such as chimeric antigen receptor T cells (CAR T), have revolutionized cancer treatment. These cell therapies can be autologous (CAR T cells manufactured using the patient's own T cells) or allogeneic (CAR T cells manufactured using T cells from a healthy donor). Currently, there are no autologous CAR T therapies approved to treat HER2-specific cancers.

[0003] Although revolutionary in their ability to treat targeted hematological cancers, challenges have arisen in the clinical use of autologous CAR-T cells since the U.S. Food and Drug Administration approved the first CAR-T therapy targeting CD19 B-cell malignancies in 2017. CAR-T cell manufacturing is a resource-intensive process, which may not produce an autologous cell therapy suitable for some patients. The average three-week manufacturing time required for autologous CAR-T cell products may be too long for severely ill patients. Finally, the complex nature of CAR-T cell manufacturing and delivery, which requires close monitoring at top-tier cancer centers and medical institutions, may limit financial and geographic accessibility to this treatment option for most patients. Importantly, even for patients who have access to this innovative therapy, CAR-T cell products carry the risk of serious and potentially fatal side effects. These side effects include cytokine release syndrome (CRS) and neurotoxicity, which can be difficult to manage or control.

[0004] Allogeneic CAR-T cell therapies, which use cells from healthy donors, can overcome some of the manufacturing and logistical challenges of autologous CAR-T cell therapies. However, these "off-the-shelf" CAR-T cell therapies also have issues, such as a potentially high risk of graft-versus-host disease (GVHD) and inefficiency due to rapid clearance by the patient's immune system.

[0005] Natural killer (NK) cells are cytolytic immune cells of the innate immune system that have the unique ability to lyse tumor cells and virus-infected cells. NK cells have the unique ability to initiate innate and multiclonal adaptive immune responses and generate long-term anti-cancer immune memory. Importantly, NK cells do not require prior antigen exposure to identify and lyse tumor cells. Receptor binding on NK cells promotes effector function by degranulating lytic granules, activating programmed cell death receptors on target cells, and secreting immunoregulatory cytokines. The effector function of natural killer cells is regulated by the balance of activating and inhibitory receptor signals. Generally, natural killer cells express CD56 + and CD3 - cells, defined as CD56 bright CD16 - Cytokine-secreting cells and CD56 dim CD16 + NK cells are subdivided into cytolytic and cytolytic cells. Binding of CD16 to antibody-labeled tumor cells is sufficient to induce cytotoxic and cytokine-releasing responses in resting NK cells. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and macrophage inflammatory protein 1 (MIP1), to signal tumors and recruit T cells. NK cells directly kill tumor cells, exposing tumor antigens for recognition by the adaptive immune system. Natural killer cells also bind to tumor cells through antibody-dependent cellular cytotoxicity (ADCC). To initiate ADCC, NK cells bind antibodies via their surface CD16 receptor.

[0006] Natural killer cells also bind to tumor cells through antibody-dependent cellular cytotoxicity (ADCC), a core component of the innate immune system. Antibody-coated target cells are killed by cells bearing Fc receptors that recognize specific regions of the bound antibody. Binding of antibody-opsonized tumor cells to CD16 (FCγRIII) is sufficient to induce cytotoxic and cytokine-releasing responses from resting NK cells. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and macrophage inflammatory protein 1 (MIP1), to signal tumors and recruit T cells. NK cells also directly kill tumor cells, exposing tumor antigens for recognition by the adaptive immune system. ADCC, particularly with antibodies belonging to the immunoglobulin G1 (IgG1) and IgG3 subclasses, is recognized as a powerful mechanism of NK cell action. To initiate ADCC, NK cells bind antibodies via the CD16 receptor.

[0007] Like T cells, allogeneic NK cells genetically modified to express CARs with antitumor activity can provide an important treatment option for cancer patients. NK cells are free from the drawbacks of allogeneic CAR-T cells, which maintain expression of endogenous T cell receptors in addition to genetically modified chimeric antigen receptors. As a result, allogeneic CAR-NK cell therapy can be safely administered to patients without many of the risks associated with allogeneic T cell therapy, including graft-versus-host disease. However, CAR-NK cells face many of the same challenges as other allogeneic cell therapies, including product sourcing, scalability, persistence, and dose-to-dose variability.

[0008] HER2, also known as human epidermal growth factor receptor 2 or ErbB2, is a receptor tyrosine kinase highly expressed on the surface of many solid tumors. Although HER2 plays an important role in cell development in normal cells, mutations or overexpression of HER2 can directly lead to tumor initiation and metastasis.

[0009] HER2 amplification, which is considered a key signal in tumorigenesis, is common in a variety of solid tumors, including 20-30% overexpression in human breast, ovarian, and gastric cancers.

[0010] Currently, eight HER2-targeted therapeutic agents are approved for cancer patients. These FDA-approved drugs include monoclonal antibodies, antibody-drug conjugates (ADCs), and small-molecule tyrosine kinase inhibitors (TKIs). In existing therapies for HER2-positive cancers, such as trastuzumab or lapatinib, downstream signaling mutations can lead to resistance to tumor growth inhibition. One of the mechanisms of action of trastuzumab is by inhibiting the MAPK and PI3K / Akt pathways, which lead to cell cycle arrest. Mutations including those that induce loss of PTEN (phosphatase and tensin homolog) and activating mutations in PIK3CA (phosphatidylinositol 3-kinase) can confer resistance to trastuzumab treatment. These mutations can constitutively activate the PI3K / Akt pathway, which induces cell proliferation. PTEN loss was observed in 36% of HER2-positive primary breast cancer specimens (stage 4), and these patients had a lower overall response rate to trastuzumab. Additionally, approximately 25% of trastuzumab-resistant patients have PIK3CA mutations. Patients with PI3KCA mutations had significantly shorter progression-free survival times after trastuzumab treatment than patients without mutations. Therefore, while many patients achieve meaningful benefit from trastuzumab, a significant number of patients eventually experience recurrence or disease progression. Once all HER2-targeted therapies have been exhausted, patients are left with only marginally effective cytotoxic chemotherapy. The lack of safe and effective treatments for patients who have exhausted all HER2-targeted treatment options continues to highlight the need for their development.

[0011] The present invention seeks to overcome these disadvantages of the prior art. Summary of the Invention

[0012] NK cells are immune cells that can bind to tumor cells through a complex array of receptors on their cell surface and antibody-dependent cellular cytotoxicity (ADCC). NK cells have several advantages over other immune cells, such as T cells, used in CAR-T cell therapy. Autologous CAR-T cells must be engineered using the patient's own cells, but this engineering process is time-consuming and can significantly progress the patient's disease during that time. Therefore, patients may require bridging therapy to maintain treatment until autologous CAR-T cells are ready. Not all patients are suitable for autologous CAR-T therapy. For example, some patients may have severe symptoms or insufficient numbers of T cells suitable for engineering purposes. Autologous CAR-T cell production does not always produce sufficient cell numbers or sufficiently active cells for therapeutic efficacy. If such production is successful, patients generally receive only a single autologous CAR-T treatment. Because the risk of acute side effects such as ICANS and CRS is greatest immediately after CAR-T cell administration, repeated administrations can potentially be dangerous if patients experience only modest benefits from a second, third, or subsequent administration. In addition, autologous CAR-T therapy is individualized to each patient, which places a heavy financial burden on the patient.

[0013] NK cells offer a key advantage as an allogeneic therapy: NK cells from a single donor can be safely used in one or more other patients without the need for HLA matching, gene editing, or other genetic manipulation. This advantage allows allogeneic CAR-NK cells to be mass-produced, cryopreserved, and distributed worldwide for administration at the point of care as needed. Therefore, allogeneic cell therapies can be administered immediately to patients without cross-linking therapy, eliminating the need to wait for the manipulation and administration of the patient's own cells. Because the allogeneic therapies described herein can be mass-produced using campaign manufacturing methods, the costs associated with manufacturing and delivering the allogeneic therapies described herein are expected to be significantly lower than those of autologous CAR-T therapies. Campaign manufacturing also reduces batch-to-batch variability, allowing a single batch of CAR-NK cells extracted from a single donor to be administered multiple times if desired by the patient.

[0014] Providing repeated administrations allows patients to achieve or maintain a higher or extended therapeutic response to treatment. For example, patients can receive response-based dosing, during which they continue to receive CAR-NK cell therapy as long as they benefit. Additionally, the number of cells administered in each dose and the number of doses can be tailored to the individual patient. In this case, patients are not limited to the number of cells as in autologous CAR-T therapy. Thus, the CAR-NK cell therapy described herein can be tailored to each patient based on their own response. In some cases, if a patient relapses, treatment can be initiated again.

[0015] Allogeneic NK cells may be an important treatment option for cancer patients. One exemplary advantage is that NK cells are well tolerated without the side effects of graft-versus-host disease, neurotoxicity, or cytokine release syndrome associated with other cell-based therapies. Another exemplary advantage is that NK cells do not require prior antigen exposure to recognize and lyse tumor cells. Yet another exemplary advantage is that NK cells have the intrinsic ability to initiate innate and polyclonal adaptive immune responses and generate long-term anti-cancer immune memory. All of these characteristics make NK cells a potential treatment option for cancer.

[0016] For example, NK cells can recruit and activate other components of the immune system. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and macrophage inflammatory protein 1 (MIP-1), to signal and recruit T cells to tumors. NK cells can also directly kill tumor cells, exposing tumor antigens for recognition by the adaptive immune system.

[0017] Furthermore, by utilizing diverse cord blood banks as a source of NK cells, cord blood units with favorable characteristics (e.g., high-affinity CD16 and KIR (Killer cell immunoglobulin-like receptor) B-haplotypes) can be selected to improve clinical activity.

[0018] Genetically engineered NK cells, such as the CAR-NK cells referred to herein, have the advantage over autologous cell therapies, such as the T cells used in CAR-T cell therapy, because they can be used as an allogeneic therapy. Thus, NK cells from one donor can be safely used in one or more other patients.

[0019] In existing Her2-positive cancer therapies, such as trastuzumab or lapatinib, mutations can lead to resistance to tumor growth inhibition. As explained above, some mutations alter downstream signaling pathways, rendering cells resistant to trastuzumab or lapatinib. Unlike trastuzumab or lapatinib, the HER2-guided CAR-NK cells and therapies such as AB-201 described herein are activated by binding to HER2 expressed on the surface of target cells. Activated CAR-NK cells kill target cells using their own cytotoxic pathways. Therefore, such killing occurs independently of the integrity of intracellular signals within the target cells. Therefore, the CAR-NK cell therapeutics described herein maintain their ability to kill HER2-positive tumor cells, even in the presence of some downstream signaling mutations that confer resistance to approved HER2 therapeutics. Other mutations may alter the epitopes to which trastuzumab or lapatinib bind, reducing the efficacy of these drugs. For example, the HER2-targeting CAR-NK scFv described herein, comprising the scFv of SEQ ID NO: 30, binds to a different domain of HER (domain I) than trastuzumab (domain IV) or lapatinib (domain II), such that the CAR-NK cells described herein can maintain their effectiveness in binding to HER2-positive cells even if other mutations reduce or inhibit the binding ability of trastuzumab or lapatinib.

[0020] Additionally, the CAR-NK cells described herein can maintain expression of CD16, including expression of the 158V / V variant of CD16. Thus, in some cases, CAR-NK cells can be used in conjunction with traditional antibody therapy. As an example, antibody therapy may consist of trastuzumab or lapatinib. Alternatively, or in combination, antibody therapy can target alternative or additional targets, including, for example, EGFR. Examples of anti-EGFR antibodies include cetuximab, panitumumab, nimotuzumab, and necitumumab. Such antibodies can bind to CD16 expressed on the surface of NK cells and induce an ADCC response from the NK cells. Thus, in some cases, therapeutic methods can include a dual-target approach that combines the use of the HER2-targeting CAR-NK cells described herein with anti-EGFR antibody therapy.

[0021] Accordingly, the present invention provides a method comprising administering to a patient between 1 million and 100 billion natural killer (NK) cells, wherein the NK cells comprise: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a polynucleotide comprising a nucleic acid encoding IL-15.

[0022] In one embodiment, the NK cells are administered in a dose comprising 1 billion to 100 billion NK cells, optionally 1 million to about 20 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 million cells, about 75 million cells, about 100 million cells, about 200 million cells, about 300 million cells, about 400 million cells, about 500 million cells, about 1 billion cells, or about 2 billion cells), about 3 billion cells, about about 4 billion cells, about 5 billion cells, about 6 billion cells, about 7 billion cells, about 8 billion cells, about 9 billion cells, about 10 billion cells, or a range defined by two of the foregoing values), about 10 million to about 20 billion cells (e.g., about 25 million cells, about 50 million cells, about 75 million cells, about 100 million cells, about 200 million cells, about 300 million cells, about 400 million cells, about 500 million cells, about 1 billion cells, about 2 billion cells, about 3 billion cells, about 4 billion cells, about 5 billion cells, or about 6 billion cells, or about 7 billion cells, or about 8 billion cells, or about 9 billion cells, or about 10 billion cells, or about 20 billion cells, or a range defined by two of the foregoing values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 1 The antibody may be administered in a dose containing about 150 million cells, about 200 million cells, about 300 million cells, about 400 million cells, about 500 million cells, about 1 billion cells, about 2 billion cells, about 3 billion cells, about 4 billion cells, about 5 billion cells, about 6 billion cells, about 7 billion cells, about 8 billion cells, about 9 billion cells, about 10 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 40 billion cells).

[0023] In one embodiment, the NK cells are at least 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 or 9×10 6 Cells, per dose, minimum 1 x 10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×107 , 7×10 7 , 8×10 7 or 9×10 7 Cells, per dose, minimum 1 x 10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 Cells, per dose, minimum 1 x 10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 Minimum 1 x 10 cells or per dose 10 or 2×10 10 The cells are administered in a dose that includes the cells.

[0024] In one embodiment, the NK cells are administered at a concentration of about 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , or 9×10 6 Cells, per dose, approximately 1 x 10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 Cells, per dose, approximately 1 x 10 8 , 2 × 10 8 , 3×10 8 , 4×1 08 , 5×10 8 , 6×10 8, 7×10 8 , 8×10 8 or 9×10 8 Cells, per dose, approximately 1 x 10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 Approximately 1 x 10 cells, or per dose 10 or 2×10 10 It is administered in cells.

[0025] In one embodiment, the NK cells are from about 1 million to about 20 billion CAR-expressing NK cells (e.g., about 5 million cells, about 25 million CAR-expressing cells, about 50 million CAR-expressing cells, about 75 million CAR-expressing cells, about 100 million CAR-expressing cells, about 200 million CAR-expressing cells, about 300 million CAR-expressing cells, about 400 million CAR-expressing cells, about 500 million CAR-expressing cells, about 1 billion ...200 million CAR-expressing cells, about 200 million CAR-expressing cells, about 200 million CAR-expressing cells, about 200 million CAR-expressing cells, about 200 million CAR-expressing cells, about In some embodiments, the CAR-expressing cells may be administered at a dose comprising about 10 billion CAR-expressing cells, about 2 billion CAR-expressing cells, about 3 billion CAR-expressing cells, about 4 billion CAR-expressing cells, about 5 billion CAR-expressing cells, about 6 billion CAR-expressing cells, about 7 billion CAR-expressing cells, about 8 billion CAR-expressing cells, about 9 billion CAR-expressing cells, about 10 billion CAR-expressing cells, or about 20 billion CAR-expressing cells, or a range defined by two of the foregoing values).

[0026] In one embodiment, the NK cells are administered at a concentration of 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 or 9×10 6 1 x 10 CAR-expressing cells per dose 7 , 2 × 10 7 , 3×10 7 , 4×107 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 1 x 10 CAR-expressing cells per dose 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 CAR-expressing cells per dose, approximately 1 x 10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 CAR-expressing cells, or approximately 1 x 10 per dose 10 or about 2 x 10 10 The patient is administered a dose containing 10 CAR-expressing cells.

[0027] In one embodiment, the NK cells are at least about 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 or 9×10 6 CAR-expressing cells, at least approximately 1 x 10 per dose 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 CAR-expressing cells, at least approximately 1 x 10 per dose 8, 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 CAR-expressing cells, at least approximately 1 x 10 per dose 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 CAR-expressing cells, at least approximately 1 x 10 per dose 10 The patient is administered a dose containing 10 CAR-expressing cells.

[0028] The present invention also provides a method of administering multiple doses of NK cells to a patient in need thereof, the NK cells comprising a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, said NK cells comprising 1×10 5 ~10×10 8 The dose is given in the form of cells / kg.

[0029] In one embodiment, the dose of NK cells is about 1 x 10 5 cells / kg ~ approx. 1×10 8 Between cells / kg, e.g., about 1.5 x 10 5 ~Approx. 1.5×10 7 cells / kg, or approximately 4 x 10 5 ~Approx. 4×10 6 cells / kg.

[0030] In one embodiment, the NK cells are 1×10 5 , 1.5×10 5 , 2 × 10 5 , 2.5×105 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 or 9.5 x 10 5 cells / kg, 1×10 6 , 1.5×10 6 , 2 × 10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 or 9.5 x 10 6 cells / kg, 1×10 7 , 1.5×10 7 , 2 × 10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 cells / kg, or 9.5 × 10 7 or about 1 x 108 , 1.5×10 8 or 2×10 8 The dose is given in the form of cells / kg.

[0031] In one embodiment, the NK cells are at least about 1 x 10 5 , 1.5×10 5 , 2 × 10 5 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 or 9.5 x 10 5 cells / kg, minimum approximately 1×10 6 , 1.5×10 6 , 2 × 10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , or 9.5 × 10 6 cells / kg, minimum approximately 1×10 7 , 1.5×10 7 , 2 × 10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×107 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 cells / kg, or 9.5 × 10 7 , or minimum or approximately 1 × 10 8 or 1.5 x 10 8 The dose is given in the form of cells / kg.

[0032] The present invention also provides a method of administering multiple doses of NK cells to a patient in need thereof, the NK cells comprising a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, the administration being carried out once every 1 to 16 weeks.

[0033] In one embodiment, the NK cells are administered every other week, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, or once every 16 weeks.

[0034] The present invention also provides a method of administering to a patient in need thereof NK cells comprising a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, wherein the patient meets one or more of the following criteria: (i) the patient has an ECOG (Eastern Cooperative Oncology Group) performance status between 0 and 4 at the time of treatment; (ii) the patient has previously received one or more systemic therapies and has been refractory or relapsed; (iii) the patient's oxygen saturation as measured by pulse oximetry is 92% or greater; (iv) the patient's left ventricular ejection fraction (LVEF) is 50% or greater; (v) the patient's absolute neutrophil count (ANC) is 1000 / mm 3 (1.0×10 9 / L) or greater; (vi) the patient's platelet count is 75,000 / mm 3 (75×10 9 / L) or greater; (vii) the patient's hemoglobin level is 8.0 g / dL or greater, regardless of previous transfusions; (viii) the patient's creatinine clearance rate is 45 mL / min or greater by the Cockcroft-Gault formula or the estimated glomerular filtration rate (eGFR) is 1.73 mL / min / 1.73 m by the Modification of Diet in Renal Disease (MDRD) formula. 2 or greater; (ix) the patient's total serum bilirubin is less than 5 mg / dL; or (x) the patient's liver transaminases (AST / ALT / ALP (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase)) are less than 5 times the upper limit of normal (ULN).

[0035] In one embodiment, the patient has an ECOG (Eastern Cooperative Oncology Group) performance status of 0, 1, 2, 3, or 4 at the time of treatment.

[0036] In one embodiment, the patient has previously received two or more systemic therapies and is refractory or has relapsed.

[0037] In one embodiment, the patient has an oxygen saturation of 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater as measured by pulse oximetry.

[0038] In one embodiment, the patient has a left ventricular ejection fraction (LVEF) of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or greater.

[0039] In one embodiment, the patient has an absolute neutrophil count (ANC) of 1000 / mm 3 (1.0×10 9 / L), 1500 / mm 3 , 2000 / mm 3 , 2500 / mm 3 , 3000 / mm 3 , 3500 / mm 3 , 4000 / mm 3 , 4500 / mm 3 , 5000 / mm 3 , 5500 / mm 3 or 6000 / mm 3 That's all.

[0040] In one embodiment, the patient has a platelet count of 75,000 / mm 3 (75×10 9 / L), 100000 / mm 3 , 125000 / mm 3 , 150000 / mm 3 , 175000 / mm 3 , 200000 / mm 3 , 225000 / mm 3 , 250000 / mm 3 , 275000 / mm 3 , 300000 / mm 3 , 325000 / mm 3 , 350000 / mm 3 , 375000 / mm 3 or 400000 / mm 3 That's all.

[0041] In one embodiment, the patient has a hemoglobin level of 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, or 20.0 g / dL or greater, with or without prior transfusions.

[0042] In one embodiment, the patient's creatinine clearance rate by the Cockcroft-Gault formula is greater than or equal to 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 mL / min or the estimated glomerular filtration rate (eGFR) by the Modification of Diet in Renal Disease (MDRD) equation is greater than or equal to mL / min / 1.73m 2 That's all.

[0043] In one embodiment, the patient has a total serum bilirubin of less than 5, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / dL.

[0044] In one embodiment, the patient's liver transaminases (AST / ALT / ALP (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase)) are less than or equal to 5, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5 or 0 times the upper limit of normal (ULN).

[0045] The present invention also provides a method of administering to a patient in need thereof NK cells comprising a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, said patient meeting one or more of the following criteria: (i) the patient has no active nervous system metastases; (ii) the patient has no history or presence of clinically relevant CNS disorders, including seizure disorders (e.g., epilepsy), cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, cerebral edema, posterior reversible encephalopathy syndrome (PRES), or autoimmune diseases arising in the CNS; (iii) the patient has no active human immunodeficiency virus (HIV) infection, as evidenced by a positive HIV polymerase chain reaction (PCR) test; (iv) the patient has no active HBV or hepatitis C virus (HCV) infection, as evidenced by laboratory test results; (v) the patient has a viral HBV load below the institutional limit of quantitation (LOQ) and is receiving stable viral suppression therapy; or (vi) the patient's HCV RNA viral load is below the institutional LOQ and has completed therapeutic antiviral therapy.

[0046] The present invention also provides a method of administering to a patient in need thereof NK cells comprising a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15; Also, the method is characterized in that the patient meets one or more of the following criteria: (i) the patient is or has been diagnosed with a HER2-positive cancer with an IHC score of 2+; (ii) the patient has a cancer that is not HER2-intoxicated but shows some degree of HER2 expression; (iii) the patient is or has been diagnosed with a HER2-low expressing cancer; (iv) the patient is or has been diagnosed with a HER2-low expressing cancer with HER2 expression grade 1+ or 2+; (v) the patient is or has been diagnosed with a HER2-low expressing cancer without HER2 amplification; (vi) the patient has a cancer containing a HER2-activating mutation; or (vii) the patient has been diagnosed with a HER2-expressing cancer with an IHC score of 1+ or IHC2+ / ISH- that has been refractory to previous treatment or has relapsed after treatment.

[0047] In one embodiment, the patient is or has been diagnosed with a HER2-low expressing cancer in the absence of HER2 amplification, with an expression grade of 1+ or 2+.

[0048] In one embodiment, the HER2-low expressing cancer possesses HER2 gain.

[0049] In one embodiment, the HER2-low expressing cancer lacks HER2 gain.

[0050] In one embodiment, the patient has been diagnosed with IHC3+ or IHC2+ / ISH+ cancer and has previously been treated with a HER2-targeted therapeutic agent in a refractory or relapsed state.

[0051] In one embodiment, the patient has been diagnosed with a HER2-expressing cancer that is IHC1+ or IHC2+ / ISH-, and the patient's cancer is unresectable, metastatic, or refractory or recurrent after previous treatment.

[0052] According to some embodiments, in any of the methods of the invention described above, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 34, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 36, a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 38, a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 44, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 46, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 48.

[0053] According to some embodiments, in any of the methods of the invention described above, the nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof encodes CDRL1 encoded by SEQ ID NO: 35; CDRL2 encoded by SEQ ID NO: 37; CDRL3 encoded by SEQ ID NO: 39; CDRH1 encoded by SEQ ID NO: 45; CDRH2 encoded by SEQ ID NO: 47; and CDRH3 encoded by SEQ ID NO: 49.

[0054] According to some embodiments, in any of the methods of the invention described above, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain variable (V) comprising SEQ ID NO: 32. L ) region and a heavy chain variable (V) region comprising SEQ ID NO: 42 H ) area.

[0055] According to some embodiments, in any of the methods of the invention described above, the nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof is a V comprising SEQ ID NO: 33. L a nucleic acid encoding a region and a V comprising SEQ ID NO: 37 H and a nucleic acid encoding the region.

[0056] According to some embodiments, in any of the methods of the invention described above, the anti-HER2 antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. L a region and a V comprising the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; H and the area.

[0057] According to some embodiments, in any of the methods of the invention described above, the anti-HER2 antibody or antigen-binding fragment is an antigen-binding fragment. In one embodiment, the antigen-binding fragment comprises a single chain Fv (scFv). In one embodiment, the V L The region is V H In one embodiment, the V L The region is V H In one embodiment, the V L The domains are connected to the V H In one embodiment, the flexible linker comprises the amino acid sequence of SEQ ID NO: 40. In one embodiment, the flexible linker is encoded by a nucleic acid comprising SEQ ID NO: 41. In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO: 30. In one embodiment, the scFv is encoded by a nucleic acid comprising SEQ ID NO: 31. In one embodiment, the scFv comprises SEQ ID NO: 30, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0058] According to some embodiments, in any of the methods of the invention described above, the anti-HER2 CAR specifically binds to human epidermal growth factor receptor 2 (HER2) protein. In one embodiment, the HER2 protein comprises the amino acid sequence of SEQ ID NO: 62.

[0059] According to some embodiments, in any of the methods of the invention described above, the CAR comprises a transmembrane domain, optionally a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 53. In one embodiment, the CD28 transmembrane domain is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0060] According to some embodiments, in any of the methods of the invention described above, the CAR additionally comprises a hinge domain between the extracellular antigen-binding domain and the transmembrane domain. In one embodiment, the hinge domain comprises at least a portion of a CD8α hinge domain. In one embodiment, the CD8α hinge domain comprises the amino acid sequence of SEQ ID NO: 50. In one embodiment, the CD8α hinge domain is encoded by a nucleic acid comprising SEQ ID NO: 51 or SEQ ID NO: 52. In one embodiment, the CD8α hinge domain comprises SEQ ID NO: 50, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0061] According to some embodiments, in any of the methods of the invention described above, the CAR comprises an intracellular signaling region, optionally comprising a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD3-zeta (CD3ζ) signaling domain. In one embodiment, the intracellular signaling region comprises a CD28 intracellular signaling domain and a CD3-zeta signaling domain.

[0062] In one embodiment, the intracellular signaling region comprises an OX40L intracellular signaling domain. In one embodiment, the OX40L intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In one embodiment, the OX40L intracellular signaling domain comprises SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the OX40L intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO:11 or SEQ ID NO:12.

[0063] In one embodiment, the intracellular signaling region comprises a CD28 intracellular signaling domain. In one embodiment, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the CD28 intracellular signaling domain comprises SEQ ID NO: 5, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the CD28 intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO: 6 or SEQ ID NO: 7.

[0064] In one embodiment, the intracellular signaling region comprises a CD3-zeta intracellular signaling domain. In one embodiment, the CD3-zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the CD3-zeta intracellular signaling domain comprises SEQ ID NO: 13, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the CD3-zeta intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO: 14 or SEQ ID NO: 15.

[0065] In one embodiment, the intracellular signal region comprises the amino acid sequence of SEQ ID NO: 25. In one embodiment, the intracellular signal region comprises SEQ ID NO: 25 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0066] According to some embodiments, in any of the methods of the invention described above, the CAR comprises the amino acid sequence of SEQ ID NO: 56. In one embodiment, the CAR is encoded by a nucleic acid comprising SEQ ID NO: 57.

[0067] According to some embodiments, in any of the methods of the invention described above, the IL-15 comprises the amino acid sequence of SEQ ID NO: 22. In one embodiment, the IL-15 is encoded by a nucleic acid comprising SEQ ID NO: 23 or SEQ ID NO: 24.

[0068] According to some embodiments, in any of the methods of the invention described above, the polynucleotide encodes a polyprotein comprising CAR and IL-15.

[0069] According to some embodiments, in any of the methods of the invention described above, the polynucleoside additionally comprises a nucleic acid encoding a self-cleaving peptide, optionally a T2A self-cleaving peptide. In one embodiment, the CAR binds to the IL-15 via the self-cleaving peptide. In one embodiment, the self-cleaving peptide can induce ribosomal skipping between the CAR and IL-15.

[0070] In some embodiments, in any of the methods of the invention described above, the polynucleotide additionally comprises a nucleic acid encoding a signal sequence. In one embodiment, the signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 27. In one embodiment, the nucleic acid encoding the signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 28.

[0071] According to some embodiments, in any of the methods of the invention described above, the polynucleotide encodes a polyprotein comprising the amino acid sequence of SEQ ID NO:59.

[0072] According to some embodiments, in any of the methods of the invention described above, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:60 or SEQ ID NO:61.

[0073] According to some embodiments, in any of the methods of the invention described above, the NK cells are cord blood NK cells.

[0074] According to some embodiments, in any of the methods of the invention described above, the NK cells comprise a KIR-B haplotype.

[0075] According to some embodiments, in any of the methods of the invention described above, the NK cells express CD16 with V / V polymorphism at F158.

[0076] According to some embodiments, in any of the methods of the present invention described above, the NK cells are administered as part of a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) a buffer.

[0077] In one embodiment, the pharmaceutical composition contains 30-50 mg / mL of human albumin, hi one embodiment, the pharmaceutical composition contains 40 mg / mL of human albumin.

[0078] In one embodiment, the pharmaceutical composition contains 20-30 mg / mL of dextran. In one embodiment, the pharmaceutical composition contains 25 mg / mL of dextran. In one embodiment, the dextran is dextran 40.

[0079] In one embodiment, the pharmaceutical composition contains 12-15 mg / mL of glucose, 12.5 mg / mL of glucose, or less than 27.5 g / L of glucose.

[0080] In one embodiment, the pharmaceutical composition contains 50-60 mg / mL DMSO. In one embodiment, the pharmaceutical composition contains 55 mg / mL DMSO. In one embodiment, the pharmaceutical composition contains 40-60% v / v buffer. In one embodiment, the buffer is phosphate buffered saline.

[0081] In one embodiment, the pharmaceutical composition comprises (a) about 40 mg / mL human albumin, (b) about 25 mg / mL dextran 40, (c) about 12.5 mg / mL glucose, (d) about 55 mg / mL DMSO, and (e) about 0.5 mL / mL phosphate buffered saline.

[0082] In one embodiment, the pharmaceutical composition additionally comprises 0.5 mL / mL of water.

[0083] According to some embodiments, in any of the methods of the invention described above, the subject has a disease or condition associated with HER2. In one embodiment, the HER2-associated disease or condition is cancer. In one embodiment, the cancer expresses HER2. In one embodiment, the cancer is a HER2+ cancer. In one embodiment, the cancer is or comprises a solid tumor. In one embodiment, the cancer is or comprises bladder cancer, breast adenocarcinoma, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, squamous cervical cancer, gastric adenocarcinoma, cholangiocarcinoma, ovarian cancer, papillary renal cell carcinoma, or a combination thereof.

[0084] In one embodiment, the cancer is or comprises breast cancer. In one embodiment, the breast cancer is non-invasive. In one embodiment, the breast cancer is invasive. In one embodiment, the breast cancer is metastatic.

[0085] In one embodiment, the cancer is or comprises gastric cancer.

[0086] In one embodiment, the cancer is or comprises ovarian cancer.

[0087] In one embodiment, the cancer is or comprises gastroesophageal cancer.

[0088] In one embodiment, the cancer is or comprises lung cancer. In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC). In one embodiment, the patient has a HER2 activating mutation.

[0089] According to some embodiments, in any of the methods of the present invention described above, the method further comprises administering a lymphodepleting chemotherapy agent to the subject prior to treatment. In one embodiment, the lymphodepleting chemotherapy agent is a non-myeloablative chemotherapy agent. In one embodiment, the lymphodepleting chemotherapy agent comprises treatment with at least one of cyclophosphamide and fludarabine. In one embodiment, the lymphodepleting chemotherapy agent comprises treatment with cyclophosphamide and fludarabine. In one embodiment, the cyclophosphamide is administered at a dose of 100-500 mg / m daily. 2 In one embodiment, the cyclophosphamide is administered at a dose of 250 mg / m daily. 2 In one embodiment, the cyclophosphamide is administered at a dose of 500 mg / m daily. 2 In one embodiment, the fludarabine is administered at a dose of 10 to 50 mg / m daily. 2 In one embodiment, the fludarabine is administered at a dose of 30 mg / m daily. 2 It is administered.

[0090] According to some embodiments, in any of the methods of the invention described above, the method further comprises administering IL-2 to the subject. In one embodiment, the IL-2 is administered to the patient at a dose of 1×10 6 IU / m 2 In one embodiment, the IL-2 is administered to the patient at a dose of 1×10 7 In one embodiment, the IL-2 is administered to a patient at a dose of 6×10 7 IU is administered. In one embodiment, the administration of IL-2 is carried out within 1 to 4 hours after administration of the cells. In one embodiment, the administration of IL-2 is carried out at least 1 to 4 hours after administration of the cells.

[0091] According to some embodiments, in any of the methods of the invention described above, the method additionally comprises administering the cells multiple times. In one embodiment, the method comprises administering the cells three, four, or eight times. In one embodiment, the method comprises administering the cells every week, every two weeks, every three weeks, or every four weeks.

[0092] According to some embodiments, in any of the methods of the present invention described above, the method additionally comprises administering pertuzumab to the subject. According to some embodiments, in any of the methods of the present invention described above, the method additionally comprises administering trastuzumab to the subject. According to some embodiments, in any of the methods of the present invention described above, the method additionally comprises administering necitumumab to the subject. According to some embodiments, in any of the methods of the present invention described above, the method additionally comprises administering margetuximab to the subject. According to some embodiments, in any of the methods of the present invention described above, the method additionally comprises administering a taxane to the subject. In one embodiment, the taxane is at least one of paclitaxel, docetaxel, and cabazitaxel. According to some embodiments, in any of the methods of the invention described above, the method further comprises administering an endocrine therapeutic agent, hi one embodiment, the endocrine therapeutic agent comprises at least one of an aromatase inhibitor, fulvestrant, and tamoxifen.

[0093] According to some embodiments, in any of the methods of the present invention described above, the method further comprises administering a checkpoint inhibitor to the subject. In one embodiment, the checkpoint inhibitor inhibits CTLA-4, PD-1, or PD-L1. In one embodiment, the checkpoint inhibitor is or comprises ipilimumab. In one embodiment, the checkpoint inhibitor is or comprises nivolumab. In one embodiment, the checkpoint inhibitor is or comprises nivolumab. In one embodiment, the checkpoint inhibitor is or comprises pembrolizumab. In one embodiment, the checkpoint inhibitor is or comprises cemiplimab. In one embodiment, the checkpoint inhibitor is or comprises atezolizumab. In one embodiment, the checkpoint inhibitor is or comprises avelumab. In one embodiment, the checkpoint inhibitor is or includes durvalumab.

[0094] According to some embodiments, in any of the methods of the invention described above, the method further comprises administering to the subject a second therapeutic component. In one embodiment, the second therapeutic component comprises a lymphodepleting chemotherapy agent. In one embodiment, the second therapeutic component comprises IL-2. In one embodiment, the second therapeutic component comprises at least one of pertuzumab, trastuzumab, necituzumab, and margetuximab. In one embodiment, the second therapeutic component comprises a checkpoint inhibitor.

[0095] The present invention also provides a polynucleotide comprising a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) and a natural killer cell expressing the same.

[0096] The present invention provides a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain comprising an anti-HER2 antibody or antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15.

[0097] In one embodiment, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 1 (CDRL1) comprising SEQ ID NO: 34, a light chain complementarity determining region 2 (CDRL2) comprising SEQ ID NO: 36, a light chain complementarity determining region 3 (CDRL3) comprising SEQ ID NO: 38, a heavy chain complementarity determining region 1 (CDRH1) comprising SEQ ID NO: 44, a heavy chain complementarity determining region 2 (CDRH2) comprising SEQ ID NO: 46, and a heavy chain complementarity determining region 3 (CDRH3) comprising SEQ ID NO: 48.

[0098] In one embodiment, the nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof encodes a CDRL1 encoded by SEQ ID NO: 35; a CDRL2 encoded by SEQ ID NO: 37; a CDRL3 encoded by SEQ ID NO: 39; a CDRH1 encoded by SEQ ID NO: 45; a CDRH2 encoded by SEQ ID NO: 47; and a CDRH3 encoded by SEQ ID NO: 49.

[0099] In one embodiment, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain variable (V) comprising SEQ ID NO: 32. L ) region and a heavy chain variable (V) region comprising SEQ ID NO: 42 H ) area.

[0100] In one embodiment, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain variable (V) comprising SEQ ID NO: 33. L ) region and a heavy chain variable (V) region comprising SEQ ID NO: 42 H ) area.

[0101] In one embodiment, the anti-HER2 antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. L a region and a V comprising the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; H and the area.

[0102] In one embodiment, the anti-HER2 antibody or antigen-binding fragment is an antigen-binding fragment.

[0103] In one embodiment, the antigen-binding fragment comprises a single chain Fv (scFv).

[0104] In one embodiment, the V L The region is V H Located at the amino-terminus of the region.

[0105] In one embodiment, the V L The region is V H Located at the carboxy-terminus of the region.

[0106] In one embodiment, the V L The domains are connected to the V H Join to the region.

[0107] In one embodiment, the flexible linker comprises the amino acid sequence of SEQ ID NO:40.

[0108] In one embodiment, the flexible linker is encoded by a nucleic acid comprising SEQ ID NO:41.

[0109] In one embodiment, the scFv comprises the amino acid sequence of SEQ ID NO:30.

[0110] In one embodiment, the scFv is encoded by a nucleic acid comprising SEQ ID NO:31.

[0111] In one embodiment, the scFv comprises SEQ ID NO: 30, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0112] In one embodiment, the anti-HER2 CAR specifically binds to the human epidermal growth factor receptor 2 (HER2) protein.

[0113] In one embodiment, the HER2 protein comprises the amino acid sequence of SEQ ID NO:62.

[0114] In one embodiment, the CAR comprises a transmembrane domain, optionally a CD28 transmembrane domain.

[0115] In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:53.

[0116] In one embodiment, the CD28 transmembrane domain is encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:54 or SEQ ID NO:55.

[0117] In one embodiment, the polynucleotide additionally comprises a hinge domain between the extracellular antigen-binding domain and the transmembrane domain.

[0118] In one embodiment, the hinge domain comprises at least a portion of a CD8α hinge domain.

[0119] In one embodiment, the CD8α hinge domain comprises the amino acid sequence of SEQ ID NO:50.

[0120] In one embodiment, the CD8α hinge domain is encoded by a nucleic acid comprising SEQ ID NO:51 or SEQ ID NO:52.

[0121] In one embodiment, the CD8α hinge domain comprises SEQ ID NO: 50, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

[0122] In one embodiment, the CAR comprises an intracellular signaling region, optionally wherein the intracellular signaling region comprises a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD3-zeta (CD3ζ) signaling domain.

[0123] In one embodiment, the intracellular signaling region comprises a CD28 intracellular signaling domain and a CD3-zeta signaling domain.

[0124] In one embodiment, the intracellular signaling region comprises an OX40L intracellular signaling domain.

[0125] In one embodiment, the OX40L intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0126] In one embodiment, the OX40L intracellular signaling domain comprises SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0127] In one embodiment, the OX40L intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO:11 or SEQ ID NO:12.

[0128] In one embodiment, the intracellular signaling region comprises a CD28 intracellular signaling domain.

[0129] In one embodiment, the CD28 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:5.

[0130] In one embodiment, the CD28 intracellular signaling domain comprises SEQ ID NO: 5, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0131] In one embodiment, the CD28 intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO:6 or SEQ ID NO:7.

[0132] In one embodiment, the intracellular signaling region comprises a CD3-zeta intracellular signaling domain.

[0133] In one embodiment, the CD3-zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:13.

[0134] In one embodiment, the CD3-zeta intracellular signaling domain comprises SEQ ID NO: 13, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

[0135] In one embodiment, the CD3-zeta intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO:14 or SEQ ID NO:15.

[0136] In one embodiment, the intracellular signal region comprises the amino acid sequence of SEQ ID NO:25.

[0137] The intracellular signal region comprises SEQ ID NO: 25 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0138] In one embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:56.

[0139] In one embodiment, the CAR is encoded by a nucleic acid comprising SEQ ID NO:57.

[0140] In one embodiment, the IL-15 comprises the amino acid sequence of SEQ ID NO:22.

[0141] In one embodiment, the IL-15 is encoded by a nucleic acid comprising SEQ ID NO:23 or SEQ ID NO:24.

[0142] In one embodiment, the polynucleotide encodes a polyprotein comprising the CAR and the IL-15.

[0143] In one embodiment, the polynucleotide may additionally comprise a nucleic acid encoding a self-cleaving peptide, optionally a T2A self-cleaving peptide.

[0144] In one embodiment, the CAR binds to IL-15 via a self-cleaving peptide.

[0145] In one embodiment, the truncated peptide is capable of inducing ribosomal skipping between the CAR and the IL-15.

[0146] In one embodiment, the polynucleotide additionally comprises a nucleic acid encoding a signal sequence.

[0147] In one embodiment, the signal sequence comprises the amino acid sequence of SEQ ID NO:27.

[0148] In one embodiment, the nucleic acid encoding the signal sequence comprises the nucleic acid sequence of SEQ ID NO:28.

[0149] In one embodiment, the polynucleotide encodes a polyprotein comprising the amino acid sequence of SEQ ID NO:59.

[0150] In one embodiment, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO:60 or SEQ ID NO:61.

[0151] The present invention also provides a vector comprising the above-described polynucleotide of the present invention.

[0152] In one embodiment, the vector is a viral vector.

[0153] In one embodiment, the viral vector is a retroviral vector or a lentiviral vector.

[0154] The present invention also provides cells comprising the above-described polynucleotides and / or vectors of the present invention.

[0155] The present invention also provides cells expressing the chimeric antigen receptor and IL-15 encoded by the polynucleotide of the present invention described above and / or the vector of the present invention described above.

[0156] In one embodiment, the cell is a lymphocyte.

[0157] In one embodiment, the lymphocytes are natural killer (NK) cells.

[0158] In one embodiment, the lymphocytes are T cells.

[0159] In one embodiment, the cell is a human cell.

[0160] In one embodiment, the cells are primary cells obtained from a subject.

[0161] In one embodiment, the cells are primary cells obtained from umbilical cord blood.

[0162] In one embodiment, the cells comprise a KIR-B haplotype.

[0163] In one embodiment, the cells express CD16 with V / V polymorphism in F158.

[0164] The present invention also provides a cell population comprising a plurality of the aforementioned cells of the present invention.

[0165] In one embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%; 95%, 96%, 97%, 98%, or 99% of the cells comprise the polynucleotide and / or vector of the invention as described above.

[0166] In one embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%; 95%, 96%, 97%, 98%, or 99% of the cells express the chimeric antigen receptor and IL-15 encoded by the polynucleotides and / or vectors of the invention described above.

[0167] The present invention also provides pharmaceutical compositions comprising the cell populations of the present invention described above.

[0168] In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0169] In one embodiment, the pharmaceutical composition additionally comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) a buffer.

[0170] In one embodiment, the pharmaceutical composition contains 30 to 50 mg / mL of human albumin.

[0171] In one embodiment, the pharmaceutical composition comprises 50 mg / mL human albumin.

[0172] In one embodiment, the pharmaceutical composition contains 20-30 mg / mL of dextran.

[0173] In one embodiment, the pharmaceutical composition comprises 25 mg / mL of dextran.

[0174] In one embodiment, the dextran is dextran 40.

[0175] In one embodiment, the pharmaceutical composition contains 12 to 15 mg / mL of glucose.

[0176] In one embodiment, the pharmaceutical composition comprises 12.5 mg / mL of glucose.

[0177] In one embodiment, the pharmaceutical composition comprises less than 27.5 g / L of glucose.

[0178] In one embodiment, the pharmaceutical composition contains 50-60 ml / mL of DMSO.

[0179] In one embodiment, the pharmaceutical composition comprises 55 mg / mL of DMSO.

[0180] In one embodiment, the pharmaceutical composition comprises 40-60% v / v buffer.

[0181] In one embodiment, the buffer is phosphate buffered saline.

[0182] In one embodiment, the pharmaceutical composition comprises: (a) about 40 mg / mL human albumin; (b) about 25 mg / mL dextran 40; (c) about 12.5 mg / mL glucose; (d) about 55 mg / mL DMSO; and (e) about 0.5 mL / mL phosphate buffered saline.

[0183] In one embodiment, the pharmaceutical composition additionally comprises 0.5 mL / mL of water.

[0184] The present invention also provides a refrigerated vial containing the composition of the present invention described above.

[0185] The present invention also provides a method of treatment comprising administering the above-described cells, cell populations or compositions of the present invention to a subject with a HER2-associated disease or condition.

[0186] The present invention also provides the use of the aforementioned cells, cell populations or compositions of the present invention for the manufacture of a medicament for treating a HER2-related disease or condition.

[0187] The present invention also provides the use of the aforementioned cells, cell populations, or compositions of the present invention for treating HER2-associated diseases or conditions. In one embodiment, the HER2-associated disease or condition is cancer.

[0188] In one embodiment, the cancer is HER2 + It's cancer.

[0189] In one embodiment, the HER2 + The cancer is or contains a solid tumor that expresses HER2.

[0190] In one embodiment, the HER2 + The cancer is or includes bladder cancer, breast cancer, colon adenocarcinoma, non-small cell lung cancer, esophageal cancer, squamous cervical cancer, gastric adenocarcinoma, cholangiocarcinoma, ovarian cancer, papillary renal cell carcinoma, or a combination thereof.

[0191] In one embodiment, the HER2 + The cancer is or includes breast cancer.

[0192] In one embodiment, the HER2 + The cancer is or includes gastric cancer.

[0193] In one embodiment, the HER2 + The cancer is or includes ovarian cancer.

[0194] In one embodiment, the method or use of the present invention additionally comprises the step of administering a lymphodepleting chemotherapy agent to the subject prior to treatment.

[0195] In one embodiment, the lymphocyte-depleting chemotherapeutic agent is a non-myeloablative chemotherapeutic agent.

[0196] In one embodiment, the lymphocyte-depleting chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.

[0197] In one embodiment, the lymphocyte-depleting chemotherapy comprises treatment with cyclophosphamide and fludarabine.

[0198] In one embodiment, the cyclophosphamide is administered at a dose of 100 to 500 mg / m daily. 2 Administer.

[0199] In one embodiment, the cyclophosphamide is administered at a dose of 250 mg / m daily. 2 Administer.

[0200] In one embodiment, the cyclophosphamide is administered at a dose of 500 mg / m daily. 2 Administer.

[0201] In one embodiment, the fludarabine is administered at a dose of 10 to 50 mg / m daily.2 Administer.

[0202] In one embodiment, the fludarabine is administered at a dose of 30 mg / m daily. 2 Administer.

[0203] In one embodiment, the method or use of the present invention additionally comprises administering IL-2 to the subject.

[0204] In one embodiment, the IL-2 is administered to a subject at 1×10 6 IU / m 2 Administer.

[0205] In one embodiment, the IL-2 is administered to a subject at 1×10 7 Administer IU.

[0206] In one embodiment, the IL-2 is administered to a subject at 6×10 7 Administer IU.

[0207] In one embodiment, the administration of IL-2 is carried out within 1 to 4 hours after the administration of the cells.

[0208] In one embodiment, the administration of L-2 occurs at least 1 to 4 hours after administration of the cells.

[0209] In one embodiment, the method or use of the present invention comprises administering the cells, populations of cells and / or pharmaceutical compositions multiple times.

[0210] In one embodiment, the method or use of the present invention comprises administering the cells, cell populations and / or pharmaceutical compositions three, four or eight times.

[0211] In one embodiment, the method or use of the present invention comprises administering the cells, cell populations and / or pharmaceutical compositions every week, every two weeks, every three weeks or every four weeks.

[0212] In one embodiment, the method or use of the present invention additionally comprises the step of administering pertuzumab to the subject.

[0213] In one embodiment, the method or use of the present invention additionally comprises the step of administering trastuzumab to the subject.

[0214] In one embodiment, the method or use of the present invention additionally comprises administering necitumumab to the subject.

[0215] In one embodiment, the method or use of the present invention additionally comprises administering margetuximab to the subject.

[0216] In one embodiment, the method or use of the present invention additionally comprises administering to the subject a taxane.

[0217] In one embodiment, the taxane is at least one of paclitaxel, docetaxel, and cabazitaxel.

[0218] In one embodiment, the method or use of the present invention additionally comprises administering to the subject an endocrine therapeutic agent.

[0219] In one embodiment, the endocrine therapeutic agent comprises at least one of an aromatase inhibitor, fulvestrant, and tamoxifen.

[0220] In one embodiment, the method or use of the present invention further comprises the step of administering a checkpoint inhibitor to the subject.

[0221] In one embodiment, the checkpoint inhibitor inhibits CTLA-4, PD-1, or PD-L1.

[0222] In one embodiment, the checkpoint inhibitor is or includes ipilimumab.

[0223] In one embodiment, the checkpoint inhibitor is or includes nivolumab.

[0224] In one embodiment, the checkpoint inhibitor is or includes pembrolizumab.

[0225] In one embodiment, the checkpoint inhibitor is or comprises cemiplimab.

[0226] In one embodiment, the checkpoint inhibitor is or includes atezolizumab.

[0227] In one embodiment, the checkpoint inhibitor is or comprises avelumab.

[0228] In one embodiment, the checkpoint inhibitor is or includes durvalumab.

[0229] The present invention also provides a method of treatment comprising administering to a subject having a disease or condition associated with HER2 the cells, cell populations and / or pharmaceutical compositions of the present invention described above; and a second therapeutic component.

[0230] In one embodiment, the second therapeutic component comprises a lymphocyte-depleting chemotherapeutic agent.

[0231] In one embodiment, the second therapeutic component comprises IL-2.

[0232] In one embodiment, the second therapeutic component comprises at least one of pertuzumab, trastuzumab, necituzumab, and margetuximab.

[0233] In one embodiment, the second therapeutic component comprises a taxane.

[0234] In one embodiment, the second therapeutic component comprises a checkpoint inhibitor.

[0235] 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 invention belongs. The experimental methods and materials herein are described for use in the present invention, and other suitable methods and materials known to those skilled in the art can also be used. The experimental methods, materials, and examples are illustrative and not limiting. All publications, patent applications, patents, sequences, databases, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0236] Other features and advantages of the present invention will be apparent from the following detailed description, drawings and claims.

[0237] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was individually and expressly indicated to be incorporated by reference. [Brief explanation of the drawings]

[0238] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be best understood from the following detailed description which sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which:

[0239] [Figure 1]1 shows an exemplary embodiment of a method for expanding and stimulating NK cells.

[0240] [Figure 2] Examples of various manufacturing systems for Master Cell Bank (MCB) and Drug Product (DP) manufacturing are presented.

[0241] [Figure 3] The phenotype of the expanded and stimulated population of NK cells is shown.

[0242] [Figure 4] We show that CAR-NKs containing a costimulatory domain containing OX40L exhibit greater cytotoxic potential than CAR-NKs without OX40L.

[0243] [Figure 5] A schematic representation of the CAR structure is shown.

[0244] [Figure 6] Figure 5 shows the growth results of the CAR construct.

[0245] [Figure 7] Figure 5 shows CAR expression of the CAR construct.

[0246] [Figure 8] Figure 5 shows CD107a expression in the CAR structures. The bars represent Mock, 2nd-CAR, and 3rd-CAR from left to right, respectively.

[0247] [Figure 9] Figure 5 shows IFN-γ expression of the CAR structures. The bars represent Mock, 2nd-CAR, and 3rd-CAR from left to right, respectively.

[0248] [Figure 10] Figure 5 shows TNF-α expression in the CAR structures. The bars represent Mock, 2nd-CAR, and 3rd-CAR from left to right, respectively.

[0249] [Figure 11] Figure 5 shows the short-term cytotoxicity of the CAR constructs.

[0250] [Figure 12] Figure 1 shows a schematic diagram of CAR structures, from top to bottom: mock GFP expressing NK (mock-NK), CAR without IL-15 (CAR-NK), truncated CAR with IL-15 (CAR(t)-IL-15-NK), and CAR with IL-15 (CAR-IL-15-NK, AB-201).

[0251] [Figure 13] CAR expression in NK cells cultured in the presence of IL-2 is shown.

[0252] [Figure 14] CAR expression in NK cells cultured in the absence of IL-2 is shown.

[0253] [Figure 15] 1 shows the proliferation of NK cells cultured in the presence and absence of IL-2.

[0254] [Figure 16] 1 shows the viability of NK cells cultured in the presence and absence of IL-2.

[0255] [Figure 17] Demonstrates NK cell cytotoxicity.

[0256] [Figure 18] IFNg production by NK cells.

[0257] [Figure 19] IL-15 production by NK cells.

[0258] [Figure 20] FIG. 1 shows that IL-15 secretion maintains bystander NK cell survival.

[0259] [Figure 21] FIG. 1 shows a schematic diagram of two different CAR structures.

[0260] [Figure 22] Figure 21 shows CAR expression over time (days) of the CAR constructs.

[0261] [Figure 23] Figure 22 shows the viability (cell number) of cells expressing the CAR constructs.

[0262] [Figure 24] The percentage viability of cells expressing the CAR constructs of Figure 21 is shown.

[0263] [Figure 25] The number of surviving NK cells after 7 days of co-culture of cells expressing the CAR constructs shown in Figure 21 with target cells in the absence of IL-2 is shown. The bars represent, from left to right, cord blood NK cells (CBNK), 2nd-CAR, and 3rd-CAR, respectively.

[0264] [Figure 26] 1 shows the in vitro killing activity of AB-201 against the breast cancer (HER2-) cell line MDA-MB-468.

[0265] [Figure 27] 1 shows the in vitro killing activity of AB-201 against the ovarian carcinoma (HER2+) cell line SKOV3.

[0266] [Figure 28] 1 shows the in vitro killing activity of AB-201 against the gastric cancer (HER2+) cell line NCI-N87.

[0267] [Figure 29] 1 shows the in vitro killing activity of AB-201 against the breast cancer (HER2+) cell line HCC1954.

[0268] [Figure 30] 1 shows the in vitro killing activity of AB-201 against the breast cancer (HER2+) cell line K562.

[0269] [Figure 31] 1 shows the results of the in vitro characterization of AB-201.

[0270] [Figure 32] 1 shows the results of the in vitro characterization of AB-201.

[0271] [Figure 33] 1 shows the results of the in vitro characterization of AB-201.

[0272] [Figure 34] 1 shows the results of the in vitro characterization of AB-201.

[0273] [Figure 35] 1 shows the results of the in vitro characterization of AB-201.

[0274] [Figure 36] 1 shows the results of the in vitro characterization of AB-201.

[0275] [Figure 37] Long-term killing analysis of NCI-N87 gastric carcinoma cells in culture (effector to target cell ratio E:T 0.3:1) using Incucyte live cell imaging.

[0276] [Figure 38] FIG. 1 shows that AB-201 provided a significant survival advantage over trastuzumab at a single dose of 1 million cells in a HER2-positive breast cancer model.

[0277] [Figure 39] This shows that a single administration of AB-201 on day 4 after establishing a HER2+ trastuzumab-resistant breast cancer model resulted in tumor regression.

[0278] [Figure 40] 1 shows the results of the in vivo characterization of AB-201.

[0279] [Figure 41] 1 shows the results of the in vivo characterization of AB-201.

[0280] [Figure 42] 1 shows the results of the in vivo characterization of AB-201.

[0281] [Figure 43] 1 shows the results of the in vivo characterization of AB-201.

[0282] [Figure 44] Cytotoxicity of primary cells (non-tumor) measured after 4 hours of co-culture of pulmonary artery endothelial cells, keratinocytes, renal epithelial cells, cardiac muscle cells, and small airway epithelial cells with AB-201 or control CB-NK cells at E:T ratios of 3:1, 1:1, or 0.3:1.

[0283] [Figure 45] Figure 1 shows tumor volume measurements in NSG mice untreated (open circles, dashed line) or treated with AB-201 (filled circles, solid line) after implantation of SKOV3-Luc tumor cells (IP). The vertical line at day 11 indicates the date of AB-201 injection.

[0284] [Figure 46] The results show that the body weight of NSG mice was measured after transplantation of SKOV3-Luc tumor cells (IP) without treatment (open circles, dashed line) or treatment with AB-201 (filled circles, solid line).

[0285] [Figure 47] 1 shows that AB-201 cells were maintained at detectable levels for at least 52 days in AB-201 treated mice.

[0286] [Figure 48] Tumor volumes in irradiated mice inoculated with NCI-N87 cells and given untreated, cord blood NK cells, or AB-201 cells are shown.

[0287] [Figure 49] The tumor volume in unconditioned mice inoculated with NCI-N87 cells and administered no treatment, cord blood NK cells, or AB-201 cells is shown.

[0288] [Figure 50] Body weight measurements for the mice in Figures 48 and 49 are shown.

[0289] [Figure 51] Tumor penetration of AB-201 is demonstrated by detecting CD56 by immunofluorescence.

[0290] [Figure 52] FIG. 1 is a schematic diagram showing the experimental course design in which NSG mice were administered 1 × 10 SK-OV-3-Luc tumor cells intraperitoneally (IP) on day 0, randomized 4 days later, and then received a single IP injection of CB-NK (5 × 10 ) or AB-201 (1 × 10 ) on day 5 or days 5 and 12.

[0291] [Figure 53] 1 shows the efficacy of AB-201 in the SK-OV-3-Luc xenograft tumor model.

[0292] [Figure 54] This shows the weight change of SK-OV-3-Luc transplanted mice. The weight change was calculated based on the body weight (BW) of the mouse on the day of NK cell injection.

[0293] [Figure 55] Figure 1 shows the presence of AB-201 in peripheral lymphoid tissues. AB-201 and CB-NK were measured by flow cytometry in peripheral blood and spleen at the indicated time points after tumor inoculation (gating on human CD45+CD56+).

[0294] [Figure 56] Expression of cell surface markers of CBNK or AB-201 after thawing is shown.

[0295] [Figure 57] Specific cytotoxicity of AB-201 and control CB-NK cells against K562, SK-OV-3, HCC1954, and NCI-N87 at various E:T ratios is shown.

[0296] [Figure 58] Cytotoxicity kinetics of AB-201 and CBNK against SK-OV-3, HCC1954, and NCI-N87. Long-term cytotoxicity of AB-201 and control CB-NK cells against various HER2-positive target tumor cell lines was measured over 120 hours using the Incucyte LiveCell Analysis System.

[0297] [Figure 59] Degranulation and cytokine secretion of AB-201 and CBNK cells after stimulation with K562, SK-OV-3, HCC1954, and NCI-N87 are shown. AB-201 or control CB-NK cells were stimulated with various target tumor cells at a 1:1 E:T ratio for 24 hours. Cells were then harvested and analyzed for degranulation (CD107a) and cytokine secretion (IFNγ, TNFα) by flow cytometry. Data are presented as the percentage of positive cells within the CD56+ gated population (n=3). (*p<0.05, **p<0.01 compared to CBNK, two-tailed t-test).

[0298] [Figure 60] IFN-γ secretion by AB-201 after stimulation with K562, SK-OV-3, HCC1954, and NCI-N87. AB-201 or control CB-NK cells were stimulated with tumor cells at a 3:1 ET ratio for 24 hours, cell-free supernatants were collected, and soluble cytokine levels were measured by ELISA for IFNγ (*p<0.05, **p<0.01 compared to CBNK, two-tailed t-test).

[0299] [Figure 61] IL-15 secretion by AB-201 after stimulation with K562, SK-OV-3, HCC1954, and NCI-N87. AB-201 or control CB-NK cells were stimulated with tumor cells at a 3:1 E:T ratio for 24 hours, and soluble cytokine levels were measured by ELISA for IL-15 after collection of cell-free supernatants (*p<0.05 compared to CBNK, two-tailed t-test). Mode for explaining the invention

[0300] The present invention relates, inter alia, to natural killer (NK) cells (e.g., CAR-NK cells), methods for producing NK cells, pharmaceutical compositions containing NK cells, and methods for treating patients with diseases such as cancer with NK cells.

[0301] Expansion and stimulation of natural killer cells In one embodiment, natural killer cells can be expanded and activated, for example, by culturing them with and stimulating them with support cells.

[0302] NK cells can be expanded and stimulated, for example, by methods described in US2020 / 0108096 or WO2020 / 101361, both of which are incorporated by reference in their entirety. Briefly, source cells can be cultured on modified HuT-78 (ATCC® TIB-161®) cells engineered to express 4-1BBL, membrane-bound IL-21, and mutant TNFα, as described in US2020 / 0108096.

[0303] Suitable NK cells can also be expanded and stimulated as described herein.

[0304] In one embodiment, NK cells are expanded and stimulated by a method comprising: (a) providing a composition comprising NK cells, e.g., CD3(-) cells; and (b) culturing in a medium comprising support cells and / or stimulatory factors to generate a population of expanded and stimulated NK cells.

[0305] Natural killer cell source In one embodiment, the NK cell source is selected from the group consisting of peripheral blood, peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PBMCs), bone marrow, umbilical cord blood, isolated NK cells, induced pluripotent stem cell-derived NK cells, embryonic stem cell-derived NK cells, and combinations thereof.

[0306] In one embodiment, the source of NK cells is a single unit of umbilical cord blood.

[0307] In one embodiment, the NK cell source (e.g., umbilical cord blood units) is about 1 x 10 7 ~Approx. 1×10 9 In one embodiment, the NK cell source (e.g., a cord blood unit) comprises about 1 x 10 total nucleated cells. 8 ~Approx. 1.5×10 8 In one embodiment, the NK cell source (e.g., umbilical cord blood unit) comprises 1 x 10 total nucleated cells. 8 In one embodiment, the NK cell source (e.g., a cord blood unit) comprises about 1 x 10 total nucleated cells. 8 In one embodiment, the NK cell source (e.g., umbilical cord blood unit) comprises 1 x 10 total nucleated cells. 9 In one embodiment, the NK cell source (e.g., a cord blood unit) comprises about 1 x 10 total nucleated cells. 9 Contains total nucleated cells.

[0308] In one embodiment, the NK cell source (eg, a cord blood unit) comprises about 20% to about 80% CD16+ cells. In one embodiment, the NK cell source (e.g., a cord blood unit) comprises about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 80%, about 60% to about 70%, or about 70% to about 80% CD16+ cells. In one embodiment, the NK cell source (e.g., umbilical cord blood units) contains 80% or less CD16+ cells. Alternatively, some NK cell sources may contain concentrations of CD16+ cells greater than 80%.

[0309] In one embodiment, the NK cell source (eg, a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% MLG2A+ cells.

[0310] In one embodiment, the NK cell source comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% NKG2C+ cells.

[0311] In one embodiment, the NK cell source (eg, a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% NKG2D+ cells.

[0312] In one embodiment, the NK cell source (eg, a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% NKp46+ cells.

[0313] In one embodiment, the NK cell source (e.g., a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% NKp30+ cells.

[0314] In one embodiment, the NK cell source (eg, a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% DNAM-1+ cells.

[0315] In one embodiment, the NK cell source (eg, a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% NKp44+ cells.

[0316] In one embodiment, the NK cell source comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% CD25+ cells.

[0317] In one embodiment, the NK cell source (e.g., a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% CD62L+ cells.

[0318] In one embodiment, the NK cell source (e.g., a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% CD69+ cells.

[0319] In one embodiment, the NK cell source (eg, a cord blood unit) comprises no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% CXCR3+ cells.

[0320] In one embodiment, the NK cell source (e.g., a cord blood unit) is composed of no more than 40%, such as no more than 30%, such as no more than 20%, such as no more than 10%, such as no more than 5% CD57+ cells.

[0321] In one embodiment, the NK cells of the NK cell source comprise a KIR B allele of the KIR receptor system, see, e.g., Hsu et al., "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism," Immunological Review 190:40-52 (2002); and Pyo et al., "Different Patterns of Evolution in the Centromeric and Telomeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-like Receptor Locus," PLoS One 5:e15115 (2010).

[0322] In one embodiment, the NK cells of the NK cell source contain the 158V / V variant of CD16 (homozygous CD16 158V polymorphism). See, e.g., Koene et al., "FcγRIIIa-158V / F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L / R / H Phenotype," Blood 90:1109-14 (1997).

[0323] In one embodiment, the NK cell source comprises all KIR B alleles of the KIR receptor system and the 158V / V variant of CD16.

[0324] In one embodiment, the NK cells of the cell source are not genetically engineered.

[0325] In one embodiment, the NK cells of the cell source do not contain the CD16 transgene.

[0326] In one embodiment, the NK cell source does not express exogenous CD16 protein.

[0327] In one embodiment, the NK cell source is depleted of CD3(+). In one embodiment, the method of the present invention includes depleting an NK cell source of CD3(+) cells. In one embodiment, depleting an NK cell source of CD3(+) cells includes contacting the NK cell source with a CD3-binding antibody or antigen-binding fragment thereof. In one embodiment, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1, and HIT3a and fragments thereof. In one embodiment, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or its antigen-binding fragment. In one embodiment, the antibody or antigen-binding fragment thereof is attached to beads, e.g., magnetic beads. In one embodiment, depleting a composition of CD3(+) cells includes contacting the composition with a CD3-targeting antibody or antigen-binding fragment thereof attached to beads and removing bead-bound CD3(+) cells from the composition. The composition can be depleted of CD3 cells by immunomagnetic selection, for example using CliniMACS T cell depletion set (LS Depletion set (162-01) Miltenyi Biotec).

[0328] In one embodiment, the NK cell source is enriched for CD56+ NK cell source, for example, by gating on CD56 expression.

[0329] In one embodiment, the NK cell source is enriched for CD56+ and depleted for CD3(+), for example, by selecting cells with CD56+CD3- expression.

[0330] In one embodiment, the NK cell source contains all KIR B alleles of the KIR receptor system and the 158V / V variant of CD16, and is + enriched and CD3(+) depleted, for example, by selecting for CD56+CD3- expressing cells.

[0331] supporting cells Support cells for NK cell expansion are described below. These support cells allow NK cells to be expanded to numbers suitable for preparation of pharmaceutical compositions of the present invention. In some cases, the support cells allow NK cells to be expanded without the loss of CD16 expression that occurs when cells are expanded using other types of support cells or other methods. In some cases, the support cells allow expanded NK cells to better tolerate freezing, allowing a greater proportion of NK cells to survive after a freeze / thaw cycle or allowing cells to survive for longer periods in a frozen state. In some cases, the support cells allow NK cells to maintain high levels of cytotoxicity, including ADCC, prolong survival, enhance persistence, and improve or maintain high levels of CD16. In some cases, the support cells allow NK cells to be expanded without significant levels of exhaustion or senescence.

[0332] Feeder cells can be used to provide substrate, growth factors and / or cytokines to stimulate NK cells and help them expand more rapidly.

[0333] NK cells can be stimulated using various types of supportive cells, including, but not limited to, peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblast cells (e.g., EBV-LCL), myeloid leukemia cells (e.g., K562), CD4(+) T cells (e.g., HuT) and their derivatives.

[0334] In one embodiment, the feeder cells are inactivated, for example, by gamma-irradiation or mitomycin-c treatment.

[0335] Support cells suitable for use in the present invention are described, for example, in US2020 / 0108096, which is incorporated herein by reference in its entirety.

[0336] In one embodiment, the support cells are inactivated CD4(+) T cells. In one embodiment, the inactivated CD4(+) T cells are HuT-78 cells (ATCC® TIB-161™) or a mutant or derivative thereof. In one embodiment, the HuT-78 derivative is H9 (ATCC® HTB-176®).

[0337] In one embodiment, the inactivated CD4(+) T cells express OX40L. In one embodiment, the inactivated CD4(+) T cells are HuT-78 cells or mutants or derivatives thereof that express OX40L (SEQ ID NO: 4).

[0338] In one embodiment, the support cells are HuT-78 cells (“eHut-78 cells”) or variants thereof engineered to express at least one gene selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and mutant TNFα (SEQ ID NO: 3).

[0339] In one embodiment, the inactivated CD4(+) T cells are HuT-78 (ATCC® TIB-161®) cells or variants or derivatives thereof that express an OX40L homolog or variant thereof. In one embodiment, the support cells are HuT-78 cells engineered to express at least one gene selected from the group consisting of a 4-1BBL homolog or variant thereof, a membrane-bound IL-21 homolog or variant thereof, and a mutant TNFα homolog or variant thereof.

[0340] In one embodiment, the support cells are HuT-78 cells ("eHut-78 cells") or variants or derivatives thereof that express OX40L (SEQ ID NO: 4) and have been engineered to express 4-1BBL (SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and mutated TNFα (SEQ ID NO: 3).

[0341] In one embodiment, the feeder cells can be expanded from cryopreservation prior to culturing with NK cells, for example, as described in Example 2.

[0342] stimulating factor NK cells can also be stimulated with one or more stimulatory factors, such as signaling factors, in addition to or in place of support cells.

[0343] In one embodiment, the stimulatory factor (e.g., signaling factor) is a component of a culture medium, as described herein. In one embodiment, the stimulatory factor (e.g., signaling factor) is a supplement to a culture medium, as described herein.

[0344] In one embodiment, the stimulatory factor is a cytokine, hi one embodiment, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, and combinations thereof.

[0345] In one embodiment, the cytokine is IL-2.

[0346] In one embodiment, the cytokine is a combination of IL-2 and IL-15.

[0347] In one embodiment, the cytokines are a combination of IL-2, IL-15 and IL-18.

[0348] In one embodiment, the cytokines are a combination of IL-2, IL-18 and IL-21.

[0349] culture NK cells can be expanded and stimulated by culturing the NK cell source with supporting cells and / or other stimulatory factors. Suitable NK cell sources, supporting cells, and stimulatory factors are described herein.

[0350] In some cases, the resulting population of expanded natural killer cells is enriched and / or differentiated after expansion. In some cases, the resulting population of expanded natural killer cells may not be enriched and / or differentiated after expansion.

[0351] The present invention also describes compositions comprising various culture compositions, e.g., compositions comprising NK cells, such as a population of expanded cord blood-derived natural killer cells comprising homozygous for the KIR-B haplotype and CD16 158V polymorphism and a plurality of engineered HuT78 cells.

[0352] Further described herein are containers, e.g., vials, freezer bags, etc., that contain the resulting population of expanded natural killer cells. In some cases, the plurality of containers can include a plurality of containers that contain a portion of the expanded natural killer cell population, e.g., at least 10, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 containers.

[0353] Also described herein are bioreactors containing various culture compositions described herein, e.g., bioreactors containing NK cells, e.g., culture media containing natural killer cells from a natural killer cell source, e.g., as described herein, and support cells, e.g., as described herein. Also described herein are bioreactors containing the resulting population of expanded natural killer cells.

[0354] Culture medium Culture media for the expansion of NK cells are described. Such culture media allow for the expansion of NK cells to numbers suitable for the preparation of pharmaceutical compositions as disclosed herein. In some cases, the culture media allow for the expansion of NK cells without the loss of CD16 expression that accompanies expansion of cells with other helper cells or other media.

[0355] In one embodiment, the culture medium is a basal culture medium, optionally supplemented with additional components as described herein.

[0356] In one embodiment, the culture medium, e.g., the basal culture medium, is a serum-free culture medium. In one embodiment, the culture medium, e.g., the basal culture medium, is a serum-free culture medium supplemented with human plasma and / or serum.

[0357] Suitable basal culture media include DMEM, RPMI 1640, MEM, DMEM / F12, SCGM (CellGenix®, 20802-0500 or 20806-0500), LGM-3® (Lonza, CC-3211), TexMACS® (Miltenyi Biotec, 130-097-196), AlyS® 505NK-AC (Cell Science and Technology Institute, Inc., 01600P02), AlyS® 505NK-EX (Cell Science and Technology Institute, Inc., 01400P10), CTS® AIM-V® SFM (ThermoFisher Scientific, A3830801), CTS® OpTmizer® (ThermoFisher Scientific, A3830801), and the like. Scientific, A1048501, ABS-001, StemXxVivo), and combinations thereof.

[0358] The culture medium can contain or be supplemented with additional components such as growth factors, signal factors, nutrients, antigen binders, etc. The culture medium can be supplemented by adding each additional component to the culture vessel before, simultaneously with, or after the medium is added to the culture vessel. The additional components can be added together or individually. If added individually, the additional components do not need to be added at the same time.

[0359] In one embodiment, the culture medium comprises plasma, e.g., human plasma. In one embodiment, the culture medium is supplemented with plasma, e.g., human plasma. In one embodiment, the plasma, e.g., human plasma, comprises an anticoagulant, e.g., trisodium citrate.

[0360] In one embodiment, the medium comprises from about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 0.5% to about 1%, from about 1% to about 10%, from about 1% to about 9%, from about 1% to about 8%, from about 1% to about 9%. from about 1% to about 7%, from about 1% to about 6%, from about 1% to about 5%, from about 1% to about 4%, from about 1% to about 3%, from about 1% to about 2%, from about 2% to about 10%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%, from about 2% to about 3%, from about 3% to about 10% from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, from about 3% to about 4%, from about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, from about 4% to about 5%, from about 5% to about 10%, from about 5% to about 9%, The culture medium may comprise about 8% or up to about 5%, about 5% or up to about 6%, about 6% or up to about 10%, about 6% or up to about 9%, about 6% or up to about 8%, about 6% or up to about 7%, about 7% or up to about 10%, about 7% or up to about 9%, about 7% or up to about 8%, about 8% or up to about 10%, about 8% or up to about 9%, or about 9% or up to about 10% v / v plasma (e.g., human plasma). In one embodiment, the culture medium may comprise or be supplemented with 0.8% to 1.2% v / v human plasma. In one embodiment, the culture medium may comprise or be supplemented with 1.0% v / v human plasma. In one embodiment, the culture medium may comprise or be supplemented with about 1.0% v / v human plasma.

[0361] In one embodiment, the culture medium comprises serum, e.g., human serum. In one embodiment, the culture medium is supplemented with serum, e.g., human serum. In one embodiment, the serum is inactivated (e.g., heat inactivated). In one embodiment, the serum is filtered (e.g., sterile filtered).

[0362] In one embodiment, the culture medium includes glutamine. In one embodiment, the culture medium is supplemented with glutamine. In one embodiment, the culture medium may include or be supplemented with about 2.0 mM or up to about 6.0 mM glutamine. In one embodiment, the culture medium may include about 2.0 mM or up to about 5.5 mM, about 2.0 mM or up to about 5.0 mM, about 2.0 mM or up to about 4.5 mM, about 2.0 mM or up to about 4.0 mM, about 2.0 mM or up to about 3.5 mM, about 2.0 mM or up to about 3.0 mM, about 2.0 mM or up to about 2.5 mM, about 2.5 mM or up to about 6.0 ...6.0 mM, about 2.5 mM or up to about 6.0 mM, about 2.0 mM or up to about 5.5 mM, about 2.0 mM or up to about 5.0 mM, about 2.0 mM or up to about 4.5 mM, about 2.0 mM or up to about 4.0 mM, about 2.0 mM or up to about 3.5 mM, about 2.0 mM or up to about 3.0 mM, about 2.0 mM or up to about 2.5 mM, about 2.5 mM or up to about 6. or up to about 5.5 mM, from about 2.5 mM to about 5.0 mM, from about 2.5 mM to about 4.5 mM, from about 2.5 mM to about 4.0 mM, from about 2.5 mM to about 3.5 mM, from about 2.5 mM to about 3.0 mM, from about 3.0 mM to about 6.0 mM, from about 3.0 mM to about 5.5 mM, from about 3.0 mM to about 5.0 mM, from about 3.0 mM to about 4 0.5 mM, from about 3.0 mM or to about 4.0 mM, from about 3.0 mM or to about 3.5 mM, from about 3.5 mM or to about 6.0 mM, from about 3.5 mM or to about 5.5 mM, from about 3.5 mM or to about 5.0 mM, from about 3.5 mM or to about 4.5 mM, from about 3.5 mM or to about 4.0 mM, from about 4.0 mM or to about 6.0 mM, from about 4.0 mM or to about 5.5 mM The culture medium may contain or be supplemented with glutamine from about 4.0 mM to about 5.0 mM, from about 4.0 mM to about 4.5 mM, from about 4.5 mM to about 6.0 mM, from about 4.5 mM to about 5.5 mM, from about 4.5 mM to about 5.0 mM, from about 5.0 mM to about 6.0 mM, from about 5.0 mM to about 5.5 mM, or from about 5.5 mM to about 6.0 mM. In one embodiment, the culture medium may contain or be supplemented with 3.2 mM to 4.8 mM glutamine. In one embodiment, the culture medium may contain or be supplemented with 4.0 mM glutamine. In one embodiment, the culture medium may contain or be supplemented with about 4.0 mM glutamine.

[0363] In one embodiment, the culture medium comprises one or more cytokines. In one embodiment, the culture medium is supplemented with one or more cytokines.

[0364] In one embodiment, the cytokine is selected from among IL-2, IL-12, IL-15, IL-18, and combinations thereof.

[0365] In one embodiment, the culture medium contains and / or is supplemented with IL-2, hi one embodiment, the culture medium contains and / or is supplemented with about 150 to about 2,500 IU / mL of IL-2.In one embodiment, the culture medium may be from about 200 to about 2,250, from about 200 to about 2,000, from about 200 to about 1,750, from about 200 to about 1,500, from about 200 to about 1,250, from about 200 to about 1,250, from about 200 to about 1,000, from about 200 to about 750, from about 200 to about 500, from about 200 to about 250, from about 250 to about 2,500, from about 250 to about 2,250, from about 250 to about 2,000, from about 250 to about 1, about 250 to about 1,750, from about 250 to about 1,500, from about 250 to about 1,250, from about 250 to about 1,000, or from about 250 to about 1,500. 0 to about 750, about 250 to about 500, about 500 to about 2,500, about 500 to about 2,500, about 500 to about 2, about 250 to about 500, about 250 to about 1,500, about 250 to about 1,500, about 250 to about 2,250, about 500 to about 2,000, about 50 0 to about 1,750, about 500 to about 1,500, about 500 to about 1,250, about 500 to about 1,250, about 500 to about 1,000, about 500 to about 750, about 750 to about 2, about 500 to about 2,000, about 500 to about 1,750, about 500 to about 1,500, about 500 ~ about 1,500, about 500 to about 1,250, about 500 to about 1,250, about 500 to about 1,250, about 500 to about 1,000, about 500 to about 1,000, about 500 to about 1,750, 250, about 750 to about 2,000, about 750 to about 1,750, about 750 to about 1,500, about 750 to about 1,250, about 750 to about 1,000, about 1,000 to about 2,500, about 2,000 to about 2,500, about 1,000 to about 2,250, about 1,000 to about 2,000, about 1,000 to about 1,750, about 1,000 to about 1,500, about 1,000 to about 1,250, about 1, It may contain and / or be supplemented with 250 to about 2,500, about 1,250 to about 2,250, about 1,250 to about 2,000, about 1,250 to about 1,750, about 1,250 to about 1,500, about 1,500 to about 2,500, about 1,500 to about 2,250, about 1,500 to about 2,000, about 2,000 to about 1,500 to about 1,750, about 1,750 to about 2,500, about 1,750 to about 2,250, about 1,750 to about 2,000, about 2,000 to about 2,500, about 2,000 to about 2,250, or about 2,250 to about 2,500 IU / mL of IL-2.

[0366] In one embodiment, the culture medium contains and / or is supplemented with 64 μg / L to 96 μg / L of IL-2. In one embodiment, the culture medium contains and / or is supplemented with 80 μg / L of IL-2 (approximately 1,333 IU / mL). In one embodiment, the culture medium contains and / or is supplemented with about 80 μg / L of IL-2.

[0367] In one embodiment, the culture medium contains and / or is supplemented with a combination of IL-2 and IL-15.

[0368] In one embodiment, the culture medium contains and / or is supplemented with a combination of IL-2, IL-15 and IL-18.

[0369] In one embodiment, the culture medium contains and / or is supplemented with a combination of IL-2, IL-18 and IL-21.

[0370] In one embodiment, the culture medium contains and / or is supplemented with glucose. In one embodiment, the culture medium contains and / or is supplemented with about 0.5 to about 3.5 g / L of glucose. In one embodiment, the culture medium contains and / or is supplemented with about 0.5 to about 3.0, about 0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.0, about 1.0 to about 3.0, about 1.0 to about 3.0, about 2.5, about 1.0 to about 2.0, about 1.0 to about 1.5, about 1.5 to about 3.0, about 1.5 to about 2.5, about 1.5 to about 2.5, about 1.5 to about 2.0, about 2.0 to about 3.0, about 2.0 to about 2.5, or about 2.5 to about 3.0 g / L of glucose. In one embodiment, the culture medium contains and / or is supplemented with 1.6-2.4 g / L of glucose. In one embodiment, the culture medium contains and / or is supplemented with 2.0 g / L of glucose. In one embodiment, the culture medium comprises about 2.0 g / L of glucose.

[0371] In one embodiment, the culture medium contains and / or is supplemented with sodium pyruvate. In one embodiment, the culture medium contains and / or is supplemented with about 0.1 to about 2.0 mM sodium pyruvate. In one embodiment, the culture medium contains and / or is supplemented with about 0.1 to about 1.8, about 0.1 to about 1.6, about 0.1 to about 1.4, about 0.1 to about 1.2, about 0.1 to about 1.0, about 0.1 to about 1.0, or about 0.1 to about 0.8, about 0.1 to about 0.6, about 0.1 to about 0.4, about 0.1 to about 0.2, about 0.2 to about 2.0, about 0.2 to about 1.8, about 0.2 to about 1.6, about 0 to about 1.6, about 0.2 to about 1.4, about 0.2 to about 1.2, about 0.2 to about 1.0, or about 0.2 to about 0. 8, about 0.2 to about 0.6, about 0.2 to about 0.4, about 0.4 to about 2.0, about 0 to about 0.4, about 0.4 to about 1.8, about 0.4 to about 1.6, about 0.4 to about 1.4, about 0.4 to about 1.2, about 0.4 to about 1.0, about 0.4 to about 0.8, about 0.4 to about 0.6, about 0.6 to about 2.0, about 0.6 to about 1.8, about 0.6 to about 1.6, about 0.6 to about 1.4, about 0.6 to about 1.2, about 0.6 to about 1.0, about 0.6 to about 0.8, about 0.8 to about 2. Containing and / or supplemented with 0, about 0.8 to about 1.8, about 0.8 to about 1.6, about 0.8 to about 1.4, about 0.8 to about 1.4, about 0.8 to about 1.2, about 0.8 to about 1.0, about 1.0 to about 2.0, about 1.0 to about 1.8, about 1.0 to about 1.6, about 1.0 to about 1.4, about 1.0 to about 1.2, about 1.2 to about 2.0, about 1.2 to about 1.8, about 1.2 to about 1.8, about 1.2 to about 1.6, about 1.2 to about 1.4, about 1.4 to about 2.0, about 1.4 to about 1.8, about 1.4 to about 1.6, about 1.6 to about 2.0, about 1.6 to about 1.8, or about 1.8 to about 2.0 mM sodium pyruvate. In one embodiment, the culture medium comprises 0.8 to 1.2 mM sodium pyruvate. In one embodiment, the culture medium comprises 1.0 mM sodium pyruvate. In one embodiment, the culture medium comprises about 1.0 mM sodium pyruvate.

[0372] In one embodiment, the culture medium contains and / or is supplemented with sodium bicarbonate. In one embodiment, the culture medium contains and / or is supplemented with about 0.5 to about 3.5 g / L of sodium bicarbonate. In one embodiment, the culture medium contains and / or is supplemented with about 0.5 to about 3.0, about 0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.0, about 1.0 to about 3.0, about 1.0 to about 2.5, about 1.0 to about 2.0, about 1.0 to about 1.5, about 1.5 to about 3.0, about 1.5 to about 2.5, about 1.5 to about 2.5, about 1.5 to about 2.0, about 2.0 to about 3.0, about 2.0 to about 2.5, or about 2.5 to about 3.0 g / L of sodium bicarbonate. In one embodiment, the culture medium contains and / or is supplemented with 1.6-2.4 g / L sodium bicarbonate. In one embodiment, the culture medium contains and / or is supplemented with 2.0 g / L sodium bicarbonate. In one embodiment, the culture medium comprises about 2.0 g / L sodium bicarbonate.

[0373] In one embodiment, the culture medium contains and / or is supplemented with albumin, e.g., human albumin, e.g., a human albumin solution described herein. In one embodiment, the culture medium contains and / or is supplemented with about 0.5% to about 3.5% of a 20% albumin solution (e.g., a 20% human albumin solution). In one embodiment, the culture medium contains and / or is supplemented with about 0.5% to about 3.0%, about 0.5% to about 2.5%, about 0.5% to about 2.0%, about 0.5% to about 1.5%, about 0.5% to about 1.0%, about 1.0% to about 3.0%, about 1.0% to about 3.0%, about 1.0% to about 2.5%, about 1.0% to about 2.0%, about 1.0% to about 1.5%, about 1.5% to about 3.0%, about 1.5% to about 2.5%, about 1 to about 1.5% to about 2.0%, about 2.0% to about 3.0%, about 2.0% to about 2.5%, or about 2.5% to about 3.0% v / v of a 20% albumin solution (e.g., a 20% human albumin solution). In one embodiment, the culture medium contains and / or is supplemented with a 20% albumin solution, e.g., 1.6% to 2.4% v / v of a 20% human albumin solution. In one embodiment, the culture medium contains and / or is supplemented with 2.0% v / v of a 20% albumin solution (e.g., a 20% human albumin solution). In one embodiment, the culture medium comprises about 2.0% v / v of a 20% albumin solution (e.g., a 20% human albumin solution).

[0374] In one embodiment, the culture medium contains and / or is supplemented with about 2 to about 6 g / L of albumin (e.g., human albumin). In one embodiment, the culture medium contains and / or is supplemented with about 2 to about 5.5, about 2 to about 5.0, about 2 to about 4.5, about 2 to about 4, about 2 to about 4, about 2 to about 3.5, about 2 to about 3, about 2 to about 3, about 2 to about 2.5, about 2 to about 6, about 2.5 to about 5.5, about 2.5 to about 5.0, about 2.5 to about 5.0, about 2.5 to about 4.5, about 2.5 to about 4.0, about 2.5 to about 3.5, about 2.5 to about 3.0, about 3 to about 6, about 3 to about 5.5, about 3 to about 5, about 3 to about 4.5, about The culture medium may contain and / or be supplemented with 3 to about 4, about 3 to about 4, about 3 to about 3.5, about 3.5 to about 6, about 3.5 to about 5.5, about 3.5 to about 5, about 3.5 to about 5, about 3.5 to about 4.5, about 3.5 to about 4.5, about 3.5 to about 4, about 4 to about 6, about 4 to about 5.5, about 4 to about 5, about 4 to about 5, about 4 to about 4.5, about 4.5 to about 6, about 4 to about 4.5, about 4 to about 4.5 to about 5.5, about 4.5 to about 5, about 5 to about 6, about 5 to about 5.5, or about 5.5 to about 6 g / L of albumin (e.g., human albumin). In one embodiment, the culture medium contains and / or is supplemented with 3.2 to 4.8 g / L of albumin (e.g., human albumin). In one embodiment, the culture medium comprises 4 g / L of albumin (e.g., human albumin). In one embodiment, the culture medium comprises about 4 g / L of albumin (e.g., human albumin).

[0375] In one embodiment, the culture medium is supplemented with poloxamer 188. In one embodiment, the culture medium contains and / or is supplemented with about 0.1 to about 2.0 g / L of poloxamer 188. In one embodiment, the culture medium contains and / or is supplemented with about 0.1 to about 1.8, about 0.1 to about 1.6, about 0.1 to about 1.4, about 0.1 to about 1.2, about 0.1 to about 1.0, about 0.1 to about 1.0, about 0.1 to about 0.8, about 0.1 to about 0.6, about 0.1 to about 0.4, about 0.1 to about 0.2, about 0.2 to about 2.0, about 0.2 to about 1.8, about 0.2 to about 1.6, about 0 to about 1.6, about 0.2 to about 1.4, about 0.2 to about 1.2, about 0.2 to about 1.0, about 0.2 to about 0.8 , about 0.2 to about 0.6, about 0.2 to about 0.4, about 0.4 to about 2.0, about 0 to about 0.4, about 0.4 to about 1.8, about 0.4 to about 1.6, about 0.4 to about 1.4, about 0.4 to about 1.2, about 0.4 to about 1.0, about 0.4 to about 0.8, about 0.4 to about 0.6, about 0.6 to about 2.0, about 0.6 to about 1.8, about 0.6 to about 1.6, about 0.6 to about 1.4, about 0.6 to about 1.2, about 0.6 to about 1.0, about 0.6 to about 0.8, about 0.8 to about 2. Containing and / or supplementing 0, about 0.8 to about 1.8, about 0.8 to about 1.6, about 0.8 to about 1.4, about 0.8 to about 1.4, about 0.8 to about 1.2, about 0.8 to about 1.0, about 1.0 to about 2.0, about 1.0 to about 1.8, about 1.0 to about 1.6, about 1.0 to about 1.4, about 1.0 to about 1.2, about 1.2 to about 2.0, about 1.2 to about 1.8, about 1.2 to about 1.8, about 1.2 to about 1.6, about 1.2 to about 1.4, about 1.4 to about 2.0, about 1.4 to about 1.8, about 1.4 to about 1.6, about 1.6 to about 2.0, about 1.6 to about 1.8, or about 1.8 to about 2.0 g / L of poloxamer 188. In one embodiment, the culture medium comprises 0.8 to 1.2 g / L of poloxamer 188. In one embodiment, the culture medium comprises 1.0 g / L of poloxamer 188. In one embodiment, the culture medium comprises about 1.0 g / L of poloxamer 188.

[0376] In one embodiment, the culture medium contains and / or is supplemented with one or more antibiotics.

[0377] The components of a first exemplary culture medium are listed in Table 1.

[0378] [Table 1]

[0379] The ingredients of a second exemplary culture medium are listed in Table 2.

[0380] [Table 2]

[0381] CD3 binding antibody In one embodiment, the culture medium contains and / or is supplemented with a CD3-binding antibody or antigen-binding fragment thereof. In one embodiment, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1, HIT3a, and variants thereof. In one embodiment, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof.

[0382] In one embodiment, the CD3 binding antibody or antigen-binding fragment thereof and support cells are added to the culture vessel prior to adding the NK cells and / or culture medium.

[0383] In one embodiment, the culture medium contains and / or is supplemented with about 5 ng / mL to about 15 ng / mL of OKT3. In one embodiment, the culture medium contains and / or is supplemented with about 5 to about 12.5, about 5 to about 10, about 5 to about 7.5, about 7 to about 7.5, about 7.5 to about 12.5, about 7.5 to about 10, about 10 to about 15, about 10 to about 12.5, or about 12.5 to about 15 ng / mL of OKT3. In one embodiment, the culture medium contains and / or is supplemented with 10 ng / mL of OKT3. In one embodiment, the culture medium contains and / or is supplemented with about 10 ng / mL of OKT3.

[0384] culture container A variety of vessels can be used in the present invention, hi one embodiment, the culture vessel is selected from the group consisting of a flask, a bottle, a dish, a multi-walled plate, a roller bottle, a bag, and a bioreactor.

[0385] In one embodiment, the culture vessel is treated to be hydrophilic. In one embodiment, the culture vessel is treated to promote attachment and / or proliferation. In one embodiment, the surface of the culture vessel is coated with serum, collagen, laminin, gelatin, poly-L-lysine, fibronectin, extracellular matrix proteins, or combinations thereof.

[0386] In some embodiments, other types of culture vessels are used depending on the culture step.

[0387] In one embodiment, the culture vessel has a volume of about 100 mL to about 1,000 L, and in one embodiment, the culture vessel has a volume of about 125 mL, about 250 mL, about 500 mL, about 1 L, about 5 L, about 10 L, or about 20 L.

[0388] In one embodiment, the culture vessel is a bioreactor.

[0389] In one embodiment, the bioreactor is a rocking bed (wave) bioreactor. In one embodiment, the bioreactor is a stirred tank bioreactor. In one embodiment, the bioreactor is a rotating wall vessel. In one embodiment, the bioreactor is a perfusion bioreactor. In one embodiment, the bioreactor is an isolation / expansion automated system. In one embodiment, the bioreactor is an automated or semi-automated bioreactor. In one embodiment, the bioreactor is a disposable bag bioreactor.

[0390] In one embodiment, the bioreactor has a volume of about 100 mL to about 1,000 L. In one embodiment, the bioreactor has a volume of about 10 L to about 1,000 L. In one embodiment, the bioreactor has a volume of about 100 L to about 900 L. In one embodiment, the bioreactor has a volume of about 10 L to about 800 L. In one embodiment, the bioreactor is about 10 L to about 700 L, about 10 L to about 600 L, about 10 L to about 500 L, about 10 L to about 400 L, about 10 L to about 300 L, about 10 L to about 200 L, about 10 L to about 100 L, about 10 L to about 90 L, about 10 L to about 80 L, about 10 L to about 70 L, about 10 L to about 60 L, about 10 L to about 50 L, about 10 L to about 40 L, about 10 L to about 30 L, about 10 L to about 20 L, about 20 L to about 1,000 L, about 20 L to about 900 L, about 20 L to about 800 L, about 20 L to about 300 L, about 10 L to about 200 L, about 1 ...10 L to about 1,000 L, about 10 L to about 1,000 L, about L~700L, 20L~600L, 20L~500L, 20L~400L, 20L~300L, 20L~200L, 20L~100L, 20L~90L, 20L~80L, 20L~70L, 2 0L~60L, 20L~50L, 20L~40L, 20L~30L, 30L~1,000L, 30L~900L, 30L~800L, 30L~700L, 30L~600L, 30L~500L, approx. 30L~400L, 30L~300L, 30L~200L, 30L~100L, 30L~90L, 30L~80L, 30L~70L, 30L~60L, 30L~50L, 30L~40L, 40 L~1,000L, approx. 40L~900L, approx. 40L~800L, approx. 40L~700L, approx. 40L~600L, approx. 40L~500L, approx. 40L~400L, approx. 40L~300L, approx. 40L~200L, approx. 40L~10 0L, approximately 40L to approximately 90L, approximately 40L to approximately 80L, approximately 40L to approximately 70L, approximately 40L to approximately 60L, approximately 40L to approximately 50L, approximately 50L to approximately 1,000L, approximately 50L to approximately 900L, approximately 50L to approximately 800L, approximately 50L to approximately 700L, approximately 50L to approximately 6 00L, approx. 50L ~ approx. 500L, approx. 50L ~ approx. 400L, approx. 50L ~ approx. 300L, approx. 50L ~ approx. 200L, approx. 50L ~ approx. 100L, approx. 50L ~ approx. 90L, approx.000L, about 60L to about 900L, about 60L to about 800L, about 60L to about 700L, about 60L to about 600L, about 60L to about 500L, about 60L to about 400L, about 60L to about 300L, about 60L to about 200L, about 60L to about 100L, about 60L to about 90L, about 60L to about 80L , about 60L to about 70L, about 70L to about 1,000L, about 70L to about 900L, about 70L to about 800L, about 70L to about 700L, about 70L to about 600L, about 70L to about 500L, about 70L to about 400L, about 70L to about 300L, about 70L to about 200L, about 70L to about 100L, about 70L to about 90L, about 70L to about 80L, about 80L to about 1,000L, about 80L to about 900L, about 80L to about 800L, about 80L to about 700L, about 80L to about 600L, about 80L to about 500L, about 80L to about 400L, about 80L to about 300L, about 80L to about 200L, about 80 L to about 100L, about 80L to about 90L, about 90L to about 1,000L, about 90L to about 900L, about 90L to about 800L, about 90L to about 700L, about 90L to about 600L, about 90L to about 500L, about 90L to about 400L, about 90L to about 300L, about 90L to about 200L, about 90L to About 100L, about 100L to about 1,000L, about 100L to about 900L, about 100L to about 800L, about 100L to about 700L, about 100L to about 600L, about 100L to about 500L, about 100L to about 400L, about 100L to about 300L, about 100L to about 200L, about 200L to about 1,000L, about 200L to about 900L, about 200L to about 800L, about 200L to about 700L, about 200L to about 600L, about 200L to about 500L, about 200L to about 400L, about 200L to about 300L, about 300L to about 1,000L, about 300L to about 900L, about 300L to about 800L, about 300L to about 700L, about 300L to about 600L, about 300L to about 500L, about 300L to about 400L, about 400L to about 1,000L, about 400L to about 900L, about 400L to about 800L, about 400L to about 700L, about 400L to about 600L, about 400L to about 50 0L, about 500L to about 1,000L, about 500L to about 900L, about 500L to about 800L, about 500L to about 700L, about 500L to about 600L, about 600L to about 1,000L, about 600L to about 900L, about 600L to about 800L, about 600L to about 700L, about 700L to about 1,The bioreactor may have a volume of about 50 L, about 700 L to about 900 L, about 700 L to about 800 L, about 800 L to about 1,000 L, about 800 L to about 900 L, or about 900 L to about 1,000 L. In one embodiment, the bioreactor has a volume of about 50 L.

[0391] In one embodiment, the bioreactor has a volume of 100 mL to 1,000 L. In one embodiment, the bioreactor has a volume of 10 L to 1,000 L. In one embodiment, the bioreactor has a volume of 100 L to 900 L. In one embodiment, the bioreactor has a volume of 10 L to 800 L. In one embodiment, the bioreactor may be 10 L to 700 L, 10 L to 600 L, 10 L to 500 L, 10 L to 400 L, 10 L to 300 L, 10 L to 200 L, 10 L to 100 L, 10 L to 90 L, 10 L to 80 L, 10 L to 70 L, 10 L to 60 L, 10 L to 50 L, 10 L to 40 L, 10 L to 30 L, 10 L to 20 L, 20 L to 1,000 L, 20 L to 900 L, 20 L to 800 L, 20 L to 700 L, 20 L to 600 L, 20 L to 500 L, 20 L to 500 L, 20 L to 1 ... L~400L, 20L~300L, 20L~200L, 20L~100L, 20L~90L, 20L~80L, 20L~70L, 20L~60L, 20L~50L, 20L~40L, 20L~30L, 30L~1,000L, 30L~900L, 30L~800L, 30L~700L, 30L~600L, 30L~500L, 30L~400L, 30L~300L, 30L~200L, 30L~100L, 30L~90L, 30L~80L, 30L~7 0L, 30L~60L, 30L~50L, 30L~40L, 40L~1,000L, 40L~900L, 40L~800L, 40L~700L, 40L~600L, 40L~500L, 40L~400L, 40L~300L, 40L~200L, 40L~100L, 40L~90L, 40L~80L, 40L~70L, 40L~60L, 40L~50L, 50L~1,000L, 50L~900L, 50L~800L, 50L~700L, 50L~6 00L, 50L~500L, 50L~400L, 50L~300L, 50L~200L, 50L~100L, 50L~90L, 50L~80L, 50L~70L, 50L~60L, 60L~1,000L, 60L~900L, 60L~800L, 60L~700L, 60L~600L, 60L~500L, 60L~400L, 60L~300L, 60L~200L, 60L~100L, 60L~90L, 60L~80L, 60L~70L, 70L~1,000L, 70L~900L, 70L~800L, 70L~700L, 70L~600L, 70L~500L, 70L~400L, 70L~300L, 70L~200L, 70L~100L, 70L~90L, 70 L~80L, 80L~1,000L, 80L~900L, 80L~800L, 80L~700L, 80L~600L, 80L~500L, 80L~400L, 80L~300L, 80L~200L, 80L~100 L, 80L~90L, 90L~1,000L, 90L~900L, 90L~800L, 90L~700L, 90L~600L, 90L~500L, 90L~400L, 90L~300L, 90L~200L, 90L ~100L, 100L~1,000L, 100L~900L, 100L~800L, 100L~700L, 100L~600L, 100L~500L, 100L~400L, 100L~300L, 100L~200L , 200L~1,000L, 200L~900L, 200L~800L, 200L~700L, 200L~600L, 200L~500L, 200L~400L, 200L~300L, 300L~1,000L, 3 00L~900L, 300L~800L, 300L~700L, 300L~600L, 300L~500L, 300L~400L, 400L~1,000L, 400L~900L, 400L~800L, 400L~7 The bioreactor has a volume of 00 L, 400 L to 600 L, 400 L to 500 L, 500 L to 1,000 L, 500 L to 900 L, 500 L to 800 L, 500 L to 700 L, 500 L to 600 L, 600 L to 1,000 L, 600 L to 900 L, 600 L to 800 L, 600 L to 700 L, 700 L to 1,000 L, 700 L to 900 L, 700 L to 800 L, 800 L to 1,000 L, 800 L to 900 L, or 900 L to 1,000 L. In one embodiment, the bioreactor has a volume of 50 L.

[0392] Cell expansion and stimulation In one embodiment, a source of natural killer cells (eg, a single unit of umbilical cord blood) is co-cultured with feeder cells to generate expanded and stimulated NK cells.

[0393] In one embodiment, the co-cultivation is carried out in a culture medium described in the present invention, for example, exemplary culture medium #1 (Table 1) or exemplary culture medium #2 (Table 2).

[0394] In one embodiment, the source of natural killer cells (e.g., single unit cord blood) is approximately 1×10 cells prior to expansion. 7 ~Approx. 1×10 9 In one embodiment, the natural killer cell source (e.g., single unit cord blood) comprises 1 x 10 total nucleated cells prior to expansion. 8 ~Approx. 1.5×10 8 In one embodiment, the natural killer cell source (e.g., single unit cord blood) comprises 1 x 10 total nucleated cells prior to expansion. 8 In one embodiment, the natural killer cell source (e.g., single unit cord blood) contains 1 x 10 total nucleated cells prior to expansion. 9 In one embodiment, the natural killer cell source (e.g., single unit cord blood) contains approximately 1 x 10 total nucleated cells prior to expansion. 9 Contains total nucleated cells.

[0395] In one embodiment, cells derived from a co-culture of a natural killer cell source (e.g., a single unit of umbilical cord blood) with feeder cells are harvested and frozen in a cryopreservation composition described herein. In one embodiment, the frozen cells from the co-culture are an infusion-ready drug product. In one embodiment, the frozen cells from the co-culture are used as a master cell bank (MCB) to produce an infusion-ready drug product, e.g., by one or more additional co-culture steps, as described herein. Thus, for example, the natural killer cell source can be expanded and stimulated as described herein to produce expanded and stimulated NK cells suitable for use in an infusion-ready drug product without producing an intermediate product. The natural killer cell source can also be expanded and stimulated as described herein to produce an intermediate product, e.g., a first master cell bank (MCB). The first MCB can be used to produce expanded and stimulated NK cells suitable for use in an infusion-ready drug product, or can be used to produce another intermediate product, e.g., a second MCB. The second MCB can be used to produce expanded and stimulated NK cells suitable for an infusion-ready drug product, or can be used to produce other intermediate products (e.g., a third MCB, etc.).

[0396] In one embodiment, the ratio of feeder cells to natural killer cell source cells or MCB cells inoculated into the co-culture is about 1:1 to 4:1. In one embodiment, the ratio of feeder cells to natural killer cell source cells or MCB cells is about 1:1 to about 3.5:1, about 1:1 to about 3:1, about 1:1 to about 2.5:1, about 1.1:1 to about 2:1, about 1:1 to about 1.5:1, about 1.5:1 to about 4:1, about 1.5:1 to about 3.5:1, about 1.5:1 to about 3:1, about 1.5:1 to about 4:1, about 1.5:1 to about 5:1, about 1.5:1 to about 5:1, about 1.5:1 to about 6:1, about 1.5:1 to about 6:1, about 1.5:1 to about 7:1, about 1.5:1 to about 8:1, about 1.5:1 to about 9:1, about 1.5:1 to about 10:1, about 1.5:1 to about 20:1, about 1.5:1 to about 3.5:1, about 1.5:1 to about 3:1, about 1.5:1 to about 4:1, about 1.5:1 to about 5:1, about 1.5:1 to about 10:1, about 1.5:1 to about 20:1, about 1.5:1 to about 3.5:1, about 1.5:1 to about 3:1, about 1.5:1 to about 20:1, about 1.5:1 to about 3.5:1, about 1.5:1 to about 4:1, about 1.5:1 to about 3.5:1, about 1.5:1 to about 3:1, about The ratio of feeder cells to natural killer cell source cells or MCB cells inoculated into the co-culture is 2.5:1 to about 2.5:1, about 1.5:1 to about 2:1, about 2:1 to about 4:1, about 2:1 to about 3.5:1, about 2:1 to about 3:1, about 2:1 to about 2.5:1, about 2.5:1 to about 4:1, about 2.5:1 to about 3.5:1, about 2.5:1 to about 3:1, about 3:1 to about 4:1, about 3:1 to about 3.5:1, or about 3.5:1 to about 4:1. In one embodiment, the ratio of feeder cells to natural killer cell source cells or MCB cells inoculated into the co-culture is 2.5:1. In one embodiment, the ratio of feeder cells to natural killer cell source cells or MCB cells inoculated into the co-culture is about 2.5:1.

[0397] In one embodiment, the co-culture is carried out in a disposable culture bag (e.g., a 1 L disposable culture bag). In one embodiment, the co-culture is carried out in a bioreactor (e.g., a 50 L bioreactor). In one embodiment, culture medium is added to the co-culture after the initial inoculation.

[0398] In one embodiment, the co-cultivation is carried out 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 or more days. In one embodiment, the co-cultivation is carried out for up to 16 days.

[0399] In one embodiment, the co-cultivation is carried out at or about 37°C.

[0400] In one embodiment, the co-cultivation is carried out at or about pH 7.9.

[0401] In one embodiment, the co-cultivation is carried out at a dissolved oxygen (DO) level of 50% or greater.

[0402] In one embodiment, exemplary culture medium #1 (Table 1) is used to produce MCBs and exemplary culture medium #2 (Table 2) is used to produce cells suitable for infusion-ready drug products.

[0403] In one embodiment, the co-culture of a natural killer cell source (e.g., a single unit cord blood) with feeder cells comprises approximately 50×10 8 ~About 50×10 12 In one embodiment, the expansion produces cells, e.g., MCB cells or infusion-ready drug product cells. In one embodiment, the expansion produces cells of about 50 x 10 8 ~Approx. 25×10 10 , about 10×10 8 ~Approx. 1×10 10 , about 50×10 8 ~Approx. 75×10 9 , about 50×10 8 ~About 50×10 9 , about 50×10 8 ~Approx. 25×10 9 , about 50×10 8 ~Approx. 1×10 9 , about 50×10 8 ~Approx. 75×10 8 , about 75×10 8 ~About 50×10 10 , about 75×10 8 ~Approx. 25×10 10 , about 75×10 8 ~Approx. 1×10 10 , about 75×10 8 ~Approx. 75×10 9 , about 75×10 8 ~About 50×10 9 , about 75×10 8 ~Approx. 25×10 9 , about 75×10 8 ~Approx. 1×10 9 , about 1×10 9 ~About 50×10 10 , about 1×10 9 ~Approx. 25×10 10 , about 1×10 9 ~Approx. 1×1010 , about 1×10 9 ~Approx. 75×10 9 , about 1×10 9 ~About 50×10 9 , about 1×10 9 ~Approx. 25×10 9 , about 25×10 9 ~About 50×10 10 , about 25×10 9 ~Approx. 25×10 10 , about 25×10 9 ~Approx. 1×10 10 , about 25×10 9 ~Approx. 75×10 9 , about 25×10 9 ~About 50×10 9 , about 50×10 9 ~About 50×10 10 , about 50×10 9 ~Approx. 25×10 10 , about 50×10 9 ~Approx. 1×10 10 , about 50×10 9 ~Approx. 75×10 9 , about 75×10 9 ~About 50×10 10 , about 75×10 9 ~Approx. 25×10 10 , about 75×10 9 ~Approx. 1×10 10 , about 1×10 10 ~About 50×10 10 , about 1×10 10 ~Approx. 25×10 10 , or approximately 25 × 10 10 ~About 50×10 10 producing cells, for example, MCB cells or infusion-prepared drug product cells.

[0404] In one embodiment, expansion produces 60 to about 100 vials each containing about 600 million to about 1 billion cells, e.g., MCB cells or infusion-ready drug product cells. In one embodiment, expansion produces 80 or about 80 vials each containing 800 million or about 800 million cells, e.g., MCB cells or infusion-ready drug product cells.

[0405] In one embodiment, expansion increases the number of cells in the natural killer cell source, eg, the number of MCB NK cells relative to the number of NK cells, by about 100 to about 500 fold. In one embodiment, expansion increases the number of cells in the natural killer cell source, e.g., the number of MCB NK cells, relative to the number of NK cells, by about 100 to about 500, about 100 to about 400, about 100 to about 300, about 100 to about 200, about 200 to about 450, about 200 to about 400, about 100 to about 350, about 200 to about 300, about 200 to about 250, about 250 to about 500, about 250 to about 450, about 200 to about 400, about 250 to about 350, about 250 to about 300, about 300 to about 500, about 300 to about 450, about 300 to about 400, about 300 to about 350, about 350 to about 500, about 350 to about 450, or about 350 to about 400 times.

[0406] In one embodiment, expansion increases the number of cells in the natural killer cell source, e.g., the number of MCB NK cells relative to the number of NK cells, by about 100-fold to about 70,000-fold. In one embodiment, expansion increases the number of cells in the natural killer cell source, e.g., the number of MCB NK cells relative to the number of NK cells, by 10,000-fold, 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold.

[0407] In one embodiment, co-culture of MCB cells with support cells produces about 500 million to about 1.5 billion cells, e.g., NK cells suitable for MCB and / or infusion-prepared drug products. In one embodiment, co-culture of MCB cells with support cells provides a yield of about 500 million to about 1.5 billion, about 500 million to about 1.25 billion, about 500 million to about 1 billion, about 500 million to about 750 million, about 750 million to about 1.5 billion, about 500 million to about 1.25 billion, about 750 million to about 1 billion, about 1 billion to about 1.5 billion, about 1 billion to about 1.25 billion, or about 1.25 billion to about 1.5 billion cells, e.g., NK cells suitable for MCB and / or infusion-prepared drug products.

[0408] In one embodiment, co-culture of MCB cells with support cells produces about 50 to about 150 vials of cells, e.g., infusion-ready drug product cells, each containing about 750 million to about 1.25 billion cells, e.g., NK cells suitable for an MCB and / or infusion-ready drug product. In one embodiment, co-culture of MCB cells with support cells produces 100 or about 100 vials, each containing 1 billion or about 1 billion cells, e.g., NK cells suitable for an MCB and / or infusion-ready drug product.

[0409] In one embodiment, expansion can increase cell numbers, e.g., MCB and / or infusion-prepared drug product cell numbers, by about 100-fold to about 500-fold. In one embodiment, the expansion yield of NK cells suitable for MCB and / or infusion-prepared drug products can be 100-fold to about 500-fold, about 100-fold to about 400-fold, about 100-fold to about 300-fold, about 100-fold to about 200-fold, about 200-fold to about 450-fold, about 200-fold to about 400-fold, about 100-fold to about 350-fold, about 200-fold to about 300-fold, about 200-fold to about 250-fold, The increase can be about 250 to about 500 times, about 250 to about 450 times, about 200 to about 400 times, about 250 to about 350 times, about 250 to about 300 times, about 300 to about 500 times, about 300 to about 450 times, about 300 to about 400 times, about 300 to about 350 times, about 350 to about 500 times, about 350 to about 450 times, or about 350 to about 400 times.

[0410] In one embodiment, expansion increases the number of cells, e.g., NK cell numbers suitable for the MCB and / or infusion-ready drug product, by about 100-fold to about 70,000-fold. In one embodiment, expansion can increase the number of cells, e.g., NK cell numbers suitable for the MCB and / or infusion-ready drug product, by at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold.

[0411] In embodiments in which cells are genetically engineered during expansion and stimulation as described herein, not all expanded and stimulated cells will necessarily be successfully engineered, e.g., transformed with a heterologous protein, e.g., a heterologous protein including CAR and / or IL-15, as described herein. Thus, the methods described herein can additionally include a step of separating engineered cells, e.g., engineered cells of the invention, from non-engineered cells.

[0412] In one embodiment, engineered cells, e.g., transformed cells, are selected from non-engineered cells, e.g., non-transformed cells, using a reagent specific for an antigen of the engineered cells, e.g., an antibody that targets an antigen of the engineered cells but not non-engineered cells. In one embodiment, the antigen of the engineered cells is a component of a CAR, e.g., a CAR described herein.

[0413] Antigen-based cell separation systems (eg, the CliniMACS® sorting system (Miltenyi Biotec)) are commercially available.

[0414] In one embodiment, engineered cells (eg, transformed cells) are sorted from non-engineered cells (eg, non-transformed cells) using flow cytometry.

[0415] In one embodiment, the sorted engineered cells are used as an MCB. In one embodiment, the sorted engineered cells are used as a component of a pharmaceutical product ready for administration.

[0416] In one embodiment, engineered cells (e.g., transformed cells) are separated from non-engineered cells (e.g., non-transformed cells) using microfluidic cell sorting methods, such as those described in Dalili et al., "A Review of Sorting, Separation and Isolation of Cells and Microbeads for Biomedical Applications: Microfluidic Approaches," Analyst 144:87 (2019).

[0417] In one embodiment, about 1% to about 99% of the expanded and stimulated cells are successfully engineered (e.g., transformed) with a vector comprising, for example, a heterologous protein, e.g., a heterologous protein comprising a CAR of the invention and / or IL-15. In one embodiment, the range is about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, about 1% to about 5%, about 5% to about 99%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 99%, about 10% ~ about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 99%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 99%, about 30% to about 90%, about 3 0% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 50%, about 30% to about 40%, about 40% to about 99%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 70%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 40% to about 50%, about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 99%, About 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 50% to about 70%, about 50% to about 60%, about 60% to about 70%, about 60% to about 80%, about 60% to about 70%, about 70% to about 99%, about 70% to about 90%, about 70% to about 80%, about 80% to about 99%, about 80% to about 90%, or about 90% to about 99% of the expanded and stimulated cells are successfully engineered (e.g., transformed).

[0418] In one embodiment, frozen cells of the first or second MCB are thawed and cultured. In one embodiment, a single vial of frozen cells of the first or second MCB, e.g., a single vial consisting of 800 million or about 800 million cells (e.g., first or second MCB cells), is thawed and cultured. In one embodiment, frozen first or second MCB cells are cultured with additional feeder cells to produce cells suitable for a second or third MCB or an infusion-ready drug product. In one embodiment, co-culture-derived cells of the first or second MCB are harvested and frozen.

[0419] In one embodiment, cells are harvested from the co-culture of the natural killer cell source, the first MCB, or the second MCB and frozen in a cryopreservation composition, e.g., a cryopreservation composition described herein. In one embodiment, the cells are washed after harvesting. Accordingly, provided herein are pharmaceutical compositions comprising activated and stimulated NK cells, e.g., activated and stimulated NK cells produced by the methods described herein, harvested and washed, and a cryopreservation composition described herein.

[0420] In one embodiment, the cells are mixed with a cryopreservation composition, e.g., as described herein, prior to freezing. In one embodiment, the cells are frozen in cryobags. In one embodiment, the cells are frozen in cryovials.

[0421] In one embodiment, the method of the invention additionally comprises the step of separating NK cells from the expanded and stimulated NK cell population.

[0422] An exemplary process for expanding and stimulating NK cells is shown in FIG.

[0423] Genetic Engineering In one embodiment, the method additionally includes engineering the NK cells to express a heterologous protein, e.g., a heterologous protein described herein, e.g., a heterologous protein comprising CAR and / or IL-15.

[0424] In one embodiment, engineering NK cells to express a heterologous protein described herein comprises, for example, stably transforming the NK cells with a vector comprising a polynucleic acid encoding a heterologous protein described herein. Suitable vectors are therefore described herein.

[0425] In one embodiment, engineering NK cells to express a heterologous protein described herein can include introducing the heterologous protein by gene editing (e.g., zinc finger nuclease (ZFN) gene editing, ARCUS gene editing, CRISPR-Cas9 gene editing, or megaTAL gene editing) in combination with adeno-associated virus (AAV) technology.

[0426] In one embodiment, the NK cells are engineered to express a heterologous protein described herein, e.g., during or after culturing the composition in medium containing support cells. For example, in some cases, the engineering (e.g., transduction) occurs during the expansion and stimulation process described herein, e.g., co-culturing the NK cell source with support cells as described herein, e.g., on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of co-culture.

[0427] In one embodiment, the method of the invention additionally comprises the step of engineering the NK cells, eg, to express, overexpress, knock out, or knock down a gene or gene product.

[0428] In one embodiment, the natural killer cells are not genetically engineered.

[0429] In one embodiment, NK cells are engineered (e.g., transduced) in culture medium supplemented with stimulatory factors (e.g., as described herein). Such stimulatory factors can be used to provide growth or survival signals to NK cells during the engineering process or to increase transcription efficiency. In one embodiment, the stimulatory factor is a cytokine. In one embodiment, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, and combinations thereof.

[0430] In one embodiment, the cytokine is IL-21. IL-21 is used at a final concentration of 10-100 ng / mL, for example, at a final concentration of about 10, 15, 20, 25, 30, 34, 40, 45, 50, 55, 60, 70, 80, 90, or 100 ng / mL, or at a final concentration of about 10, 15, 20, 25, 30, 34, 40, 45, 50, or 10-100 ng / mL. In one embodiment, the cytokine is IL-2. In one embodiment, the cytokine is a combination of IL-2 and IL-21. In one embodiment, the cytokine is a combination of IL-2, IL-18, and IL-21.

[0431] In one embodiment, the stimulatory factor is added to the culture medium at the time of manipulation (e.g., transduction). In one embodiment, the stimulatory factor is added to the culture medium after the time of manipulation (e.g., transduction), for example, 1 to 48 hours after manipulation, for example, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 12 to 28 hours, 12 to 36 hours, 12 to 24 hours, 24 to 48 hours, 24 to 36 hours, or 36 to 48 hours after manipulation. In one embodiment, the stimulatory factor is added to the culture medium at the time of transduction and all times after the time of manipulation (e.g., 1 to 48 hours after transduction).

[0432] In one embodiment, the culture medium is supplemented with stimulatory factors after culturing in medium containing feeder cells. Thus, in some cases, the culture medium can include feeder cells at the time of manipulation (e.g., transduction). In some cases, the feeder cells are removed from the culture medium before supplementing or manipulating with stimulatory factors. In some cases, the feeder cells are not removed from the culture medium before supplementing or manipulating with stimulatory factors. In some cases, no additional feeder cells are added to the culture medium during manipulation, regardless of whether or not residual feeder cells are removed. In some cases, all additional feeder cells and stimulatory factors are added to the culture medium during manipulation. In some cases, no additional feeder cells are added during manipulation, but stimulatory factors are added to the culture medium during manipulation.

[0433] Characteristics of expanded and stimulated NK cells For example, after ex vivo expansion and stimulation as described herein, the expanded and stimulated NK cell population not only has a number / density (e.g., as described above) that does not naturally occur in the human body, but also differs in phenotypic characteristics (e.g., gene expression and / or surface protein expression) from the starting source material or other naturally occurring NK cell populations.

[0434] In some cases, the starting NK cell source is a sample extracted from a single individual (e.g., a single umbilical cord blood unit not expanded in vitro). Thus, in some cases, the expanded and stimulated NK cells share a common lineage, i.e., share a genotype due to the clonal expansion of a cell population derived from a single organism, since they all arise by expansion of the starting NK cell source. However, they would not arise naturally at the densities achieved by in vitro expansion and have different phenotypic characteristics from the starting NK cell source.

[0435] In some cases, the population of expanded stimulated NK cells comprises a minimum of 100 million or more expanded natural killer cells, e.g., 200 million, 250 million, 300 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 9 billion, 10 billion, 20 billion, 25 billion, 30 billion, 40 billion, 50 billion, 60 billion, 70 billion, 80 billion, 90 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

[0436] In one embodiment, the expanded and stimulated NK cells comprise at least 80%, eg, at least 90%, at least 95%, at least 99% or 100% CD56+CD3- cells.

[0437] In one embodiment, the expanded and stimulated NK cells are not genetically engineered.

[0438] In one embodiment, the expanded and stimulated NK cells do not contain the CD16 transforming gene.

[0439] In one embodiment, the expanded and stimulated NK cells do not express exogenous CD16 protein.

[0440] For example, expanded and stimulated NK cells express one or more of CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44 on their surface.

[0441] In the present invention, surface protein expression levels can be achieved in some cases without positive selection for a particular surface protein. For example, in some cases, the NK cell source (e.g., a single cord blood unit) contains all KIR B alleles of the KIR receptor system and the 158V / V variant of CD16, and is enriched for CD56+CD3- expression and depleted of CD3(+), but without selection for other surface protein expression during expansion and stimulation.

[0442] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKG2D+ cells.

[0443] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp46+ cells.

[0444] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp30+ cells.

[0445] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% DNAM-1+ cells.

[0446] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells.

[0447] In one embodiment, the expanded and stimulated NK cells, e.g., by expansion and stimulation of a single cord blood unit as described above, can comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% CD94+(KLRD1) cells; in one embodiment, the expanded and stimulated NK cells, e.g., by expansion and stimulation of a single cord blood unit as described above, can comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, or 100% CD94+(KLRD1) cells.

[0448] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, comprise 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD3+ cells.

[0449] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD14+ cells.

[0450] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD19+ cells.

[0451] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CXCR+ cells.

[0452] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD122+(IL2RB) cells.

[0453] As described herein, the inventors have surprisingly demonstrated that NK cells expanded and stimulated by the methods described herein express high levels of CD16 throughout the expansion and stimulation process, producing a cell population with high CD16 expression. The high expression of CD16 obviates the need to engineer the expanded cells to express CD16, which is important for initiating ADCC, thereby providing an unexpected advantage of the expansion and stimulation methods disclosed herein. Thus, in one embodiment, for example, by expansion and stimulation of a single cord blood unit, e.g., as described above, the expanded and stimulated NK cells comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0454] In one embodiment, for example, by expansion and stimulation of a single cord blood unit as described above, the expanded and stimulated NK cells comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of all CD16+ NK cells bearing KIR B alleles and CD16 and 158V / V variants of the KIR receptor system.

[0455] In one embodiment, e.g., from expansion and stimulation of a single cord blood unit as described above, the proportion of expanded and stimulated NK cells expressing, e.g., CD16, is the same as or higher than the proportion of NK cells in the cord blood-derived seed cells.

[0456] In one embodiment, e.g., from expansion and stimulation of a single cord blood unit as described above, the proportion of expanded and stimulated NK cells expressing, e.g., NKG2D, is the same as or higher than the proportion of natural killer cells in the cord blood-derived source cells.

[0457] In one embodiment, e.g., from expansion and stimulation of a single cord blood unit as described above, the proportion of expanded and stimulated NK cells that express, e.g., NKp30, is the same as or higher than the proportion of NK cells in the cord blood-derived source cells.

[0458] In one embodiment, for example, the proportion of expanded and stimulated NK cells expressing DNAM-1 from expansion and stimulation of a single cord blood unit (e.g., as described above) is the same as or higher than the proportion of NK cells in cord blood-derived source cells.

[0459] In one embodiment, the proportion of expanded and stimulated NK cells, e.g., expressing NKp44, from expansion and stimulation of a single cord blood unit (e.g., as described above) is the same as or higher than the proportion of NK cells in the cord blood-derived source cells.

[0460] In one embodiment, the proportion of expanded and stimulated NK cells, e.g., expressing NKp46, from expansion and stimulation of a single cord blood unit (e.g., as described above) is the same as or higher than the proportion of NK cells in the cord blood-derived source cells.

[0461] As described herein, the inventors have also surprisingly demonstrated that NK cells expanded and stimulated by the methods described herein express low levels of CD38, which is an effective target for certain cancer therapies (e.g., multiple myeloma and acute myeloid leukemia) (see Jiao et al., "CD38: Targeted Therapy in Multiple Myeloma and Therapeutic Potential for Solid Cancers," Expert Opinion on Investigational Drugs 29(11):1295-1308 (2020)).

[0462] Thus, in one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, contain no more than 80% or the same CD38+ cells, e.g., no more than 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% CD38+ cells.

[0463] In one embodiment, the NK cells expanded and stimulated, e.g., by expansion and stimulation of a single cord blood unit as described above, contain all KIR B alleles of the KIR receptor system and the 158V / V variant of CD16, and comprise no more than 80% CD38+ cells, e.g., no more than 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% CD38+ cells.

[0464] In one embodiment, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, the expanded and stimulated NK cells contain all KIR B alleles of the KIR receptor system and the 158V / V variant of CD16, and are composed of up to 80% or identical CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells and 50% or more (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells).

[0465] In one embodiment, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, the expanded and stimulated NK cells contain all KIR B alleles of the KIR receptor system and 158V / V mutations of CD16, and include: i) 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells; and / or ii) less than or equal to 80% CD38+ cells, e.g., 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% CD38+ cells; and / or iii) at least 60%, e.g., at least 70% CD38+ cells. 0%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells; and / or iv) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells; and / or v) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp30+ cells; and / or vi) at least 60%, e.g., at least 70%, at least 80%, at least 90% and / or vii) at least 95%, minimum 99%, or 100% DNAM-1+ cells; and / or vii) minimum 60%, e.g., minimum 70%, minimum 80%, minimum 90%, minimum 95%, minimum 99%, or 100% NKp44+ cells; and / or viii) minimum 60%, e.g., minimum 70%, minimum 80%, minimum 90%, minimum 95%, minimum 99%, or 100% CD94+ (KLRD1) cells; and / or ix) 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD94+ (KLRD1) cells. and / or x) 20% or less, e.g., 10% or less, 5% or less, 1% or 0% CD14+ cells; and / or xi) 20% or less, e.g., 10% or less, 5% or less, 1% or 0% CD19+ cells; and / or xii) 20% or less, e.g., 10% or less, 10% or less, 5% or less, 1% or 0% or less CXCR+ cells; and / or xiii) 20% or less (e.g., 10% or less, 5% or less, 1% or 0% or less CD122+ (IL2RB) cells.

[0466] In one embodiment, the supporting cells do not persist with the expanded and stimulated NK cells, but residual signals of the supporting cells can be detected, for example, by the presence of residual cells (e.g., detection of cells with specific surface protein expression) or detection of residual nucleic acids and / or proteins expressed by the supporting cells.

[0467] For example, in some cases, the methods of the invention include expanding and stimulating NK cells using support cells, e.g., the eHuT-78 support cells described above, engineered to express a sequence not expressed by cells of the NK cell source, including the NK cells. For example, the support cells can be engineered to express at least one gene selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and mutant TNFα (SEQ ID NO: 3) or a variant thereof ("eHut-78 cells").

[0468] Such supporting cells may not remain in the expanded and stimulated NK cells, but the expanded and stimulated NK cells may contain detectable amounts of cells, proteins, and / or nucleic acids from the feeding cells. Thus, their residual presence can be detected in the expanded and stimulated NK cells, for example, by detecting the cells themselves (e.g., by flow cytometry), proteins expressed by the cells, and / or nucleic acids expressed by the cells.

[0469] Thus, the present specification also describes populations of expanded and stimulated NK cells that contain feeder cells (living or dead cells) or residual feeder cell impurities (e.g., residual feeder cell proteins or portions thereof and / or genetic material such as nucleic acids or portions thereof), and in some cases, the expanded and stimulated NK cells can contain more than 0% but less than 0.3% residual feeder cells (e.g., eHuT-78 feeder cells).

[0470] In some cases, the expanded and stimulated NK cells comprise residual feeder cell nucleic acid, e.g., residual 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and / or mutated TNFα (SEQ ID NO: 3), or portions thereof. In some cases, the membrane-bound IL-21 comprises the CD8 transmembrane domain.

[0471] In some cases, the expanded and stimulated NK cells comprise a proportion of residual supporting cells that is greater than or equal to 0% and less than or equal to 0.2%, e.g., as measured by the relative proportion of a particular protein or nucleic acid sequence of supporting cells in the sample (i.e., a protein or nucleic acid sequence not expressed by natural killer cells). For example, this can be measured by qPCR, as described above.

[0472] In one embodiment, the residual support cells are CD4(+) T cells. In one embodiment, the residual support cells are engineered CD4(+) T cells. In one embodiment, the residual support cells are engineered to express at least one gene selected from 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and mutated TNFα (SEQ ID NO: 3) ("eHut-78 cells") or variants thereof. Thus, in some cases, the support cell-specific protein is 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and / or mutated TNFα (SEQ ID NO: 3). Thus, the support cell-specific nucleic acid is a nucleic acid encoding 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and mutated TNFα (SEQ ID NO: 3), or a portion thereof. In some cases, the membrane-bound IL-21 comprises a CD8 transmembrane domain.

[0473] A variety of methods can be used to analyze and detect the presence of nucleic acid or protein gene products in biological samples. As used herein, the term "detection" refers to a method used to discover, determine, or confirm the presence or absence of a compound and / or substance (e.g., a cell, a protein, and / or a nucleic acid). In one embodiment, the detection method can be used to detect proteins. In one embodiment, detection can involve chemiluminescent or fluorescent techniques. In one embodiment, detection can involve immunological-based methods (e.g., quantitative enzyme-linked immunosorbent assays (ELISA), Western blotting, or dot blotting) in which antibodies are used to specifically react with whole proteins or specific epitopes of proteins. In one embodiment, detection can involve immunoprecipitation of proteins (Jungblut et al., J Biotechnol. 31;41(2-3):111-20 (1995); Franco et al., Eur J Morphol. 39(1):3-25 (2001)). In one embodiment, the detection method can be used to detect nucleic acids (e.g., DNA and / or RNA). In one embodiment, detection can involve Northern blot analysis, nuclease protection assay (NPA), in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR) (Raj et al., Nat. Methods 5, 877-879 (2008); Jin et al., J Clin Lab Anal. 11(1):2-9 (1997); Ahmed, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 20(2):77-116 (2002)).

[0474] Accordingly, methods for detecting a population of expanded and stimulated NK cells expanded and stimulated by the methods of the invention, e.g., co-cultured with eHuT-78 feeder cells described herein, are also described herein.

[0475] Anti-HER2 CAR-NK Provided herein are engineered cells, e.g., engineered natural killer cells, e.g., CAR-NK cells, e.g., anti-HER2 CAR-NK cells. In one embodiment, the CAR-NK cells are engineered to express IL-15.

[0476] In one embodiment, the natural killer cells are transformed, e.g., during expansion and stimulation as described herein. In one embodiment, the natural killer cells are manipulated during expansion and stimulation as described herein, e.g., during production of MCB. In one embodiment, the natural killer cells are manipulated during expansion and stimulation, e.g., during production of NK cells suitable for an infusion-ready drug product and / or production of MCB, as described above. Thus, in one embodiment, the NK cells provided herein are host cells, NK host cells that express a heterologous protein.

[0477] In one embodiment, the natural killer cells are manipulated before expansion and stimulation. In one embodiment, the natural killer cells are manipulated after expansion and stimulation.

[0478] In one embodiment, the NK cells are engineered by transformation with a vector. Suitable vectors include, for example, lentiviral vectors, e.g., lentiviral vectors comprising heterologous proteins as described herein. In one embodiment, the NK cells are transformed during production of the first MCB, as described herein.

[0479] In one embodiment, NK cells are transformed at an MOI (multiplicity of infection) of about 1 to about 40 viral particles per cell. In one embodiment, NK cells are transformed at an MOI of about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, or about 40 viral particles per cell.

[0480] Chimeric Antigen Receptor In one embodiment, the heterologous protein is a fusion protein, for example, a fusion protein comprising a chimeric antigen receptor (CAR) is introduced into the NK cells during expansion and stimulation.

[0481] In one embodiment, the CAR comprises one or more of a signal sequence, an extracellular domain, a hinge, a transmembrane domain, and one or more intracellular signal domain sequences. In one embodiment, the CAR additionally comprises a spacer sequence.

[0482] In one embodiment, the CAR comprises (from N-terminus to C-terminus) a signal sequence, an extracellular domain, a hinge, a spacer, a transmembrane domain, a first signal domain sequence, a second signal domain sequence, and a third signal domain sequence.

[0483] In one embodiment, the CAR comprises (from N-terminus to C-terminus) a signal sequence, an extracellular domain, a hinge, a transmembrane domain, a first signal domain sequence, a second signal domain sequence, and a third signal domain sequence.

[0484] The signal sequence can be cleaved from the mature CAR protein. Such cleavage can be mediated by a signal peptidase and can occur during or after translocation is complete to produce the mature protein. Thus, in one embodiment, the CAR comprises (from N- to C-terminus) an extracellular domain, a hinge, a spacer, a transmembrane domain, a first signal domain sequence, a second signal domain sequence, and a third signal domain sequence.

[0485] In one embodiment, the CAR comprises (from N-terminus to C-terminus) an extracellular domain, a hinge, a transmembrane domain, a first signal domain sequence, a second signal domain sequence, and a third signal domain sequence.

[0486] In one embodiment, the extracellular domain comprises an antibody or antigen-binding fragment thereof.

[0487] In one embodiment, one or more of the intracellular signal domain sequences is a CD28 intracellular signal domain sequence. In one embodiment, the CD28 intracellular signal domain sequence comprises or consists of SEQ ID NO:5.

[0488] In one embodiment, one or more of the intracellular signal domain sequences is an OX40L signal sequence (see, e.g., Matsumura et al., "Intracellular Signaling of gp34, the OX40 Ligand: Induction of c-jun and c-fos mRNA Expression Through gp34 upon Binding of Its Receptor, OX40," J. Immunol 163:3007-11 (1999), incorporated herein by reference in its entirety). In one embodiment, the OX40L intracellular signal domain sequence comprises or consists of SEQ ID NO: 8, 9, or 10.

[0489] In one embodiment, one or more of the intracellular signal sequences is a CD3ζ intracellular signal domain sequence. In one embodiment, the CD3ζ intracellular signal domain sequence comprises or consists of SEQ ID NO:13.

[0490] In one embodiment, the CAR comprises a CD28 intracellular signal domain sequence (SEQ ID NO: 5), an OX40L intracellular signal domain sequence (SEQ ID NO: 8, 9, or 10), and a CD3ζ intracellular signal domain sequence (SEQ ID NO: 13).

[0491] In one embodiment, the CAR comprises an intracellular signaling region comprising or consisting of SEQ ID NO:25.

[0492] In one embodiment, the CAR does not comprise the OX40L intracellular signal domain sequence.

[0493] In one embodiment, the CAR comprises the CD28 intracellular signal domain sequence (SEQ ID NO: 5) and the CD3ζ intracellular signal domain sequence (SEQ ID NO: 13), but does not comprise the OX40L intracellular signal domain sequence. In one embodiment, the signal sequence is the CD8α signal sequence. In one embodiment, the signal sequence comprises or consists of SEQ ID NO: 27.

[0494] In one embodiment, the extracellular domain comprises a single-chain variable fragment (scFv). In one embodiment, the extracellular domain comprises an anti-HER2 antibody or antigen-binding fragment thereof. In one embodiment, the extracellular domain comprises an anti-HER2 scFv.

[0495] In one embodiment, the anti-HER2 scFv comprises a CDRL1 domain comprising or consisting of SEQ ID NO: 34, a CDRL2 domain comprising or consisting of SEQ ID NO: 36, a CDRL3 domain comprising or consisting of SEQ ID NO: 38, a CDRH1 domain comprising or consisting of SEQ ID NO: 44, a CDRH2 domain comprising or consisting of SEQ ID NO: 46, and a CDRH3 domain comprising or consisting of SEQ ID NO: 48.

[0496] In one embodiment, the anti-HER2 scFv comprises a VL domain comprising or consisting of SEQ ID NO:32 and a VH domain comprising or consisting of SEQ ID NO:42.

[0497] In one embodiment, the anti-HER2 scFv comprises a VL domain comprising or consisting of SEQ ID NO: 32, a linker comprising or consisting of SEQ ID NO: 40, and a VH domain comprising or consisting of SEQ ID NO: 42.

[0498] In one embodiment, the anti-HER2 scFv comprises or consists of SEQ ID NO:30.

[0499] In one embodiment, the hinge comprises or consists of a CD8α hinge. In one embodiment, the CD8α hinge comprises or consists of SEQ ID NO: 50.

[0500] In one embodiment, the transmembrane domain is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane comprises or consists of SEQ ID NO:53.

[0501] In one embodiment, the fusion protein comprises or consists of SEQ ID NO:56.

[0502] IL-15 In one embodiment, NK cells are engineered to express IL-15, e.g., human IL-15 (UniProtKB#P40933; NCBI Gene ID#3600), e.g., soluble human IL-15, or an analog or any of the aforementioned variants. In one embodiment, IL-15 is expressed as part of a fusion protein that additionally includes a cleavage site. In one embodiment, IL-15 is expressed as part of a polyprotein that includes a self-cleaving peptide, such as a T2A ribosomal skipping sequence site (also known as a self-cleavage site) (Radcliffe & Mitrophanous, "Multiple Gene Products from a Single Vector: 'Self-Cleaving' 2A Peptides," Gene Therapy 11:1673-4 (2004); Liu et al., "Systematic Comparison of 2A Peptides for Cloning Multi-Genes in a Polycistronic Vector," Scientific Reports 7(1):2193 (2017)).

[0503] In one embodiment, the IL-15 comprises or consists of SEQ ID NO:22.

[0504] In one embodiment, the self-cleaving peptide is a 2A self-cleaving peptide. In one embodiment, the self-cleaving peptide is a T2A, P2A, E2A, or F2A self-cleaving peptide. In one embodiment, the self-cleaving peptide comprises SEQ ID NO: 16. In one embodiment, the self-cleaving peptide comprises or consists of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0505] In one embodiment, the T2A cleavage site comprises or consists of SEQ ID NO:17.

[0506] In one embodiment, IL-15 is expressed as part of a fusion protein comprising a CAR, e.g., a CAR described herein.

[0507] In one embodiment, the fusion protein comprises (N- to C-terminus) CAR, a cleavage site, and IL-15. In one embodiment, the fusion protein comprises SEQ ID NO: 26. In one embodiment, the fusion protein comprises or consists of SEQ ID NO: 59.

[0508] Inhibitory Receptors In one embodiment, the NK cells are engineered to have altered (e.g., decreased) expression of one or more inhibitory receptor genes. In one embodiment, the inhibitory receptor gene is an HLA-specific inhibitory receptor. In one embodiment, the inhibitory receptor gene is a non-HLA-specific inhibitory receptor. In one embodiment, the inhibitory receptor gene is selected from the group consisting of KIR, CD94 / NKG2A, LILRB1, PD-1, Irp60, Siglec-7, LAIR-1, and combinations thereof.

[0509] Polynucleic acids, vectors and host cells The present invention also provides polynucleotide sequences encoding the fusion proteins or portions thereof, e.g., polynucleotide sequences encoding the polypeptides described in the present invention, as set forth in the sequence listing provided herein.

[0510] Additionally, the present invention also provides vectors containing the polynucleic acids and cells, eg, NK cells, containing the vectors.

[0511] In one embodiment, the vector is a lentiviral vector (see, e.g., Milone et al. "Clinical Use of Lentiviral Vectors," Leukemia 32:1529-41 (2018)). In one embodiment, the vector is a retroviral vector. In one embodiment, the vector is a gamma retroviral vector. In one embodiment, the vector may be a non-viral vector, such as a piggyback non-viral vector (PB transposon, see, e.g., Wu et al., "piggyback is a Flexible and Highly Active Transposon as Compared to Sleeping Beauty, Tol2, and Mos1 in Mammalian Cells," PNAS 103(41):15008-13 (2006)), a Sleeping Beauty non-viral vector (SB transposon, see, e.g., Hudecek et al., "Going Non-Viral: The Sleeping Beauty Transposon System Breaks on Through to the Clinical Side," Critical Reviews in Biochemistry and Molecular Biology 52(4):355-380 (2017)), or an mRNA vector.

[0512] Cryopreservation Cryopreservation composition Provided herein are cryopreserved compositions, e.g., cryopreserved compositions suitable for intravenous administration, e.g., intravenous administration of the NK cells described herein. In one embodiment, a pharmaceutical composition comprises the cryopreserved composition and cells, e.g., the NK cells described herein.

[0513] albumin In one embodiment, the cryopreserved composition comprises an albumin protein, e.g., human albumin (UniProtKB Accession P0278, SEQ ID NO: 63), or a variant thereof. In one embodiment, the cryopreserved composition comprises an albumin protein, e.g., a homologue of a human albumin protein, or a variant thereof. In one embodiment, the cryopreserved composition comprises a biologically active portion of an albumin protein, e.g., human albumin, or a variant thereof.

[0514] In one embodiment, the albumin, e.g., human albumin, is provided as a solution, sometimes referred to herein as an albumin solution or a human albumin solution. Thus, in one embodiment, the cryopreservation composition is or comprises an albumin solution, e.g., a human albumin solution. In one embodiment, the albumin solution is a serum-free albumin solution.

[0515] In one embodiment, the albumin solution is suitable for intravenous injection.

[0516] In one embodiment, the albumin solution contains about 40 to about 200 g / L of albumin, or about 40 to about 50 g / L of albumin. In one embodiment, the albumin solution contains about 40 to about 50 g / L of albumin (e.g., human albumin). In one embodiment, the albumin solution contains about 200 g / L of albumin (e.g., human albumin). In one embodiment, the albumin solution contains 200 g / L of albumin (e.g., human albumin).

[0517] In one embodiment, the albumin solution comprises a protein composition that is 95%, particularly 96%, 97%, 98%, or 99% or more albumin protein (eg, human albumin protein).

[0518] In one embodiment, the albumin solution further contains sodium. In one embodiment, the albumin solution contains about 100 to about 200 mmol of sodium. In one embodiment, the albumin solution contains about 130 to about 160 mmol of sodium.

[0519] In one embodiment, the albumin solution additionally comprises potassium. In one embodiment, the albumin solution comprises up to 3 mmol of potassium. In one embodiment, the albumin solution further comprises up to 2 mmol of potassium.

[0520] In one embodiment, the albumin solution further comprises one or more stabilizers. In one embodiment, the stabilizers are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as acetyltryptophan, N-acetyl-L-tryptophan, or acetyl-L-tryptophan), and 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-acetyltryptophan, or N-acetyl-DL-tryptophan). In one embodiment, the solution comprises less than 0.1 mmol of each of the one or more stabilizers per gram of protein in the solution. In one embodiment, the solution comprises about 0.05 to about 0.1, e.g., about 0.064 to about 0.096 mmol of each stabilizer per gram of protein in the solution. In one embodiment, the solution comprises less than 0.1 mmol of total stabilizers per gram of protein in the solution. In one embodiment, the solution comprises about 0.05 to about 0.1, eg, about 0.064 to about 0.096 mmol of total stabilizer per gram of protein in the solution.

[0521] In one embodiment, the albumin solution is comprised of a protein composition that is 95% or more albumin protein, sodium, potassium, and a stabilizer selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(11H-indol-3-yl)propanoic acid (also referred to as acetyltryptophan, N-acetyl-L-tryptophan, or acetyl-L-tryptophan), and 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-acetyltryptophan, or N-acetyl-DL-tryptophan).

[0522] In one embodiment, the cryopreservation composition comprises about 10% v / v to about 50% v / v of an albumin solution, eg, an albumin solution described herein. In one embodiment, the cryopreserved composition is about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, or about 20% to about 25% , about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 50%, about 30% to about 45%, about 30% to about 45%, about 30% to about 45%, about 30% to about 45%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, about 40% to about 45%, or about 45% to about 50% v / v of the albumin solution described herein. In one embodiment, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v / v of an albumin solution described herein. In one embodiment, the cryopreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v / v of an albumin solution described herein.

[0523] In one embodiment, the cryopreservation composition contains about 20 to about 100 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreservation composition contains about 20 to about 100, about 20 to about 90, about 20 to about 80, about 20 to about 70, about 20 to about 60, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, The albumin concentration may be about 40 to about 60, about 40 to about 50, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about 100, about 60 to about 90, about 60 to about 80, about 60 to about 70, about 70 to about 100, about 70 to about 90, about 70 to about 80, about 80 to about 100, about 80 to about 90, or about 90 to about 100 g / L of albumin (e.g., human albumin).

[0524] In one embodiment, the cryopreservation composition comprises 20 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreservation composition comprises 40 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreservation composition comprises 70 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreservation composition comprises 100 g / L of albumin (e.g., human albumin).

[0525] In one embodiment, the cryopreserved composition comprises about 20 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreserved composition comprises about 40 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreserved composition comprises about 70 g / L of albumin (e.g., human albumin). In one embodiment, the cryopreserved composition comprises about 100 g / L of albumin (e.g., human albumin).

[0526] In one embodiment, the cryopreservation composition further comprises a stabilizer, e.g., an albumin stabilizer. In one embodiment, the stabilizer is selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(11H-indol-3-yl)propanoic acid (also referred to as acetyltryptophan, N-acetyl-L-tryptophan, or acetyl-L-tryptophan), and 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to as N-acetyltryptophan, DL-acetyltryptophan, or N-acetyl-DL-tryptophan). In one embodiment, the cryopreservation composition comprises less than 0.1 mmol of each of one or more stabilizers per gram of protein in the composition, e.g., per gram of albumin protein. In one embodiment, the cryopreservation composition comprises about 0.05 to about 0.1 mmol, e.g., about 0.064 to about 0.096 mmol, of each stabilizer per gram of protein, e.g., per gram of albumin protein in the composition. In one embodiment, the cryopreservation composition comprises less than 0.1 millimoles of total stabilizer per gram of protein, e.g., per gram of albumin protein in the cryopreservation composition. In one embodiment, the cryopreservation composition comprises about 0.05 to about 0.1, e.g., about 0.064 to about 0.096 mmol of total stabilizer per gram of protein, e.g., per gram of albumin protein in the cryopreservation composition.

[0527] Dextran In one embodiment, the cryopreservation composition comprises dextran or a derivative thereof. Dextran is a dextran having approximately 95% α-D-(1-6) linkages (CH 10 O5) n Dextran is an anhydroglucose polymer composed of dextran copolymers (denoted by ). Dextran fractions are available in molecular weights ranging from about 1,000 Da to about 2,000,000 Da. They are designated by a number (Dextran X), such as Dextran 1, Dextran 10, Dextran 40, and Dextran 70, where X is the average molecular weight divided by 1,000 Da. For example, the average molecular weight of Dextran 40 is about 40,000 Da.

[0528] In one embodiment, the average molecular weight of the dextran is between about 1,000 Da and about 2,000,000 Da or is about 2,000,000 Da. In one embodiment, the average molecular weight of the dextran is about 40,000 Da or is about 40,000 Da. In one embodiment, the average molecular weight of the dextran is about 70,000 Da or is about 70,000 Da.

[0529] In one embodiment, the dextran is selected from the group consisting of dextran 40, dextran 70, and combinations thereof. In one embodiment, the dextran is dextran 40.

[0530] In one embodiment, the dextran (e.g., Dextran 40) is provided as a solution, also referred to herein as a dextran solution or a Dextran 40 solution. Thus, in one embodiment, the composition comprises a dextran solution (e.g., a Dextran 40 solution).

[0531] In one embodiment, the dextran solution is suitable for intravenous injection.

[0532] In one embodiment, the dextran solution contains about 5% to about 50% w / w dextran (e.g., dextran 40). In one embodiment, the dextran solution contains about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, or about 10% to about 45% , about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 30%, about 15% to about 25%, About 15% to about 20%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 50%, About 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, about 40% to about 45%, or about 45% to about 50% comprises about 50% w / w dextran, e.g., Dextran 40. In one embodiment, the dextran solution comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50% w / w dextran, e.g., Dextran 40. In one embodiment, the dextran solution comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% w / w dextran, e.g., dextran 40.

[0533] In one embodiment, the dextran solution contains about 25 g / L to about 200 g / L of dextran (e.g., dextran 40). In one embodiment, the dextran solution contains about 35 to about 200, about 25 to about 175, about 25 to about 150, about 25 to about 125, about 25 to about 100, about 25 to about 75, about 25 to about 50, about 50 to about 200, about 50 to about 175, about 50 to about 150, about 50 to about 125, about 50 to about 100, about 50 to about 75, about 75 to about 200, about 75 to about 175, about 75 to about 1 The dextran solution contains 50, about 75 to about 125, about 75 to about 100, about 100 to about 200, about 100 to about 175, about 100 to about 150, about 100 to about 150, about 100 to about 125, about 125 to about 200, about 125 to about 175, about 125 to about 150, about 150 to about 200, about 150 to about 175, or about 175 to about 200 g / L of dextran (e.g., dextran 40). In one embodiment, the dextran solution contains 25, 50, 75, 100, 125, 150, 175, or 200 g / L of dextran (e.g., dextran 40). In one embodiment, the dextran solution contains 100 g / L of dextran (e.g., dextran 40). In one embodiment, the dextran solution comprises about 25, about 50, about 75, about 100, about 125, about 150, about 175, or about 200 g / L of dextran (e.g., Dextran 40). In one embodiment, the dextran solution comprises about 100 g / L of dextran (e.g., Dextran 40).

[0534] In one embodiment, the dextran solution additionally comprises glucose (also known as dextrose). In one embodiment, the dextran solution comprises about 10 g / L to about 100 g / L of glucose. In one embodiment, the dextran solution may be about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, about 20 to about 100, about 20 to about 90, about 20 to about 90, about 20 to about 80, about 20 to about 70, about 20 to about 60, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 60, about 30 to about 70, about 30 to about 60, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 80, about 30 to about 90, about 30 to about 60, about 30 to about 80, about 30 to about 90, about 30 to about 60, about 30 to about 90, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 9 ...90, about 30 to about 80, about 30 to about The glucose concentration ranges from 0 to about 50, from about 30 to about 40, from about 40 to about 100, from about 40 to about 90, from about 40 to about 80, from about 40 to about 70, from about 40 to about 60, from about 40 to about 50, from about 50 to about 100, from about 50 to about 90, from about 50 to about 80, from about 50 to about 70, from about 50 to about 60, from about 60 to about 100, from about 60 to about 90, from about 60 to about 80, from about 60 to about 70, from about 70 to about 100, from about 70 to about 90, from about 70 to about 80, from about 80 to about 90, from about 80 to about 100, from about 80 to about 90, from about 90 to about 100 g / L. In one embodiment, the dextran solution contains 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g / L of glucose. In one embodiment, the dextran solution contains 50 g / L of glucose. In one embodiment, the dextran solution contains about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 g / L of glucose. In one embodiment, the dextran solution contains 50 g / L of glucose.

[0535] In one embodiment, the dextran solution is composed of dextran (eg, dextran 40) and glucose dissolved in water.

[0536] In one embodiment, the cryopreservation composition comprises about 10% v / v to about 50% v / v of the dextran solution described herein. In one embodiment, the cryopreservation composition comprises about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 50%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 50%, about 40% to about 45%, or about 45% to about 50% v / v dextran solutions, including, for example, the dextran solutions described herein. In one embodiment, the cryopreservation composition comprises a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v / v dextran solution, such as a dextran solution described herein. In one embodiment, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v / v dextran solution, such as a dextran solution described herein.

[0537] In one embodiment, the cryopreservation composition contains about 10 to about 50 g / L of dextran (e.g., dextran 40). In one embodiment, the cryopreservation composition contains about 10 to about 50, about 10 to about 45, about 10 to about 40, about 10 to about 35, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 15, about 15 to about 50, about 15 to about 45, about 15 to about 40, about 15 to about 35, about 15 to about 30, about 15 to about 25, about 15 to about 20, about 20 to about 50, about 20 to about 45, about 20 to about 45, about 20 to about 4 The cryopreservation composition comprises 0, about 20 to about 30, about 20 to about 25, about 25 to about 50, about 25 to about 45, about 25 to about 40, about 25 to about 35, about 25 to about 30, about 30 to about 50, about 30 to about 45, about 30 to about 40, or about 30 to about 35, about 35 to about 50, about 35 to about 45, about 35 to about 40, about 40 to about 50, about 40 to about 45, or about 45 to about 50 g / L of dextran, e.g., dextran 40. In one embodiment, the cryopreservation composition comprises 10, 15, 20, 25, 30, 30, 35, 40, 45, or 50 g / L of dextran (e.g., dextran 40). In one embodiment, the cryopreservation composition comprises about 10, about 15, about 20, about 25, about 30, about 30, about 35, about 40, about 45, or about 50 g / L of dextran (e.g., Dextran 40).

[0538] glucose In one embodiment, the cryopreservation composition contains glucose (D-glucose and / or L-glucose). In one embodiment, as described above, the cryopreservation composition contains a dextran solution containing glucose. In one embodiment, the cryopreservation composition contains a dextran solution that does not contain glucose. For example, in one embodiment, if the dextran solution does not contain glucose, glucose is added separately to the cryopreservation composition.

[0539] In one embodiment, the cryopreservation composition contains about 5 to about 25, about 5 to about 20, about 5 to about 15, about 5 to about 10, about 10 to about 25, about 10 to about 20, about 10 to about 15, about 15 to about 25, about 15 to about 20, or about 20 to about 25 g / L of glucose, or about 15 to about 25, about 15 to about 20, or about 20 to about 25 g / L of glucose. In one embodiment, the cryopreservation composition contains 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, or 25 g / L of glucose. In one embodiment, the cryopreservation composition contains 12.5 g / L of glucose. In one embodiment, the cryopreservation composition contains about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 g / L of glucose. In one embodiment, the cryopreservation composition comprises about 12.5 g / L of glucose.

[0540] In one embodiment, the cryopreservation composition comprises less than 2.75% w / v glucose. In one embodiment, the cryopreservation composition comprises less than 27.5 g / L glucose. In one embodiment, the cryopreservation composition comprises less than 2% w / v glucose. In one embodiment, the cryopreservation composition comprises less than 1.5% w / v glucose. In one embodiment, the cryopreservation composition comprises about 1.25% w / v or less glucose.

[0541] Dimethyl sulfoxide In one embodiment, the cryopreservation composition comprises dimethyl sulfoxide (DMSO, also known as methyl sulfoxide or methylsulfinylmethane).

[0542] In one embodiment, DMSO is provided as a solution, sometimes referred to herein as a DMSO solution. Thus, in one embodiment, the cryopreservation composition comprises a DMSO solution.

[0543] In one embodiment, the DMSO solution is suitable for intravenous injection.

[0544] In one embodiment, the DMSO solution comprises 1.1 g / mL DMSO. In one embodiment, the DMSO solution comprises about 1.1 g / mL DMSO.

[0545] In one embodiment, the cryopreservation composition comprises about 1% to about 10% v / v DMSO solution. In one embodiment, the cryopreservation composition comprises about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 8% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 6%, about 3% to about 5%, about Contains 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, about 5% to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, 8% to about 10%, about 8% to about 9%, and about 9% to about 10% v / v DMSO solution. In one embodiment, the cryopreservation composition comprises a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% v / v DMSO solution. In one embodiment, the cryopreservation composition comprises a 5% DMSO solution. In one embodiment, the cryopreservation composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% v / v DMSO solution. In one embodiment, the cryopreservation composition comprises about a 5% DMSO solution.

[0546] In one embodiment, the cryopreservation composition contains about 11 to about 110 g / L of DMSO. In one embodiment, the cryopreservation composition contains about 11 to about 110 g / L of DMSO. In one embodiment, the cryopreservation composition contains about 11 to about 110 g / L of DMSO. In one embodiment, the cryopreservation composition contains about 11 to about 110 g / L of DMSO. In one embodiment, the cryopreservation composition contains about 11 to about 9 ... , about 33 to about 66, about 33 to about 55, about 33 to about 44, about 44 to about 110, about 44 to about 99, about 44 to about 88, about 44 to about 77, about 44 to about 66, about 44 to about 55, about 55 to about 110, about 55 to about 99, about 55 to about 88, about 55 to about 77, about 55 to about 66, about 66 to about 110, about 66 to about 99, about 66 to about 88, about 66 to about 77, about 77 to about 119, about 77 to about 88, about 88 to about 110, about 88 to about 99, or about 99 to about 110 g / L of DMSO. In one embodiment, the cryopreservation composition comprises 11, 22, 33, 44, 55, 66, 77, 88, 99, or 110 g / L of DMSO. In one embodiment, the cryopreservation composition comprises 55 g / L DMSO. In one embodiment, the cryopreservation composition comprises about 11, about 22, about 33, about 44, about 55, about 66, about 77, about 88, about 99, or about 110 g / L DMSO. In one embodiment, the cryopreservation composition comprises about 55 g / L DMSO.

[0547] Buffer (buffer solution) In one embodiment, the cryopreserved composition comprises a buffer solution, for example, a buffer solution suitable for intravenous administration.

[0548] Buffer solutions include, but are not limited to, phosphate buffered saline (PBS), Ringer's solution, Tyrode's buffer, Hank's balanced salt solution, Earle's balanced salt solution, saline, and Tris. In one embodiment, the buffer solution is phosphate buffered saline (PBS).

[0549] Exemplary Cryopreservation Compositions In one embodiment, the cryopreservation composition comprises or consists of: 1) albumin (e.g., human albumin), 2) dextran (e.g., dextran 40), 3) DMSO, and 4) buffer. In one embodiment, the cryopreservation composition further comprises glucose. In one embodiment, the cryopreservation composition consists of 1) albumin (e.g., human albumin), 2) dextran (e.g., dextran 40), 3) glucose, 4) DMSO, and 5) buffer.

[0550] In one embodiment, the cryopreservation composition comprises: 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.

[0551] In one embodiment, the cryopreservation composition is composed of: 1) an albumin solution described herein, 2) a dextran solution described herein, 3) a DMSO solution described herein, and 4) a buffer solution.

[0552] In one embodiment, the cryopreservation composition does not include cell culture medium.

[0553] In one embodiment, the cryopreservation composition comprises about 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO.

[0554] In one embodiment, the cryopreservation composition comprises about 40 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO, and 0.5 mL / mL 100% PBS dissolved in water.

[0555] In one embodiment, the cryopreservation composition comprises about 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO.

[0556] In one embodiment, the cryopreservation composition comprises approximately 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, 55 mg / mL DMSO, and 0.54 mL / mL 100% PBS dissolved in water.

[0557] Exemplary cryopreservation compositions are summarized in Tables 3, 4, 5 and 6 below.

[0558] [Table 3]

[0559] [Table 4]

[0560] [Table 5]

[0561] [Table 6]

[0562] Cryopreservation method The cryopreservation compositions described herein can be used to cryopreserve cells, e.g., therapeutic cells, e.g., natural killer (NK) cells, e.g., NK cells described herein.

[0563] In one embodiment, the cell is an animal cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is an immune cell. In one embodiment, the immune cell is selected from among a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and combinations thereof.

[0564] In one embodiment, the immune cells are natural killer (NK) cells. In one embodiment, the natural killer cells are expanded and stimulated by the methods described herein. In one embodiment, the NK cells are CAR-NK cells, e.g., CAR-NK cells described herein.

[0565] In one embodiment, the process of cryopreserving cells includes mixing the cells with a cryopreservation composition described herein or components thereof to form a composition, e.g., a pharmaceutical composition, and freezing the mixture.

[0566] In one embodiment, the process of cryopreserving cells comprises mixing a composition comprising the cells with a cryopreservation composition or components thereof described herein to form a composition, e.g., a pharmaceutical composition, and freezing the mixture. In one embodiment, the composition comprising the cells comprises the cells and a buffer. Suitable buffers are described herein.

[0567] In one embodiment, cryopreservation of cells comprises mixing a composition comprising cells and a buffer (e.g., PBS) with a composition comprising albumin, dextran, and DMSO, and freezing the mixture, e.g., as described herein.

[0568] In one embodiment, cryopreservation of cells involves mixing a composition comprising cells and a buffer, such as PBS 1:1 with 40 mg / mL albumin (e.g., human albumin), 25 mg / mL dextran (e.g., dextran 40), 12.5 mg / mL glucose, and 55 mg / mL DMSO.

[0569] In one embodiment, a composition comprising cells and a buffer (e.g., PBS) contains approximately 2×10 7 ~about 2×10 9 In one embodiment, a composition comprising cells and a buffer (e.g., PBS) contains 2 x 10 cells / mL. 8In one embodiment, a composition comprising cells and a buffer (e.g., PBS) contains about 2 x 10 cells / mL. 8 Contains cells / mL.

[0570] In one embodiment, the process for cryopreserving cells includes mixing cells, a buffer (e.g., PBS), albumin (e.g., human albumin), dextran (e.g., dextran 40), and DMSO, and freezing the mixture.

[0571] In one embodiment, the mixture comprises about 1×10 7 ~Approx. 1×10 9 In one embodiment, the mixture contains 1 x 10 cells / mL. 8 In one embodiment, the mixture contains about 1 x 10 cells / mL. 8 Contains cells / mL.

[0572] Suitable ranges for albumin, dextran and DMSO are set forth above.

[0573] In one embodiment, the composition is frozen at -135°C or below.

[0574] In one embodiment, the composition is frozen at a controlled rate.

[0575] Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein.

[0576] In some cases, the dosage unit contains between 100 million and 1.5 billion cells, e.g., 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, or 1.5 billion cells.

[0577] Pharmaceutical compositions typically contain a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes substances suitable for administration of pharmaceuticals, such as saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc.

[0578] In one embodiment, the pharmaceutical composition comprises: a) the natural killer cells described in the present invention; and b) a cryopreserved composition.

[0579] Suitable cryopreservation compositions are described in the present invention.

[0580] In one embodiment, the composition is frozen, hi one embodiment, the composition is frozen for at least 3 months, e.g., at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0581] In one embodiment, at least 60% of the natural killer cells are viable after thawing, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.

[0582] In one embodiment, the pharmaceutical composition comprises: a) a cryopreserved composition described herein; and b) therapeutic cells.

[0583] In one embodiment, the therapeutic cells are animal cells. In one embodiment, the therapeutic cells are human cells. In one embodiment, the therapeutic cells are immune cells. In one embodiment, the immune cells are selected from among basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.

[0584] In one embodiment, the immune cells are natural killer (NK) cells. In one embodiment, natural killer cells, such as the CAR-NK described herein, are expanded and stimulated by the methods described herein.

[0585] In one embodiment, the pharmaceutical composition additionally comprises c) a buffer solution. Suitable buffer solutions, for example, buffer solutions suitable for cryopreservation compositions, are described herein.

[0586] In one embodiment, the pharmaceutical composition comprises about 1×10 7 ~Approx. 1×10 9 In one embodiment, the pharmaceutical composition comprises 1 x 10 cells / mL. 8 In one embodiment, the pharmaceutical composition comprises about 1 x 10 cells / mL. 8 Contains cells / mL.

[0587] In one embodiment, the pharmaceutical composition additionally comprises an antibody or antigen-binding fragment thereof, eg, an antibody described herein.

[0588] Pharmaceutical compositions are generally prepared to be compatible with their intended route of administration, including parenteral (e.g., intravenous, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0589] Methods for preparing suitable pharmaceutical compositions are known to those skilled in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, 21 sted., 2005; and references in Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and a tonicity adjusting ingredient such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0590] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders, or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable vehicles for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In most cases, it is preferable to include isotonic agents (e.g., sugars, polyalcohols such as mannitol, sorbitol, sodium chloride) in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0591] Sterile injectable solution can be prepared by mixing the active compound in the required amount with one or combination of the above-listed components in a suitable solvent, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile solvent that contains a basic dispersion medium and other necessary components from the above-listed components.For the preparation of sterile powder for sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying, which obtains the powder of active ingredient and the previously sterile-filtered solution that contains the additional desired components.

[0592] Treatment method The NK cells described herein, for example, the CAR-NK cells described herein, are used to treat cancer or other proliferative disorders.

[0593] Thus, the present invention also provides a method of treating a patient suffering from a disease, e.g., a disease associated with cancer, e.g., a HER2-positive cancer, comprising administering an NK cell described in the present invention, e.g., a CAR-NK cell described in the present invention.

[0594] The present invention also provides a method for preventing, reducing, and / or inhibiting the recurrence, growth, proliferation, migration, and / or metastasis of a cancer cell or population of cancer cells, comprising administering an NK cell described in the present invention, e.g., a CAR-NK cell described in the present invention.

[0595] The present invention also provides a method of enhancing, improving, and / or increasing response to anti-cancer therapy in a subject in need thereof, comprising administering an NK cell described in the present invention, e.g., a CAR-NK cell described herein.

[0596] Furthermore, the present invention provides a method for inducing the immune system in a subject in need thereof, comprising administering the NK cells described in the present invention, for example, the CAR-NK cells described in the present invention.

[0597] The methods described herein include methods for treating diseases associated with abnormal cell death or differentiation processes, e.g., cell proliferative or cell differentiative diseases, e.g., cancers, including solid tumors and hematological cancers. Generally, the methods involve administering a therapeutically effective amount of a therapeutic agent, as described herein, to a subject in need of or determined to be in need of such treatment. In one embodiment, the method involves administering a therapeutically effective amount of a therapeutic agent comprising NK cells, e.g., CAR-NK cells, as described herein.

[0598] As used herein, the terms "treatment" and "treating" refer to reversing, mitigating, slowing, or inhibiting the progression of a disease associated with abnormal cell death or differentiation processes. For example, treatment may result in a decrease in tumor size or growth rate. Administration of a therapeutically effective amount of a composition described herein for treating a disease associated with abnormal cell death or differentiation processes can result in a decrease in tumor size or growth rate, a decrease in the risk or frequency of recurrence, a delay in recurrence, a decrease in metastasis, an increase in survival, a decrease in morbidity and mortality, and the like. In one embodiment, a therapeutic agent can be administered after one or more symptoms have developed. In other examples, a therapeutic agent can be administered in the absence of symptoms. For example, a therapeutic agent can be administered to a susceptible individual (e.g., based on a history of the condition and / or genetic or other susceptibility factors) before symptoms develop. Treatment can also be continued after symptoms have resolved to prevent or delay recurrence.

[0599] As used herein, the term "inhibit" refers to inhibiting the growth, division, maturation or survival of cancer cells through cytotoxicity, nutrient deprivation or induction of cell death in relation to cancer and / or cancer cell proliferation, or inducing the death of cancer cells, either individually or collectively with other cancer cells.

[0600] As used herein, "delaying" a disease or disorder or one or more symptoms means delaying, inhibiting, slowing, retarding, stabilizing, and / or postponing the onset of the disease, disorder, or symptom. Such delay may occur for a variety of periods depending on the history of the disease and / or the subject being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can include prevention, in that the subject does not actually develop the disease, disorder, or its symptoms. For example, a method for "delaying" the onset of cancer is one that reduces the probability of developing the disease in a given period of time or reduces the severity of the disease in a given period of time compared to not using the method. Such comparisons can be based on clinical studies involving a statistically significant number of subjects.

[0601] As used herein, the terms "prevention" or "preventing" refer to a therapy that prevents the onset of a disease or disorder and prevents the clinical symptoms of the disease from appearing. Thus, "prevention" refers to administering a therapy (e.g., administering a therapeutic agent) to a subject before symptoms of the disease are detected and / or prior to a specific stage of the disease (e.g., administering a therapeutic agent to a subject with cancer that has not yet metastasized). A subject may be an individual at risk of developing a disease or disorder or at risk for disease progression (e.g., cancer metastasis). For example, an individual may have a mutation associated with the onset or progression of cancer. It should be understood that prevention does not completely prevent the onset of a disease or disorder. In some cases, prevention includes reducing the risk of developing a disease or disorder. Such a reduction in risk may not completely eliminate the risk of developing a disease or disorder.

[0602] "Increase" or "enhancement" (e.g., with respect to anti-cancer response, metastasis of cancer cells) means an increase of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 fold or more (e.g., 100, 500, 1000 fold) (including all integers and decimal points, e.g., 2.1, 2.2, 2.3, 2.4, etc.). It may also include an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of the amounts or levels described herein.

[0603] A "decrease" or "reduction" or "lesser" amount (e.g., in relation to tumor size, cancer cell proliferation or growth) can mean a decrease of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more (e.g., 1, 100, 500, 1000 fold) (including all integers and decimal points, e.g., 1.5, 1.6, 1.7, 1.8, etc.). It can also include a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of the amounts or levels described herein.

[0604] disease The methods and compositions disclosed herein can be used to target a variety of diseases, such as cell proliferative disorders. An advantage of the approaches of the present invention is that they target specific cells using allogeneic cells. Unlike previous treatments, such as chemotherapy and radiation therapy, the approaches and pharmaceutical compositions disclosed herein can specifically target cells exhibiting potentially harmful proliferative activity without the need for systemic drug or toxin administration, which indiscriminately affect proliferating cells.

[0605] Examples of cell proliferative and / or differentiative disorders include cancer (e.g., carcinoma, sarcoma), metastatic disease, or hematopoietic neoplastic disorders (e.g., leukemia). Metastatic tumors can arise from a variety of primary tumor types, including, but not limited to, prostate, colon, lung, breast, and liver.

[0606] As used herein, the terms "cancer," "hyperproliferative," and "tumor" refer to cells capable of autonomous growth, i.e., an abnormal stage or condition characterized by rapid cell proliferation. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., those that characterize or constitute a disease state, or non-pathological, i.e., those that deviate from normal but are not associated with a disease state. The terms are used to include all types of cancer growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. An example of non-pathological hyperproliferative cells is the proliferation of cells associated with wound healing.

[0607] As used herein, the term "cancer" or "neoplasm" includes malignant tumors of various organ systems, such as lung, breast, thyroid, lymphatic, gastrointestinal and genitourinary tract, and adenocarcinomas, including colorectal cancer, renal cell carcinoma, prostate and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.

[0608] As used herein, the term "carcinoma" is an art-recognized term and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory carcinoma, gastrointestinal carcinoma, genitourinary carcinoma, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine carcinoma, and melanoma. In one embodiment, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming in tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma originating from glandular tissue or in which the tumor cells form distinguishable glandular structures.

[0609] As used herein, the term "sarcoma" is an art-recognized term that refers to a malignant tumor of intermediate lobe origin.

[0610] Further examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorder" includes diseases occurring in hyperplastic / neoplastic cells of hematopoietic origin, e.g., myeloid, lymphoid, or erythroid lineages, or their precursor cells. Preferably, such diseases occur in poorly differentiated acute leukemias, such as erythroid leukemia and acute adenytoblastic leukemia. Further examples of myeloid diseases include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL) (including B-lineage ALL and T-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), giant granulocytic lymphoma (LGG), Hodgkin's disease, and Reed-Sternberg disease.

[0611] In one embodiment, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, cecal cancer, astrocytoma, typical teratoid rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid, cardiac tumor, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, bile duct carcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, craniopharyngioma, cutaneous T-cell lymphoma, or the like. melanoma, ductal carcinoma in situ, germinal tumor, endometrial cancer, epididymoma, esophageal cancer, aesthesioneurocytoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (e.g., intraocular melanoma or retinoblastoma), trumpet duct carcinoma, osteofibrous histiocytoma, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, cardiac tumor, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, renal (renal cell) carcinoma, Langerhans cell histiocytosis , laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, pleuropulmonary blastoma, organ bronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic carcinoma, metastatic squamous neck cancer, intermediate duct carcinoma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, fungal infections, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine Urinary tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, uterine and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g., childhood rhabdomyosarcoma, childhood vascular tumor, Ewing's sarcoma, Kaposi's sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the head and neck, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer,Selected from the group consisting of nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, thymoma and thymic carcinoma, thyroid carcinoma, bronchial tumor, metastatic cell carcinoma of the renal pelvis and ureter, urethral carcinoma, uterine carcinoma, uterine sarcoma, vaginal carcinoma, vascular tumor, vulvar carcinoma and Wilms' tumor.

[0612] In one embodiment, the cancer is a solid tumor.

[0613] In one embodiment, the cancer is metastatic.

[0614] In one embodiment, the cancer is a HER2-positive cancer.

[0615] In one embodiment, the HER2-positive cancer is selected from the group consisting of bladder cancer, breast adenocarcinoma, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, squamous cervical cancer, gastric adenocarcinoma, cholangiocarcinoma, ovarian cancer, papillary renal cell carcinoma, and combinations thereof.

[0616] In one embodiment, the HER2-positive cancer is selected from the group consisting of breast cancer, gastric cancer, and ovarian cancer.

[0617] In one embodiment, the HER2-positive cancer is breast cancer. In one embodiment, the HER2-positive cancer is gastric cancer. In one embodiment, the HER2-positive cancer is ovarian cancer.

[0618] patient Suitable patients for the compositions and methods of the present invention include those suffering from, diagnosed with, or suspected of having a cell proliferative and / or differentiative disorder (e.g., cancer). Patients amenable to the techniques disclosed herein generally respond better to the methods and compositions herein, in part because the pharmaceutical compositions are allogeneic and do not target generally proliferating cells, but rather target cells identified by the antigen-binding domain. As a result, there are fewer off-target effects, and patients are more likely to complete treatment without harm from target-off effects.

[0619] In one embodiment, the therapeutic methods provided herein can be used to treat a subject (e.g., a human, monkey, dog, cat, mouse) diagnosed with or suspected of having a cell proliferative and / or differentiative disorder (e.g., cancer). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

[0620] As used herein, a subject refers to a mammal, including, for example, a human.

[0621] In one embodiment, the mammal is selected from the group consisting of an armadillo, donkey, bat, bear, beaver, cat, chimpanzee, cow, coyote, deer, dog, dolphin, elephant, fox, panda, gibbon, giraffe, goat, gopher, hedgehog, hippopotamus, horse, humpback whale, jaguar, kangaroo, koala, leopard, deer, llama, lynx, mole, monkey, mouse, narwhal, orangutan, killer whale, otter, cow, pig, polar bear, hedgehog, puma, rabbit, raccoon dog, rat, rhinoceros, sheep, squirrel, tiger, sea elephant, weasel, wolf, zebra, goat, horse, and combinations thereof.

[0622] In one embodiment, the mammal is a human.

[0623] The subject, e.g., a human subject, may be a child between the ages of 0 and about 14. The subject may be an adolescent, e.g., between the ages of about 15 and about 24. The subject may be an adult, e.g., between the ages of about 25 and about 64. The subject may be an elderly person, e.g., 65 years of age or older.

[0624] In one embodiment, the subject may be a human exhibiting one or more symptoms associated with a cell proliferative and / or differentiative disorder, e.g., cancer, e.g., a tumor. The therapeutic methods provided herein can be used to treat various stages of cancer. For example, cancer stages include, but are not limited to, early stage, advanced, locally advanced, in remission, refractory, post-remission relapse, and progressive. In one embodiment, the subject is in an early stage of cancer. In one embodiment, the subject is in an advanced stage of cancer. In various examples, the subject has stage 1, stage 2, stage 3, or stage 4 cancer. The therapeutic methods described herein can promote tumor reduction or regression, reduce or inhibit tumor growth or cancer cell proliferation, and / or induce, increase, or promote tumor cell death. In one embodiment, the subject is in cancer remission. The therapeutic methods described herein can prevent or delay cancer metastasis or recurrence.

[0625] In one embodiment, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, 2, 3, or 4 at the time of treatment. In one embodiment, if the subject has an ECOG performance status of 0-4, 0-3, 0-2, 0-1, 1-4, 1-3, 1-2, 2-4, 2-3, or 3-4 at the time of treatment, the ECOG performance status at the time of treatment is 0-4, 0-2, 0-3, or 3-4.

[0626] In one embodiment, the subject has been diagnosed with, or has not been diagnosed with, a cell proliferative and / or differentiative disorder (eg, cancer) but is at risk or otherwise predisposed to developing such a disorder.

[0627] As used herein, an "at-risk" individual refers to an individual who is at risk of developing a disease being treated (e.g., a cell proliferative and / or differentiative disorder such as cancer). Generally, an "at-risk" subject may or may not have detectable disease, and may or may not exhibit detectable disease prior to the treatment methods described herein. "At-risk" refers to an individual having one or more so-called risk factors, which are measurable parameters associated with the disease or disease onset and known to those skilled in the art. For example, an at-risk subject may have one or more risk factors, which are measurable parameters that correlate with the onset of cancer. Subjects with one or more such risk factors have a higher probability of developing cancer than individuals without such risk factors. Generally, risk factors can include, for example, age, sex, race, diet, history of previous disease, presence of precursor diseases, genetic factors, and environmental exposures. In one embodiment, a subject at risk of developing cancer includes, for example, a subject who has a relative who has experienced the disease or whose risk has been determined by analysis of genetic or biochemical markers.

[0628] The subject may also be undergoing one or more standard therapies, such as chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof. Accordingly, the one or more kinase inhibitors may be administered before, during, or after the administration of chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof.

[0629] In certain examples, the subject can be (i) substantially refractory to at least one chemotherapy treatment, (II) relapsed after chemotherapy treatment, or both (i) and (II). In one embodiment, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapy).

[0630] In one embodiment, the subject has a cancer that expresses HER2.

[0631] In one embodiment, the patient is or has been diagnosed with HER2-positive cancer.

[0632] In one embodiment, the patient is or has been diagnosed with a HER2-positive cancer by immunohistochemical staining of a cancer biopsy or surgical sample. In one embodiment, the patient is or has been diagnosed with a HER2-positive cancer by fluorescent in situ hybridization of a cancer biopsy or surgical sample.

[0633] In one embodiment, the patient is or has been diagnosed with HER2-positive cancer according to ASCO® guidelines (e.g., the 2018 ASCO® guidelines), and in this regard, reference may be made to Wolff et al., "Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer," Arch Pathol Lab Med 142:1364-82 (2018), the entire contents of which are incorporated herein by reference. In one embodiment, the patient is or has been diagnosed with HER2+ cancer according to ASCO® guidelines (e.g., the 2018 ASCO® guidelines), for example, as described in Wolff et al., "Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer," Arch Pathol Lab Med 142:1364-82 (2018), with an IHC score of 2+. In one embodiment, the patient is or has been diagnosed with HER2-positive cancer with an IHC score of 2+ within 12 months (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month) of treatment.

[0634] In one embodiment, the patient has a cancer that is not HER2-poisoned but does exhibit some degree of HER2 expression. In one embodiment, the patient is or has been diagnosed with a HER2-low-expressing cancer. In one embodiment, a HER2-low-expressing cancer is a cancer that has a HER2 expression classification of 1+ or 2+ according to, for example, the 2018 ASCO® guidelines (e.g., as described in Wolff et al., "Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer," Arch Pathol Lab Med 142:1364-82 (2018)), but lacks HER2 amplification. In one embodiment, a HER2-low-expressing cancer possesses HER2 gain. In one embodiment, a HER2-low-expressing cancer does not possess HER2 gain.

[0635] In one embodiment, the patient is or has been diagnosed with HER2-positive cancer by genetic analysis (eg, identifying HER2-mutated cancers such as somatic mutations in the HER2 (ERBB2) gene).

[0636] In one embodiment, the patient has a cancer comprising one or more mutations set forth in Table 7, an insertion or deletion polymorphism in the HER2 gene, a copy number mutation in the HER2 gene, a methylation mutation in the HER2 gene, or a combination thereof. In one embodiment, the mutation is a HER2 activating mutation.

[0637] In one embodiment, the patient has a chromosomal translocation associated with the cancer (e.g., a HER2+ cancer). In one embodiment, the patient has a fusion gene associated with the cancer (e.g., a HER+ cancer).

[0638] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8]

[0639] In one embodiment, the HER2-positive cancer is HER2-positive breast cancer, gastric cancer, gastroesophageal (GEJ) cancer, or lung cancer.

[0640] In one embodiment, the breast cancer is in situ (non-invasive). In one embodiment, the breast cancer is invasive. In one embodiment, the breast cancer is metastatic.

[0641] In one embodiment, the breast cancer is a carcinoma (e.g., an adenocarcinoma). In one embodiment, the breast cancer is a ductal carcinoma. In one embodiment, the breast cancer is an invasive lobular carcinoma. In one embodiment, the breast cancer is inflammatory breast cancer. In one embodiment, the breast cancer is Paget's disease of the breast. In one embodiment, the breast cancer is a phyllodes tumor.

[0642] In one embodiment, the gastric cancer is an adenocarcinoma. In one embodiment, the gastric cancer is an intestinal adenocarcinoma. In one embodiment, the gastric cancer is a diffuse adenocarcinoma. In one embodiment, the gastric cancer is a lymphoma. In one embodiment, the gastric cancer is a mucosa-associated lymphoid tissue lymphoma. In one embodiment, the gastric cancer is a gastrointestinal stromal tumor. In one embodiment, the gastric cancer is a gastric carcinoid tumor. In one embodiment, the gastric cancer is a type 1, type 2, or type 3 ECL cell breast carcinoma. In one embodiment, the gastric cancer is hereditary diffuse gastric cancer.

[0643] In one embodiment, the GEJ cancer is a GEJ adenocarcinoma.

[0644] In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC, e.g., metastatic NSCLC). In one embodiment, the patient has non-small cell lung cancer with a HER2 activating mutation.

[0645] In one embodiment, the patient is an individual who is refractory to or has relapsed after treatment, such as trastuzumab or a biosimilar thereof.

[0646] In one embodiment, the patient has previously received one or more systemic therapies and is refractory or has relapsed, hi one embodiment, the patient has previously received two or more systemic therapies and is refractory or has relapsed.

[0647] In one embodiment, the patient has been diagnosed with IHC3+ or IHC2+ / ISH+ cancer according to ASCO® guidelines (e.g., Wolff et al., “Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer,” Arch Pathol Lab Med 142:1364-82 (2018)), and has previously been treated with a HER2-targeted therapeutic agent and is in a refractory or relapsed state.

[0648] In one embodiment, the patient is diagnosed with a HER2-expressing cancer (e.g., breast cancer) that is IHC1+ or IHC2+ / ISH- according to ASCO® guidelines (e.g., Wolff et al., “Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer,” Arch Pathol Lab Med 142:1364-82 (2018)), and the cancer is unresectable, metastatic, refractory to previous treatment, or has recurred.

[0649] In one embodiment, the patient is refractory or has relapsed after treatment with pertuzumab (or an FDA-approved biosimilar), trastuzumab (or an FDA-approved biosimilar), and docetaxel (or a pharmaceutically acceptable salt thereof). In one embodiment, pertuzumab (or its FDA-approved biosimilar) is administered at 840 mg intravenous injection on day 1, followed by 420 mg intravenous injection. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at 7 mg / kg intravenous injection on day 1, followed by 6 mg / kg intravenous injection on day 1 every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-OSC injection are administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration. In one embodiment, docetaxel (or a pharmaceutically acceptable salt thereof) is administered at 75-100 mg / m 2 cyclically once every 21 days. 2 It is given IV on day 1.

[0650] In one embodiment, the patient has refractory or relapsed disease after treatment with pertuzumab (or an FDA-approved biosimilar), trastuzumab (or an FDA-approved biosimilar), and paclitaxel (or a pharmaceutically acceptable salt thereof). In one embodiment, pertuzumab (or its FDA-approved biosimilar) is administered at 840 mg intravenous injection on day 1, followed by 420 mg intravenous injection every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at 4 mg / kg intravenous injection on day 1, followed by 2 mg / kg intravenous weekly, or at 8 mg / kg intravenous injection on day 1, followed by 6 mg / kg intravenous injection on day 1 every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-OSC injection are administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration. In one embodiment, paclitaxel (or a pharmaceutically acceptable salt thereof) is administered at 80 mg / m once weekly. 2 Intravenous injection or 175 mg / m cyclically every 21 days 2 It is administered.

[0651] In one embodiment, the patient is refractory or has relapsed after treatment with tucatinib (or a pharmaceutically acceptable salt thereof), trastuzumab (or an FDA-approved biosimilar), and capecitabine (or a pharmaceutically acceptable salt thereof). In one embodiment, tucatinib (or its FDA-approved biosimilar) is administered orally at 300 mg twice daily on days 1-21. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered as an 8 mg / kg intravenous injection on day 1, followed by 6 mg / kg intravenous injection on day 1 every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-OSC injection are administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration. In one embodiment, capecitabine (or its FDA-approved biosimilar) is administered at 1000 mg / m twice daily on days 1-14. 2In one embodiment, the administration of tucatinib (or an FDA-approved biosimilar thereof), trastuzumab (or an FDA-approved biosimilar thereof), and capecitabine (or a pharmaceutically acceptable salt thereof) is cyclical every 21 days.

[0652] In one embodiment, the patient is refractory or has relapsed after treatment with ado-trastuzumab emtansine (T-DM1) (or an FDA-approved biosimilar thereof). In one embodiment, ado-trastuzumab emtansine (T-DM1) (or an FDA-approved biosimilar thereof) is administered at 3.6 mg / kg IV on day 1 of a rotation every 21 days.

[0653] In one embodiment, the patient has been refractory to or relapsed after treatment with fam-trastuzumab deruxtecan-nxki (or an FDA-approved biosimilar). In one embodiment, fam-trastuzumab deruxtecan-nxki is administered at 5.4 mg / kg IV on day 1, cycling every 21 days.

[0654] In one embodiment, the patient is refractory or has relapsed after treatment with paclitaxel / carboplatin (or a pharmaceutically acceptable salt thereof) and trastuzumab (or an FDA-approved biosimilar thereof). In one embodiment, carboplatin / paclitaxel (or a pharmaceutically acceptable salt thereof) is administered every 21 days at a dose of 175 mg / m on day 1 of carboplatin and 175 mg / m on day 1 of paclitaxel. 2In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at 4 mg / kg intravenously on day 1 followed by 2 mg / kg intravenously every week, or at 8 mg / kg intravenously on day 1 followed by 6 mg / kg intravenously every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-OSC injection is administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration.

[0655] In one embodiment, the patient is refractory or has relapsed after paclitaxel / carboplatin (or a pharmaceutically acceptable salt thereof) and trastuzumab (or an FDA-approved biosimilar thereof). In one embodiment, carboplatin / paclitaxel (or a pharmaceutically acceptable salt thereof) is administered at an AUC2 IV carboplatin and 80 mg / m on days 1, 8, and 15 cycled every 28 days. 2 In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at 4 mg / kg intravenously on day 1 followed by 2 mg / kg intravenously every week, or at 8 mg / kg intravenously on day 1 followed by 6 mg / kg intravenously every 21 days. In one embodiment, trastuzumab is administered subcutaneously in the form of trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-OSC injectable.

[0656] In one embodiment, the patient is refractory or has relapsed after treatment with trastuzumab (or an FDA-approved biosimilar thereof) and paclitaxel (or a pharmaceutically acceptable salt thereof). In one embodiment, paclitaxel (or a pharmaceutically acceptable salt thereof) is administered at 175 mg / m once daily cyclically every 21 days. 2 Intravenous or 80–90 mg / m once daily every week 2It is administered intravenously. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at a dose of 4 mg / kg intravenously on day 1 followed by 2 mg / kg intravenously every week, or at a dose of 8 mg / kg intravenously on day 1 followed by 6 mg / kg intravenously every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-osc injection is administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration.

[0657] In one embodiment, the patient is refractory or has relapsed after treatment with trastuzumab (or an FDA-approved biosimilar thereof) and docetaxel (or a pharmaceutically acceptable salt thereof). In one embodiment, docetaxel (or a pharmaceutically acceptable salt thereof) is administered at 80-100 mg / m cyclically every 21 days. 2 IV on day 1 and 35 mg / m on days 1, 8, and 15 weekly 2 IV is administered on day 1. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at a dose of 4 mg / kg intravenous on day 1 followed by 2 mg / kg intravenous every week, or at a dose of 8 mg / kg intravenous on day 1 followed by 6 mg / kg intravenous every 21 days. In one embodiment, trastuzumab (or an FDA-approved biosimilar) and hyaluronidase-OSC injection is administered with trastuzumab (or an FDA-approved biosimilar) for subcutaneous administration.

[0658] In one embodiment, the patient is refractory or has relapsed after treatment with trastuzumab (or an FDA-approved biosimilar thereof) and vinorelbine (or a pharmaceutically acceptable salt thereof). In one embodiment, vinorelbine (or a pharmaceutically acceptable salt thereof) is administered at a dose of 25 mg / m once weekly. 2 20–35 mg / m IV or cyclically on days 1 and 8 every 21 days 2 Intravenous or cyclically every 28 days, 25–30 mg / m on days 1, 8, and 15 2It is administered intravenously. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at 4 mg / kg intravenously on day 1 followed by 2 mg / kg intravenously every week, or at 8 mg / kg intravenously on day 1 followed by 6 mg / kg intravenously every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-OSC injection is administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration.

[0659] In one embodiment, the patient is refractory or has relapsed after treatment with trastuzumab (or an FDA-approved biosimilar thereof) and capecitabine (or a pharmaceutically acceptable salt thereof). In one embodiment, capecitabine (or a pharmaceutically acceptable salt thereof) is administered at 1000-1250 mg / m twice daily on 1-14 day cycles every 21 days. 2 It is administered PO. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at a dose of 4 mg / kg intravenous injection on day 1 followed by 2 mg / kg intravenous injection every week, or at a dose of 8 mg / kg intravenous injection on day 1 followed by 6 mg / kg intravenous injection every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-osc injection is administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration.

[0660] In one embodiment, the patient is refractory or has relapsed after treatment with lapatinib (or a pharmaceutically acceptable salt thereof) and capecitabine (or a pharmaceutically acceptable salt thereof). In one embodiment, lapatinib (or a pharmaceutically acceptable salt thereof) is administered at a dose of 1250 mg / m daily on days 1-21. 2 In one embodiment, capecitabine (or a pharmaceutically acceptable salt thereof) is administered at 1000 mg / m PO twice daily on days 1-14. 2 It is administered PO and given periodically every 21 days.

[0661] In one embodiment, the patient is refractory or has relapsed after treatment with trastuzumab (or an FDA-approved biosimilar thereof) and lapatinib (or a pharmaceutically acceptable salt thereof). In one embodiment, lapatinib (or a pharmaceutically acceptable salt thereof) is administered at a dose of 1000 mg / m daily. 2 It is administered PO. In one embodiment, trastuzumab (or its FDA-approved biosimilar) is administered at a dose of 4 mg / kg intravenous injection on day 1 followed by 2 mg / kg intravenous injection every week, or at a dose of 8 mg / kg intravenous injection on day 1 followed by 6 mg / kg intravenous injection every 21 days. In one embodiment, trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-osc injection is administered with trastuzumab (or its FDA-approved biosimilar) for subcutaneous administration.

[0662] In one embodiment, the patient is refractory or has relapsed after treatment with neratinib and capecitabine (or a pharmaceutically acceptable salt thereof). In one embodiment, neratinib is administered at 240 mg / m daily on days 1-21. 2 In one embodiment, capecitabine is administered at 750 mg / m PO twice daily on days 1-14. 2 It is given cyclically every 21 days PO.

[0663] In one embodiment, the patient has an oxygen saturation of at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured by pulse oximetry within 1 minute, 5 minutes, 10, 15, 20, 30, 45, 60, or 90 minutes, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 18 hours, or within 1 day, 1 week, or 1 month after treatment.

[0664] In one embodiment, the patient has a left ventricular ejection fraction (LVEF) of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% or greater measured within 1 day, 1 week or 1 month of treatment.

[0665] In one embodiment, the patient has an absolute neutrophil count (ANC) of at least 1000 / mm measured within 1 day, 1 week, or 1 month after treatment. 3 (1.0×10 9 / L), 1500 / mm 3 , 2000 / mm 3 , 2500 / mm 3 , 3000 / mm 3 , 3500 / mm 3 , 4000 / mm 3 , 4500 / mm 3 , 5000 / mm 3 , 5500 / mm 3 or 6000 / mm 3 In one embodiment, the patient has an ANC of 1000 / mm or greater measured within 1 day, 1 week, or 1 month after treatment. 3 ~6000 / mm 3 , 1000 / mm 3 ~5500 / mm 3 , 1000 / mm 3 ~5000 / mm 3 , 1000 / mm 3 ~4500 / mm 3 , 1000 / mm 3 ~4000 / mm 3 , 1000 / mm 3 ~3500 / mm 3 , 1000 / mm 3 ~3000 / mm 3 , 1000 / mm 3 ~2500 / mm 3 , 1000 / mm 3 ~2000 / mm 3 , 1000 / mm 3 ~1500 / mm 3 , 1500 / mm 3 ~6000 / mm 3 , 1500 / mm 3 ~5500 / mm 3 , 1500 / mm 3 ~5000 / mm 3 , 1500 / mm 3 ~4500 / mm 3 , 1500 / mm 3 ~4000 / mm 3 , 1500 / mm 3~3500 / mm 3 、1500 / mm 3 ~3000 / mm 3 、1500 / mm 3 ~2500 / mm 3 、1500 / mm 3 ~2000 / mm 3 、2000 / mm 3 ~6000 / mm 3 、1500 / mm 3 ~8000 / mm 3 、2000 / mm 3 ~5500 / mm 3 、2000 / mm 3 ~5000 / mm 3 、2000 / mm 3 ~4500 / mm 3 、2000 / mm 3 ~4000 / mm 3 、2000 / mm 3 ~3500 / mm 3 、2000 / mm 3 ~3000 / mm 3 、2000 / mm 3 ~2500 / mm 3 、2500 / mm 3 ~6000 / mm 3 、2000 / mm 3 ~6000 / mm 3 、2500 / mm 3 ~5500 / mm 3 、2500 / mm 3 ~5000 / mm 3 、2500 / mm 3 ~4500 / mm 3 、2500 / mm 3 ~4000 / mm 3 、2500 / mm 3 ~3500 / mm 3 、2500 / mm 3 ~3000 / mm 3 、3000 / mm 3 ~6000 / mm 3 、3000 / mm 3 ~5500 / mm 3 、3000 / mm 3 ~5000 / mm 3 、3000 / mm3 ~4500 / mm 3 , 3000 / mm 3 ~4000 / mm 3 , 3000 / mm 3 ~3500 / mm 3 , 3500 / mm 3 ~6000 / mm 3 , 3500 / mm 3 ~5500 / mm 3 , 3500 / mm 3 ~5000 / mm 3 , 3500 / mm 3 ~4500 / mm 3 , 3500 / mm 3 ~4000 / mm 3 , 4000 / mm 3 ~6000 / mm 3 , 4000 / mm 3 ~5500 / mm 3 , 4000 / mm 3 ~5000 / mm 3 , 4000 / mm 3 ~4500 / mm 3 , 4500 / mm 3 ~6000 / mm 3 , 3500 / mm 3 ~6000 / mm 3 , 4500 / mm 3 ~5500 / mm 3 , 4500 / mm 3 ~5000 / mm 3 , 5000 / mm 3 ~6000 / mm 3 , 5000 / mm 3 ~5500 / mm 3 or 5500 / mm 3 ~6000 / mm 3 This is the case.

[0666] In one embodiment, the patient's platelet count is at least 75,000 / mm3 as measured within one day, one week, or one month after treatment. 3 (75×10 9 / L), 100000 / mm 3 , 125000 / mm 3 , 150000 / mm 3, 175000 / mm 3 , 200000 / mm 3 , 225000 / mm 3 , 250000 / mm 3 , 275000 / mm 3 , 300000 / mm 3 , 325000 / mm 3 , 350000 / mm 3 , 375000 / mm 3 or 400000 / mm 3 In one embodiment, the patient's platelet count is at least 75,000 / mm3 as measured within 1 day, 1 week, or 1 month after treatment. 3 ~400000 / mm 3 , 75000 / mm 3 ~375000 / mm 3 , 75000 / mm 3 ~350000 / mm 3 , 75000 / mm 3 ~300000 / mm 3 , 75000 / mm 3 ~275000 / mm 3 , 75000 / mm 3 ~250000 / mm 3 , 75000 / mm 3 ~225000 / mm 3 , 75000 / mm 3 ~200000 / mm 3 , 75000 / mm 3 ~175000 / mm 3 , 75000 / mm 3 ~150000 / mm 3 , 75000 / mm 3 ~125000 / mm 3 , 75000 / mm 3 ~100000 / mm 3 , 100000 / mm 3 ~400000 / mm 3 , 100000 / mm 3 ~375000 / mm 3 , 100000 / mm 3 ~350000 / mm 3 , 100000 / mm 3 ~325000 / mm 3、100000 / mm 3 ~300000 / mm 3 、100000 / mm 3 ~275000 / mm 3 、100000 / mm 3 ~250000 / mm 3 、100000 / mm 3 ~225000 / mm 3 、100000 / mm 3 ~200000 / mm 3 、100000 / mm 3 ~175000 / mm 3 、100000 / mm 3 ~150000 / mm 3 、100000 / mm 3 ~125000 / mm 3 、125000 / mm 3 ~400000 / mm 3 、125000 / mm 3 ~375000 / mm 3 、125000 / mm 3 ~350000 / mm 3 、125000 / mm 3 ~325000 / mm 3 、125000 / mm 3 ~300000 / mm 3 、125000 / mm 3 ~275000 / mm 3 、125000 / mm 3 ~25000 / mm 3 、125000 / mm 3 ~250000 / mm 3 、125000 / mm 3 ~225000 / mm 3 、125000 / mm 3 ~200000 / mm 3 、125000 / mm 3 ~175000 / mm 3 、125000 / mm 3 ~150000 / mm 3 、150000 / mm 3 ~400000 / mm 3 、150000 / mm 3 ~375000 / mm3 、150000 / mm 3 ~350000 / mm 3 、150000 / mm 3 ~325000 / mm 3 、150000 / mm 3 ~300000 / mm 3 、150000 / mm 3 ~275000 / mm 3 、150000 / mm 3 ~250000 / mm 3 、150000 / mm 3 ~225000 / mm 3 、150000 / mm 3 ~200000 / mm 3 、150000 / mm 3 ~175000 / mm 3 、175000 / mm 3 ~400000 / mm 3 、175000 / mm 3 ~375000 / mm 3 、175000 / mm 3 ~350000 / mm 3 、175000 / mm 3 ~325000 / mm 3 、175000 / mm 3 ~300000 / mm 3 、175000 / mm 3 ~275000 / mm 3 、175000 / mm 3 ~250000 / mm 3 、175000 / mm 3 ~225000 / mm 3 、175000 / mm 3 ~200000 / mm 3 、200000 / mm 3 ~400000 / mm 3 、200000 / mm 3 ~375000 / mm 3 、200000 / mm 3 ~350000 / mm 3 、200000 / mm 3 ~325000 / mm 3 、200000 / mm 3~300000 / mm 3 、200000 / mm 3 ~275000 / mm 3 、200000 / mm 3 ~250000 / mm 3 、200000 / mm 3 ~225000 / mm 3 、225000 / mm 3 ~400000 / mm 3 、225000 / mm 3 ~375000 / mm 3 、225000 / mm 3 ~350000 / mm 3 、225000 / mm 3 ~325000 / mm 3 、225000 / mm 3 ~300000 / mm 3 、225000 / mm 3 ~275000 / mm 3 、225000 / mm 3 ~250000 / mm 3 、250000 / mm 3 ~400000 / mm 3 、250000 / mm 3 ~375000 / mm 3 、250000 / mm 3 ~350000 / mm 3 、275000 / mm 3 ~325000 / mm 3 、225000 / mm 3 ~375000 / mm 3 、275000 / mm 3 ~300000 / mm 3 、300000 / mm 3 ~400000 / mm 3 、300000 / mm 3 ~375000 / mm 3 、300000 / mm 3 ~350000 / mm 3 、300000 / mm 3 ~325000 / mm 3 、325000 / mm 3 ~400000 / mm 3 、350000 / mm3 ~400000 / mm 3 , 350000 / mm 3 ~375000 / mm 3 or 375000 / mm 3 ~400000 / mm 3 is.

[0667] In one embodiment, the patient's hemoglobin level is at least 8.0, 9.0, 10.0, 11.0, 12.0, 130, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0 or 20.0 g / dL, measured within 1 day, 1 week or 1 month after treatment, with or without prior transfusion. In one embodiment, the patient's hemoglobin level, measured within 1 day, 1 week, or 1 month after treatment, with or without prior transfusion, is a minimum of 8.0 to 20.0, 8.0 to 19.0, 8.0 to 18.0, 8.0 to 17.0, 8.0 to 16.0, 8.0 to 15.0, 8.0 to 14.0, 8.0 to 13.0, 8.0 to 12.0, 8.0 to 11.0, 8.0 to 10.0, 8.0 to 9.0, 9.0 to 20.0, 9.0 to 19.0, 9.0 to 18.0, 9.0 to 17.0, 9 ...9.0 to 17.0, 9.0 to 18.0 0~16.0, 9.0~15.0, 9.0~14.0, 9.0~13.0, 9.0~12.0, 9.0~11.0, 9.0~10.0, 10.0~20.0, 10.0~19.0, 10.0~18.0, 10.0~17.0, 10.0~16.0, 10.0~15.0, 10.0~14.0, 10.0~13.0, 10.0~12.0, 10.0~11.0, 11.0~20.0, 11.0~19.0, 11.0~18.0, 11.0~17.0, 11.0~1 6.0, 11.0-15.0, 11.0-14.0, 11.0-13.0, 11.0-12.0, 12.0-20.0, 12.0-19.0, 12.0-18.0, 12.0-17.0, 12.0-16.0, 12.0-15.0, 12.0-14.0, 12.0-13.0, 13.0-20.0, 13.0-19.0, 13.0-18.0, 13.0-17.0, 13.0-16.0, 13.0-15.0, 13.0-14.0, 14.0-20.0, 14 .0-19.0, 14.0-18.0, 14.0-17.0, 14.0-16.0, 14.0-15.0, 15.0-20.0, 15.0-19.0, 15.0-18.0, 15.0-17.0, 15.0-16.0, 16.0-20.0, 16.0-19.0, 16.0-18.0, 16.0-17.0, 17.0-20.0, 17.0-19.0, 17.0-18.0, 18.0-20.0, 18.0-19.0, or 19.0-20.0 g / dL.

[0668] In one embodiment, the patient's creatinine clearance rate by the Cockcroft-Gault formula is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 mL / min as measured within one day, one week, or one month after treatment, or an estimated glomerular filtration rate (eGFR) by the Modification of Diet in Renal Disease (MDRD) formula is at least mL / min / 1.73 m 2 is. In one embodiment, the patient's total serum bilirubin is less than 5, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, 0.4, 0.3, 0.2 or 0.1 mg / dL as measured within 1 day, 1 week or 1 month after treatment. In one embodiment, the patient's total serum bilirubin, as measured within 1 day, 1 week, or 1 month after treatment, is between 0.1 and 5.0, 0.1 and 4.5, 0.1 and 4.0, 0.1 and 3.5, 0.1 and 3.0, 0.1 and 2.5, 0.1 and 2.0, 0.1 and 1.5, 0.1 and 0.5, 0.1 and 0.4, 0.1 and 0.3, 0.1 and 0.2, 0.2 and 5.0, 0.2 and 4.5, 0.2 and 4.0, 0.2 and 3.5, 0.2 and 3.0, 0.2 and 2.5 ... 2.0, 0.2~1.5, 0.2~1.0, 0.2~0.5, 0.2~0.4, 0.2~0.3, 0.3~5.0, 0.3~4.5, 0.3~4.0, 0.3~3.5, 0.3~3.0, 0.3~2.5, 0.3~2.0, 0.3~1.5, 0.3~1.0, 0.3~0.5, 0.3~0.4, 0.4~5.0, 0.4~4.5, 0.4~4.0, 0.4~3.5, 0.4~3.0, 0.4~2.5, 0.4~2.0, 0.4~1 .5, 0.4~1.0, 0.4~0.5, 0.5~5.0, 0.5~4.5, 0.5~4.0, 0.5~3.5, 0.5~3.0, 0.5~2.5, 0.5~2.0, 0.5~1.5, 0.5~1.0, 1.0~5.0, 1.0~4.5, 1.0~4.0, 1.0~3.5, 1.0~3.0, 1.0~2.5, 1.0~2.0, 1.0~1.5, 1.5~5.0, 1.5~4.5, 1.5~4.0, 1.5~4.0, 1.5~3. 5, 1.5-3.0, 1.5-2.5, 1.5-2.0, 2.0-5.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.5-5.0, 3.5-4.5, 3.5-4.0, 4.0-5.0, 4.0-4.5, 4.5-5.0 mg / dL.

[0669] In one embodiment, if the patient has Gilbert's Syndrome, the patient's total serum bilirubin is less than 5, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, 0.4, 0.3, 0.2 or 0.1 mg / dL as measured within 1 day, 1 week or 1 month after treatment. In one embodiment, if the patient has Gilbert's syndrome, the patient's total serum bilirubin, as measured within 1 day, 1 week, or 1 month after treatment, is 0.1 to 5, 0.1 to 4.5, 0.1 to 4.0, 0.1 to 3.5, 0.1 to 3.0, 0.1 to 2.5, 0.1 to 2.0, 0.1 to 1.5, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3, 0.1 to 0.2, 0.2 to 5.0, 0.2 to 4.5, 0.2 to 4.0, 0.2 / 3.5, 0.2 / 3.0, 0.2~2.5, 0.2~2.0, 0.2~1.5, 0.2~1.0, 0.2~0.5, 0.2~0.4, 0.2~0.3, 0.3~5.0, 0.3~4.5, 0.3~4.0, 0.3~3.5, 0.3~3.0, 0.3~2.5, 0.3~2.0, 0.3~1.5, 0.3~1.0, 0.3~0.5, 0.3~0.4, 0.4~5.0, 0.4~4.5, 0.4~4.0, 0.4 / 3.5, 0.4 / 3.0, 0.4~2.5, 0.4~2.0, 0.4 / 1.5, 0.4~1.0, 0.4~0.5, 0.5~5.0, 0.5~4.5, 0.5~4.0, 0.5~3.5, 0.5~3.0, 0.5~2.5, 0.5~2.0, 0.5~1.5, 0.5~1.0, 1.0~5.0, 1.0~4.5, 1.0~4.0, 1.0~3.5, 1.0~3.0, 1.0~2.5, 1.0~2.0, 1.0~1.5, 1.5~5.0, 1.5~4.5, 1.5~4.0, 1.5~4.0, 1.5~3.5, 1.5-3.5, 1.5-3.0, 1.5-2.5, 1.5-2.0, 2.0-5.0, 2.0-4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.0, 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.5-5.0, 3.5-4.5, 3.5-4.0, 4.0-5.0, 4.0-4.5, 4.5-5.0 mg / dL.

[0670] In one embodiment, if the patient does not have Gilbert's syndrome, the patient's total serum bilirubin is less than 2.5, 2.0, 1.5, 1.0, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / dL when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, if the patient does not have Gilbert's syndrome, the patient's total serum bilirubin is less than 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.4, 0.1-0.3, 0.1-0.2, 0.2-2.5, 0.2-2.0, 0.2-1.5, 0.2-1.0, 0.2-0.5, 0.2-0.4, 0.2-0. 0.3, 0.3-2.0.5, 0.3-2.0, 0.3-1.5, 0.3-1.0, 0.3-0.5, 0.3-0.4, 0.4-2.5, 0.4-2.0, 0.4-1.5, 0.4-1.0, 0.4-0.5, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, 1.0-2.5, 1.0-2.0, 1.0-1.5, 1.5-2.5, 1.5-2.0, or 2.0-2.5 mg / dL.

[0671] In one embodiment, the patient's hepatic transaminases (AST / ALT / ALP (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase)) are 5, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, or 0 times the upper limit of normal (ULN) when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, the patient's hepatic transaminases (AST / ALT / ALP) are 5.0 times the upper limit of normal when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, the patient's hepatic transaminases (AST / ALT / ALP) are 0-5.0, 0-4.5, 0-4.0, 0 / 3.5, 0 / 3.0, 0-2.5, or 0-2.0 when measured within 1 day, 1 week, or 1 month after treatment. 0, 0~1.5, 0~1.0, 0~0.5, 0.5~5.0, 0.5~4.5, 0.5~4.0, 0.5~3.5, 0.5~3.0, 0.5~2.5, 0.5~2.0, 0.5~1.5, 0.5~1.0, 1.0~5.0, 1.0~4.5, 1.0~4.0, 1.0~3.5, 1.0~3.0, 1.0~2.5, 1.0~2.0, 2.0, 1.0~1.5, 1.5~5.0, 1.5~4.5, 1.5~4.0, 1.5~3.5, 1.5~3.0, 1 0.5-2.5, 1.5-2.0, 2.0-5.0, 2.0-2.0, 4.5, 2.0-4.0, 2.0-3.5, 2.0-3.0, 2.0-2.5, 2.5-5.0, 2.5-4.5, 2.5-4.0, 2.5-3.5, 2.5-3.5, 2.5-3.0, 3.0-5.0, 3.0-4.5, 3.0-4.0, 3.0-3.5, 3.5-5.0, 3.5-4.5, 3.5-4.0, 4.0-5.0, 4.0-4.5 or 4.5-5.0 times.

[0672] In one embodiment, if the patient has liver metastases, the patient's liver transaminases (AST / ALT / ALP) are less than or equal to 5, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, or 0 times the upper limit of normal (ULN) when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, if the patient has liver metastases, the patient's liver transaminases (AST / ALT / ALP) are less than or equal to 5.0 times the upper limit of normal when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, if the patient has liver metastases, the patient's liver transaminases (AST / ALT / ALP) are below the upper limit of normal as measured within 1 day, 1 week, or 1 month after treatment, and are within the range of 0 to 5.0, 0 to 4.5, 0 to 4.0, 0 to 3.5, 0 to 3.0, 0 to 2.5, 0 to 2.0, 0 to 1.5, 0 to 1.0, 0 to 0.5, 0.5 to 5.0, 0.5 to 4.5, 0.5 to 4.0, 0.5, 0.3.5, 0.5 to 3.0, 0.5 to 2.5, 0.5 to 2.0, 0.5 to 1.5, 0.5 to 1.0, 1.0 to 5.0, 1.0 to 4.5, 1.0 to 4.0, 1.0 to 3.5, 1.0 to 3 .... 0~2.5, 1.0~2.0, 1.0~1.5, 1.5~5.0, 1.5~4.5, 1.5~4.0, 1.5~3.5, 1.5~3.0, 1.5~2.5, 1.5~2.0, 2.0~5.0, 2.0~4.5, 2.0~4.0, 2.0~3.5, 2.0~3.0, 2.0~2.5, 2.5~ 5.0, 2.5 to 4.5, 2.5 to 4.0, 2.5 to 3.5, 2.5 to 3.0, 3.0 to 5.0, 3.0 to 4.5, 3.0 to 4.0, 3.0 to 3.5, 3.5 to 5.0, 3.5 to 4.5, 3.5 to 4.5, 4.0 to 5.0, 4.5 to 5.0, 4.0 to 4.5 or 4.5 to 5.0 times.

[0673] In one embodiment, if the patient does not have liver metastases, the patient's liver transaminases (AST / ALT / ALP) are less than or equal to 2.5, 2.0, 1.5, 1.0, 0.5, or 0 times the upper limit of normal (ULN) when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, if the patient does not have liver metastases, the patient's liver transaminases (AST / ALT / ALP) are less than or equal to the upper limit of normal and less than 5.0 times the upper limit of normal when measured within 1 day, 1 week, or 1 month after treatment. In one embodiment, if the patient does not have liver metastases, the patient's liver transaminases (AST / ALT / ALP) are 0-2.5, 0-2.0, 0-1.5, 0-1.0, 0-0.5, 0-1.0, 0-0.5, 0.5-2.5, 0.5-2.0, 0.5-1.5, 0.5-1.0, 1.0-2.5, 1.0-2.0, 1.0-1.5, 1.5-2.5, 1.5-2.0 or 2.0-2.5 times.

[0674] In one embodiment, the patient does not have active nervous system metastases. In one embodiment, the patient has active nervous system metastases.

[0675] In one embodiment, the patient has no history or presence of a clinically relevant CNS disorder such as seizure disorder (e.g., epilepsy), cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, cerebral edema, posterior reversible encephalopathy syndrome (PRES), or a CNS-related autoimmune disease.

[0676] In one embodiment, the patient is free of active human immunodeficiency virus (HIV) infection, as evidenced, for example, by a positive HIV polymerase chain reaction (PCR) test result within one day, one week, or one month after treatment.

[0677] In one embodiment, the patient is free of active HBV or hepatitis C virus (HCV) infection, e.g., within 1 day, 1 week, or 1 month of treatment, as determined by laboratory test results. In one embodiment, the patient's viral HBV load is below the institutional limit of quantitation (LOQ) and they are receiving stable viral suppression therapy. In one embodiment, the patient's HCV RNA viral load is below the institutional LOQ and they have completed therapeutic antiviral therapy.

[0678] Lymphocyte depletion In one embodiment, the patient was lymphocyte-depleted prior to treatment. Exemplary informative biomarkers associated with lymphocyte-depleting chemotherapy are described in WO2016 / 191756 and WO2019 / 079564, which are incorporated herein by reference. In certain embodiments, lymphocyte-depleting chemotherapy includes administering to the patient cyclophosphamide (200 mg / m 2 / day~2000mg / m 2 / day) and fludarabine (20 mg / m 2 / day~900mg / m 2 / day).

[0679] In one embodiment, lymphodepletion is performed with about 250 to about 500 mg / m cyclophosphamide. 2 , for example, about 250 to about 500, 250, 400, 500, about 250, about 400, or about 500 mg / m 2 The method includes administering cyclophosphamide.

[0680] In one embodiment, lymphocyte depletion is with fludarabine at about 20 mg / m 2 / day~about 40mg / m 2 / day (e.g. approx. 30mg / m 2 / day).

[0681] In one embodiment, lymphocyte depletion comprises administering both cyclophosphamide and fludarabine. 2 / day) and fludarabine (30 mg / m 2 Patients undergo lymphocyte depletion with intravenous administration of 100mg / day.

[0682] In one embodiment, cyclophosphamide (500 mg / m 2 / day) and fludarabine (30 mg / m 2 Patients undergo lymphocyte depletion with intravenous administration of 100mg / day.

[0683] In one embodiment, lymphodepletion does not occur 5 days prior to the first administration of NK cells. In one embodiment, lymphodepletion does not occur 7 days prior to the first administration of NK cells.

[0684] In one embodiment, lymphodepletion is performed daily for three consecutive days (i.e., from day -5 to day -3) beginning five days before the first administration of NK cells. In one embodiment, lymphodepletion is performed on days -5, -4, and -3.

[0685] In one embodiment involving repeated or multiple administrations, the patient is lymphodepleted before some or all of the administrations, as described above, hi one embodiment, the patient is lymphodepleted 2, 3, 4, 5, or 6 times.

[0686] Administration NK cells In one embodiment, the NK cells described herein, e.g., the CAR-NK cells described herein, are administered to a patient as part of a pharmaceutical composition, e.g., a pharmaceutical composition described herein. The cells are administered after thawing, in some cases without further manipulation if the cryoprotectant is suitable for immediate administration. Therapeutic regimens for a particular individual often involve administering various samples or doses of NK cells over an extended period of time, including doses taken from a common batch or donor.

[0687] NK cells can be administered by any suitable means, such as, for example, bolus injection, injection, e.g., intravenous injection, or subcutaneous, intraocular, peribulbar, subretinal, intravitreal, transseptal, subscleral, intrachoroidal, intrachoroidal, intraocular, subconjunctival, subtenon, retrobulbar, periscleral, or posterior scleral delivery. In one embodiment, NK cells are administered parenterally, intrapulmonary, and intranasally, with intraductal administration being used if desired for localized treatment. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In one embodiment, a given dose is administered by a single bolus of cells. In one embodiment, a given dose is administered by multiple boluses of cells or continuous infusion of cells over a period of, e.g., 3 days or less. In one embodiment, administration of the cell dose or any additional therapy (e.g., lymphocyte-depleting therapy, allergy therapy, and / or combination therapy) is performed via outpatient transport.

[0688] In the context of adoptive cell therapy, administration of a given "dose" includes administering a given amount or number of cells as a single composition and / or as a single uninterrupted administration (e.g., a single injection or continuous injections). A given "dose" can also include administering a given amount or number of cells as divided doses or multiple compositions, e.g., multiple compositions provided in multiple individual compositions or injections over a specified period of three days or less. Thus, in some situations, a dose is a single or continuous administration for a specified number of cells provided or initiated at a single time point. However, in some situations, a dose is administered as multiple injections over a period of three days or less, such as once daily for three days, or as multiple injections every two days, or multiple times per day.

[0689] In one embodiment, NK cells are administered to a patient in the range of about 1 million to about 100 billion. In one embodiment, the NK cells, e.g., the NK cells described herein, e.g., the CAR-NK cells described herein, are administered in a dose of about 5 x 10 6 ~Approx. 1×10 9In one embodiment, the NK cells are administered at about 5 x 10 per dose. 6 , about 1×10 7 , about 3×10 7 , about 1×10 8 , about 3×10 8 , about 1×10 9 , about 5×10 6 or about 1 x 10 9 In one embodiment, the NK cells are administered in a concentration of 1 million to about 20 billion cells (e.g., about 5 million cells, about 25 million cells, about 50 million cells, about 75 million cells, about 100 million cells, about 200 million cells, about 300 million cells, about 400 million cells, about 500 million cells, about 1 billion cells, or about 2 billion cells), about 3 billion cells, about 4 billion cells, about 5 billion cells, about 6 billion cells, about 7 billion cells, or about 8 billion cells. cells, about 8 billion cells, about 9 billion cells, about 10 billion cells, or a range defined by two of the foregoing values), about 10 million to about 20 billion cells (e.g., about 25 million cells, about 50 million cells, about 75 million cells, about 100 million cells, about 200 million cells, about 300 million cells, about 400 million cells, about 500 million cells, about 1 billion cells, about 2 billion cells, about 3 billion cells, about 4 billion cells, cells, about 5 billion cells, or about 6 billion cells, or about 7 billion cells, or about 8 billion cells, or about 9 billion cells, or about 10 billion cells, or about 20 billion cells, or a range defined by any two of the foregoing values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 150 million cells, about 200 million cells, about 300 million cells, about 400 million cells, about 500 million cells, about 1 billion cells, about 2 billion cells, about 3 billion cells, about 4 billion cells, about 5 billion cells, about 6 billion cells, about 7 billion cells, about 8 billion cells, about 9 billion cells, about 10 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, about 40 billion cells), or a range defined by any two of the foregoing values.

[0690] Thus, in one embodiment, NK cells are administered at a concentration of about 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6, 6×10 6 , 7×10 6 , 8×10 6 , or 9×10 6 Cells, per dose, approximately 1 x 10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 Cells, per dose, approximately 1 x 10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 Approximately 1 x 10 cells, or per dose 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 Approximately 1 x 10 cells, or per dose 10 or about 2 x 10 10 It is administered in cells.

[0691] Thus, in one embodiment, NK cells are administered at a concentration of at least about 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , or 9×10 6 Cells, at least approximately 1 x 10 per dose 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7, 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 Cells, at least approximately 1 x 10 per dose 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 Cells, at least approximately 1 x 10 per dose 9 cells, 2 x 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 cells, or at least approximately 1 x 10 per dose 10 or 2×10 10 It is administered in cells.

[0692] In one embodiment, the NK cells are administered in a dose comprising about 1 million to about 20 billion CAR-expressing NK cells (e.g., about 5 million cells, about 25 million CAR-expressing cells, about 50 million CAR-expressing cells, about 75 million CAR-expressing cells, about 100 million CAR-expressing cells, about 200 million CAR-expressing cells, about 300 million CAR-expressing cells, about 400 million CAR-expressing cells, about 500 million CAR-expressing cells, about 1 billion CAR-expressing cells, about 2 billion CAR-expressing cells, about 3 billion CAR-expressing cells, about 400 million CAR-expressing cells, about 500 million CAR-expressing cells, about 1 billion CAR-expressing cells, about 2 billion CAR-expressing cells, about 3 billion CAR-expressing cells, about 4 billion CAR-expressing cells, about 5 billion CAR-expressing cells, about 6 billion CAR-expressing cells, about 7 billion CAR-expressing cells, about 8 billion CAR-expressing cells, about 9 billion CAR-expressing cells, about 10 billion CAR-expressing cells, or about 20 billion CAR-expressing cells, or a range defined by two of the foregoing values).

[0693] Thus, in one embodiment, NK cells are administered at a concentration of 1×10 per dose. 6 , 2 × 10 6 , 3×106 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 or 9×10 6 1 x 10 CAR-expressing cells per dose 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 1 x 10 CAR-expressing cells per dose 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 CAR-expressing cells per dose, approximately 1 x 10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 CAR-expressing cells, or approximately 1 x 10 per dose 10 or about 2 x 10 10 The patient is administered a dose containing 10 CAR-expressing cells.

[0694] Thus, in one embodiment, NK cells are administered at a concentration of at least about 1 x 10 per dose. 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 or 9×10 6CAR-expressing cells, at least approximately 1 x 10 per dose 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 CAR-expressing cells, at least approximately 1 x 10 per dose 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 or 9×10 8 CAR-expressing cells, at least approximately 1 x 10 per dose 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 or 9×10 9 CAR-expressing cells, at least approximately 1 x 10 per dose 10 The patient is administered a dose containing 10 CAR-expressing cells.

[0695] In one embodiment, the dose of cells is a uniform or fixed dose of cells that is not based on or adheres to the patient's body surface area or weight.

[0696] In one embodiment, the dose of cells is administered based on the patient's weight. For example, the dose can be determined per kilogram of the patient's weight. In one embodiment, the dose of cells is about 1 x 10 cells / kg. 5 and approximately 1 × 10 8 For example, approximately 1.5 x 10 cells / kg 5 and approximately 1.5 x 10 cells / kg 7 or about 4 x 10 5 and approximately 4 x 10 cells / kg6 It can include between.

[0697] Thus, in one embodiment, NK cells are present at 1×10 5 , 1.5×10 5 , 2×1 05 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 or 9.5 x 10 5 cells / kg, 1×10 6 , 1.5×10 6 , 2 × 10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 or 9.5 x 10 6 cells / kg, 1×10 7 , 1.5×10 7 , 2 × 10 7 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×107 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 cells / kg, or 9.5 × 10 7 or about 1 x 10 8 , 1.5×10 8 or 2×10 8 Cells / kg administered.

[0698] In one embodiment, the NK cells are at least about 1 x 10 5 , 1.5×10 5 , 2 × 10 5 , 2.5×10 5 , 3×10 5 , 3.5×10 5 , 4×10 5 , 4.5×10 5 , 5×10 5 , 5.5×10 5 , 6×10 5 , 6.5×10 5 , 7×10 5 , 7.5×10 5 , 8×10 5 , 8.5×10 5 , 9×10 5 or 9.5 x 10 5 cells / kg, at least approximately 1 x 10 6 , 1.5×10 6 , 2 × 10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 , 7×10 6 , 7.5×10 6 , 8×10 6 , 8.5×10 6 , 9×10 6 , or 9.5 × 10 6 cells / kg, at least approximately 1 x 10 7 , 1.5×10 7 , 2 × 107 , 2.5×10 7 , 3×10 7 , 3.5×10 7 , 4×10 7 , 4.5×10 7 , 5×10 7 , 5.5×10 7 , 6×10 7 , 6.5×10 7 , 7×10 7 , 7×10 7 , 7.5×10 7 , 8×10 7 , 8.5×10 7 , 9×10 7 cells / kg, or 9.5 × 10 7 , or at least about 1 × 10 8 or 1.5 x 10 8 Cells / kg administered.

[0699] In one embodiment, the dose of cells, e.g., recombinant receptor-expressing NK cells, is administered to a subject in a single dose or administered only once within a period of 2 weeks, 1 month, 3 months, 6 months, 1 year or more.

[0700] The ability to provide repeated administrations allows patients to experience or maintain a deeper or longer-lasting response from treatment. Thus, in one embodiment, a patient is administered NK cells via multiple administrations, e.g., two or more administrations or at least one subsequent administration. In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, or 10 administrations are provided to a subject. In one embodiment, at least one subsequent dose comprises a second dose. In one embodiment, at least one subsequent dose comprises a second and a third dose. In one embodiment, at least one subsequent dose comprises a second, third, and fourth dose. In one embodiment, at least one subsequent dose comprises a second, third, fourth, and fifth dose. In one embodiment, at least one subsequent dose comprises a second, third, fourth, fifth, and sixth dose. In one embodiment, at least one subsequent dose comprises a second, third, fourth, fifth, sixth, and seventh dose. In one embodiment, the at least one subsequent dose includes a second, third, fourth, fifth, sixth, seventh, and eighth dose. In one embodiment, a patient can receive response-based dosing, with the CAR-NK cell therapy being administered continuously while the patient is experiencing therapeutic benefit. The number of doses and the number of cells per dose can be tailored to the individual patient. In one embodiment, the number of cells administered to a subject in a follow-up or subsequent dose is the same or similar to that of the first dose. Thus, the CAR-NK cell therapy described in the present invention can be tailored to each patient based on their own response. In some cases, if a patient does not achieve any further benefit from CAR-NK cell therapy, treatment can be discontinued. In some cases, treatment can be restarted if the patient relapses.

[0701] In one embodiment involving multiple or repeated administrations, the NK cells are administered weekly, every other week, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, or once every 16 weeks. For example, a patient may receive a first, second, and third administration, each of which may be separated by 6 weeks or approximately 6 weeks. In one embodiment, the NK cells are administered monthly. In one embodiment, the NK cells are administered every other month or every 3 months. In one embodiment, the NK cells are administered every 8 weeks or approximately 8 weeks.

[0702] In one embodiment, the administration schedule may be different depending on the course of treatment.For example, the patient may receive a first series of administration every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks, and a second series of administration every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 weeks, and the time between the first series of administration and the second series of administration may be different.In one embodiment, the patient may receive 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses in the first administration series, and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses in the second administration series, and the number of doses in the first administration series and the second administration series may be the same or different.For example, the patient may receive 4 doses every other week in the first administration series, and 4 doses every 12 weeks in the second administration series.

[0703] In one embodiment, the NK cells are administered 1 to 4 times over a 9 month period.

[0704] In one embodiment, the NK cells are cryopreserved in an infusion-ready medium, such as a cryopreservation composition suitable for intravenous administration, as described herein.

[0705] In one embodiment, the NK cells are present at about 1 x 10 per vial. 7 ~Approx. 1×10 9The vials containing the cells are cryopreserved. In one embodiment, the NK cells are cryopreserved in vials containing a single dose.

[0706] In one embodiment, the cells are thawed, for example in a 37° C. water bath, prior to administration.

[0707] In one embodiment, a thawed NK cell vial is aseptically transferred, for example, using a vial adapter and a sterile syringe, to a single-dose container (e.g., a dosing bag) where the NK cells can be administered to a patient via intravenous infusion by gravity through a Y-type blood / solution set filter.

[0708] In one embodiment, the NK cells are administered as soon as possible, preferably within less than 90 minutes, eg, within 80, 70, 60, 50, 40, 30, 20, or 10 minutes after thawing.

[0709] In one embodiment, the NK cells are administered within 30 minutes of thawing.

[0710] In one embodiment, the pharmaceutical composition is administered intravenously via a syringe. In one embodiment, 1 mL, 4 mL, or 10 mL of the drug is administered intravenously via a syringe to a patient.

[0711] In one embodiment, the patient is administered acetaminophen prior to administration of the NK cell infusion. In one embodiment, the patient is administered 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1750, 1800, 1900, or 2000 mg of acetaminophen. In one embodiment, the acetaminophen is administered to the patient immediately prior to administration of the NK cells. In one embodiment, acetaminophen is administered 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 150, or 180 minutes prior to administration of the NK cells. In one embodiment, acetaminophen is administered orally.

[0712] In one embodiment, the patient receives diphenhydramine prior to administration of the NK cell infusion. In one embodiment, the patient is administered 5, 10, 12.5, 15, 17.5, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, or 100 mg of diphenhydramine. In one embodiment, the diphenhydramine is administered to the patient immediately before the NK cells are administered. In one embodiment, the diphenhydramine is administered 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 150, or 180 minutes before the administration of the NK cells. In one embodiment, acetaminophen is administered orally.

[0713] In one embodiment, the patient is monitored for a period of time before and after administration of NK cells. For example, the patient's vital signs can be monitored. This may include body temperature, respiratory rate, heart rate, blood pressure, and oxygen saturation (SaO2) via pulse oximetry. Optionally, at least one vital sign is measured 5, 10, 15, 20, 25, or 30 minutes before administration of the NK cells. After administration of the NK cells, at least one vital sign can be monitored continuously for 1, 2, 3, 4, or 5 hours, or at regular or irregular intervals, including about 5, 10, 15, 20, 25, or 30 minutes. Optionally, after administration of the NK cells, vital signs can be monitored until the patient is stabilized.

[0714] cytokines In one embodiment, a cytokine is administered to the patient. In one embodiment, the cytokine is administered together with the NK cells as part of a pharmaceutical composition. In one embodiment, the cytokine is administered separately from the NK cells, for example, as part of a separate pharmaceutical composition.

[0715] In one embodiment, the cytokine is IL-2.

[0716] In one embodiment, IL-2 is administered subcutaneously.

[0717] In one embodiment, IL-2 is administered between about 1 and 4 hours after administration of NK cells is completed. In one embodiment, IL-2 is administered at least 1 hour after administration of NK cells is completed. In one embodiment, IL-2 is administered no later than 4 hours after administration of NK cells is completed. In one embodiment, IL-2 is administered at least 1 hour but not later than 4 hours after administration of NK cells is completed.

[0718] In one embodiment, IL-2 is 10 million IU / M 2 , e.g., up to 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million or 10 million IU / m 2It is administered up to

[0719] In one embodiment, IL-2 is about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million IU / M 2 It is administered.

[0720] In one embodiment, IL-2 is administered at 1×10 6 IU / M2 or approximately 1 x 10 6 IU / M 2 In one embodiment, IL-2 is administered at a dose of 2×10 6 IU / M 2 or about 2 x 10 6 IU / M 2 It is administered.

[0721] In one embodiment, IL-2 is administered at 1×10 6 IU / M 2 Less than 100 mg of acetaminophen is administered to patients.

[0722] In one embodiment, the patient is administered a fixed dose of IL-2. In one embodiment, the patient is administered a fixed dose of 6 million IU or about 6 million IU.

[0723] In one embodiment, IL-2 is not administered to the patient.

[0724] dose An "effective amount" refers to an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic dose is an amount capable of achieving a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is an amount necessary to prevent the onset of a disease or disease symptom. An effective amount can be administered in one or more administrations, dosages, or doses. The therapeutically effective amount (i.e., effective dosage) will vary depending on the therapeutic compound selected. The composition can be administered one or more times per week, including once or more times per day to once every other day. One of skill in the art will recognize that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other existing illnesses, can affect the dosage and timing required to effectively treat a subject. Additionally, treating a subject with a therapeutically effective amount of a therapeutic composition described herein can include a single treatment or a series of consecutive treatments.

[0725] The dosage, toxicity, and therapeutic efficacy of therapeutic compounds can be determined by standard control procedures in cell cultures or experimental animals, e.g., to determine the LD50 (the dose that is lethal to 50% of a population) and the ED50 (the dose that is therapeutically effective in 50% of a population). The dose ratio between toxicity and therapeutic effect is referred to as the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions that exhibit high therapeutic indices are preferred. Compositions that exhibit toxic side effects can be used, but care must be taken to design a delivery system that targets such compositions to infected tissue sites in order to minimize potential damage to uninfected cells and reduce side effects.

[0726] Data obtained from cell culture assays and animal studies can be used to formulate various dosages for use in humans. The dosage of such compounds can lie within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage within this range may vary depending on the dosage form and route of administration used. For all compounds used in the methods of the present invention, the therapeutically effective amount can be initially estimated from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test substance that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma concentrations can be measured, for example, by high-performance liquid chromatography.

[0727] Combination therapy In one embodiment, the method can include administering the NK cells described herein, e.g., the CAR-NK cells described herein, in combination with another therapeutic agent, e.g., an antibody, an NK cell engager, an antibody-drug conjugate (ADC), a chemical drug, e.g., a small molecule drug, an immune checkpoint inhibitor, and combinations thereof, which can be administered before, after, or simultaneously with the administration of the NK cells.

[0728] antibody In one embodiment, the other therapeutic agent is an antibody.

[0729] In one embodiment, the antibody is selected from the group consisting of CD20, HER-2, EGFR, CD38, SLAMF7, GD2, ALK1, AMHR2, CCR2, CD137, CD19, CD26, CD32b, CD33, CD37, CD70, CD73, CD74, CD248, CLDN6, Clever-1, c-MET, CSF-1R, CXCR4, DKK1, DR5, Epha3, FGFR2b, FGFR3, FL The antibody binds to a target selected from the group consisting of T3, FOLR1, Globo-H, Glypican3, GM1, Grp78, HER-3, HGF, IGF-1R, IL1RAP, IL-8R, ILT4, integrin alpha V, M-CSF, mesothelin, MIF, MUC1, MUC16, MUC5AC, myostatin, NKG2A, NOTCH, NOTCH2 / 3, PIGF, PRL3, PSMA, ROR1, SEMA4D, Sialyl Lewis A, Siglec15, TGF-b, TNFR3, TRAIL-R2, VEGF, VEGFR1, VEGFR2, vimentin, and combinations thereof.

[0730] Suitable antibodies include, but are not limited to, those listed in Table 8.

[0731] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10]

[0732] Small molecule / chemotherapy drugs In one embodiment, the additional therapeutic agent is a small molecule drug. In one embodiment, the additional therapeutic agent is a chemotherapy drug. In one embodiment, the additional therapeutic agent is a small molecule chemotherapy drug. Such small molecule drugs can include existing standard of care therapies to which adoptive NK cell therapy is added. In some cases, the use of NK cells described herein can improve the effectiveness of small molecule drugs, such as by improving efficacy, reducing the amount of small molecule drug needed to achieve a desired effect, or reducing the toxicity of the small molecule drug.

[0733] In one embodiment, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-acetyloxy-1,9,12-trihydroxy-15-[(2R,3S)-2-hydroxy-3-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoxyl]oxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0 3,10 .0 4,7 ]heptadec-13-en-2-yl]benzoate (docetaxel) or a pharmaceutically acceptable salt thereof.

[0734] In one embodiment, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4,12-diacetyloxy-15-[(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoxyl]oxy-1,9-dihydroxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0 3,10 .0 4,7]heptadec-13-en-2-yl]benzoate (paclitaxel) or a pharmaceutically acceptable salt thereof.

[0735] In one embodiment, the drug is 6-N-(4,4-dimethyl-5H-1,3-oxazol-2-yl)-4-N-[3-methyl-4-([1,2,4]triazolo[1,5-a]pyridin-7-oxy)phenyl]quinazoline-4,6-diamine (tucatinib) or a pharmaceutically acceptable salt thereof.

[0736] In one embodiment, the drug is pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) or a pharmaceutically acceptable salt thereof.

[0737] In one embodiment, the drug is an azanide; cyclobutane-1,1-dicarboxylic acid; platinum(2+) (carboplatin) or a pharmaceutically acceptable salt thereof.

[0738] In one embodiment, the drug is methyl(1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-4-[(12S,14R)-16-ethyl-12-methoxycarbonyl-1,10-diazatetracyclo[12.3.1.0 3,11 .0 4,9 ]octadeca-3(11),4,6,8,15-pentaen-12-yl]-10-hydroxy-5-methoxy-8-methyl-8,16-diazapentacyclo[10.6.1.0 1,9 .0 2,7 .0 16,19 ]nonadeca-2,4,6,13-tetraene-10-carboxylate (vinorelbine) or a pharmaceutically acceptable salt thereof.

[0739] In one embodiment, the drug is N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]furan-2-yl]quinazolin-4-amine (lapatinib) or a pharmaceutically acceptable salt thereof.

[0740] In one embodiment, the drug is (E)—N-[4-[3-chloro-4-(pyridin-2-ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide (neratinib) or a pharmaceutically acceptable salt thereof.

[0741] In one embodiment, the drug is 6-acetyl-8-cyclopentyl-5-methyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one (palbociclib) or a pharmaceutically acceptable salt thereof.

[0742] In one embodiment, the drug is 7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide (ribociclib) or a pharmaceutically acceptable salt thereof.

[0743] In one embodiment, the drug is N-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine (abemaciclib) or a pharmaceutically acceptable salt thereof.

[0744] In one embodiment, the drug is (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 ]Hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone (everolimus) or a pharmaceutically acceptable salt thereof.

[0745] In one embodiment, the drug is (2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide (alpelisib) or a pharmaceutically acceptable salt thereof.

[0746] In one embodiment, the drug is 4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one (olaparib) or a pharmaceutically acceptable salt thereof.

[0747] In one embodiment, the drug is (11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-2,3,10-triazatricyclo[7.3.1.0 5,13 ]trideca-1,5(13),6,8-tetraen-4-one (talazoparib) or a pharmaceutically acceptable salt thereof.

[0748] In one embodiment, the drug is N-[2-[2-(dimethylamino)ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]propyl-2-enamide (osimertinib) or a pharmaceutically acceptable salt thereof.

[0749] In one embodiment, the drug is N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib) or a pharmaceutically acceptable salt thereof. In one embodiment, the drug is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib) or a pharmaceutically acceptable salt thereof.

[0750] In one embodiment, the drug is (E)—N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide (afatinib) or a pharmaceutically acceptable salt thereof.

[0751] In one embodiment, the drug is azan; dichloroplatinum (cisplatin, platinol) or a pharmaceutically acceptable salt thereof.

[0752] In one embodiment, the drug is an azanide; cyclobutane-1,1-dicarboxylic acid; platinum(2+) (carboplatin) or a pharmaceutically acceptable salt thereof.

[0753] In one embodiment, the drug is 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one (gemcitabine) or a pharmaceutically acceptable salt thereof.

[0754] In one embodiment, the drug is (2S)-2-[[4-[2-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid (pemetrexed) or a pharmaceutically acceptable salt thereof.

[0755] In one embodiment, the drug is N,N-bis(2-chloroethyl)-2-oxo-1,3,2λ5 -oxazaphosphinan-2-amine (cyclophosphamide) or a pharmaceutically acceptable salt thereof.

[0756] In one embodiment, the drug is (2R,3S,4S,5R)-2-(6-amino-2-fluoropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol (fludarabine) or a pharmaceutically acceptable salt thereof.

[0757] In one embodiment, the drug is (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione (doxorubicin) or a pharmaceutically acceptable salt thereof.

[0758] In one embodiment, the drug is methyl(1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-4-[(13S,15S,17S)-17-ethyl-17-hydroxy-13-methoxycarbonyl-1,11-diazatetracyclo[13.3.1.0 4,12 .0 5,10 ]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5-methoxy-8,16-diazapentacyclo[10.6.1.0 1,9 .0 2,7 .0 16,19 ]nonadeca-2,4,6,13-tetraene-10-carboxylate (vincristine) or a pharmaceutically acceptable salt thereof.

[0759] In one embodiment, the drug is (8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,12,14,15,16-octahydrocyclopenta[a]phenanthrene-3,11-dione (prednisone) or a pharmaceutically acceptable salt thereof.

[0760] In one embodiment, the drug is N,3-bis(2-chloroethyl)-2-oxo-1,3,2λ 5 -oxazaphosphinan-2-amine (ifosfamide) or a pharmaceutically acceptable salt thereof.

[0761] In one embodiment, the drug is (5S,5aR,8aR,9R)-5-[[(2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one (etopside) or a pharmaceutically acceptable salt thereof.

[0762] In one embodiment, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one (dexamethasone) or a pharmaceutically acceptable salt thereof.

[0763] In one embodiment, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one (cytarabine) or a pharmaceutically acceptable salt thereof.

[0764] In one embodiment, the CAR-NK cells described herein, e.g., NK cells such as AB-201 cells, are administered together with an antibody, e.g., a monoclonal antibody, an antibody-drug conjugate (ADC), a kinase inhibitor, a CDK4 / 5 inhibitor, an mTOR inhibitor, a PI3K inhibitor, a PARP inhibitor, or a combination thereof.

[0765] In one embodiment, the antibody is selected from the group consisting of trastuzumab, pertuzumab, margetuximab, and combinations thereof.

[0766] In one embodiment, the antibody-drug conjugate is selected from the group consisting of ado-trastuzumab emtansine, fam-trastuzumab deruxtecan, sacituzumab govitecan, and combinations thereof.

[0767] In one embodiment, the kinase inhibitor is selected from the group consisting of lapatinib, neratinib, tucatinib, and combinations thereof.

[0768] In one embodiment, the CDK4 / 6 inhibitor is selected from the group consisting of palbociclib, ribociclib, abemaciclib, and combinations thereof.

[0769] In one embodiment, the mTOR inhibitor is everolimus.

[0770] In one embodiment, the PI3K inhibitor is alpelisib.

[0771] In one embodiment, the PARP inhibitor is selected from the group consisting of olaparib, talazoparib, and combinations thereof.

[0772] In one embodiment, the CAR-NK cells described herein, e.g., NK cells such as AB-201 cells, are administered together with pertuzumab (or an FDA-approved biosimilar thereof), trastuzumab (or an FDA-approved biosimilar thereof), and docetaxel (or a pharmaceutically acceptable salt thereof). In one embodiment, pertuzumab (or an FDA-approved biosimilar thereof) is administered at 840 mg intravenously on day 1, followed by 420 mg intravenously. In one embodiment, trastuzumab (or an FDA-approved biosimilar thereof) is administered at 7 mg / kg intravenously on day 1, followed by 6 mg / kg intravenously on day 1 every 21 days. In one embodiment, trastuzumab (or an FDA-approved biosimilar) is administered in the form of a trastuzumab and hyaluronidase-osc injectable formulation for subcutaneous administration. In one embodiment, docetaxel (or a pharmaceutically acceptable salt thereof) is administered at 75-100 mg / ...

Claims

1. A composition comprising 1 million to 100 billion natural killer (NK) cells as an active ingredient, wherein the NK cells are characterized by comprising polynucleotides including the following: a) Nucleic acids encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an anti-HER2 antibody or an extracellular antigen-binding domain containing an antigen-binding fragment thereof; and b) The nucleic acid that codes for IL-15.

2. A composition comprising NK cells as an active ingredient, wherein the NK cells comprise polynucleotides including the following: a) Nucleic acids encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an anti-HER2 antibody or an extracellular antigen-binding domain containing an antigen-binding fragment thereof; and b) Nucleic acid encoding IL-15, The aforementioned NK cells are 1 × 10 ５ ~10 x 10 ８ A composition characterized by being administered to patients requiring such administration in a dose containing cells / kg.

3. A composition containing NK cells as an active ingredient, wherein the NK cells comprise a polynucleotide comprising the following: a) Nucleic acids encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an anti-HER2 antibody or an extracellular antigen-binding domain containing an antigen-binding fragment thereof; and b) Nucleic acid encoding IL-15, Furthermore, the NK cells are administered multiple times to patients who require their administration. The composition is characterized in that the administration is performed once every 1 to 16 weeks.

4. A composition containing NK cells as an active ingredient, wherein the NK cells comprise a polynucleotide comprising the following: a) Nucleic acids encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an anti-HER2 antibody or an extracellular antigen-binding domain containing an antigen-binding fragment thereof; and b) Nucleic acid encoding IL-15, Furthermore, the composition is characterized in that the NK cells are administered to patients who meet one or more of the following criteria: (i) If the patient's ECOG (Eastern Cooperative Oncology Group) performance status is between 0 and 4 at the time of treatment; (ii) If the patient has previously received one or more systemic treatments and is refractory or has relapsed; (iii) When the patient's oxygen saturation, as measured by a pulse and oxygen meter, is 92% or higher; (iv) If the patient's left ventricular ejection fraction (LVEF) is 50% or greater; (v) The patient's absolute neutrophil count (ANC) is 1000 / mm³ ３ (1.0 x 10 ９ If it is L or higher; (vi) The patient's platelet count is 75,000 / mm³ ３ (75 x 10 ９ If it is L or higher; (vii) Regardless of whether the patient has received a blood transfusion before, if the patient's hemoglobin level is 8.0 g / dL or higher; (viiii) The patient's creatinine removal rate according to the Cockcroft-Gault formula is 45 mL / min or higher, or the estimated glomerular filtration rate (eGFR) according to the Modification of Diet in Renal Disease (MDRD) formula is mL / min / 1.73 m ２ If the above applies; (ix) If the patient's total serum bilirubin is less than 5 mg / dL; or (x) If the patient's liver transaminase (AST / ALT / ALP (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase)) is 5 times or less the upper limit of normal (ULN) of the test.

5. A composition containing NK cells as an active ingredient, wherein the NK cells comprise a polynucleotide comprising the following: a) Nucleic acids encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an anti-HER2 antibody or an extracellular antigen-binding domain containing an antigen-binding fragment thereof; and b) Nucleic acid encoding IL-15, Furthermore, the composition is characterized in that the NK cells are administered to patients who meet one or more of the following criteria: (i) If the patient does not have metastases to the active nervous system; (ii) The patient has no history of or presence of clinically relevant CNS disorders, including seizure disorders (e.g., epilepsy), cerebrovascular ischemia / hemorrhage, dementia, cerebellar disorders, cerebral edema, posterior reversible encephalopathy syndrome (PRES), or autoimmune disorders occurring in the CNS; (iii) If the patient is not infected with active human immunodeficiency virus (HIV), such as by being confirmed positive by an HIV polymerase chain reaction (PCR) test; (iv) If the patient does not have active HBV or hepatitis C virus (HCV) infection based on laboratory test results, etc. (v) The patient has a viral HBV load below the institutional limit of quantity (LOQ) and is receiving stable viral suppression therapy; or (vi) The patient's HCV RNA viral load is below the institutional LOQ and has completed therapeutic antiviral therapy.

6. A composition containing NK cells as an active ingredient, wherein the NK cells comprise a polynucleotide comprising the following: a) Nucleic acids encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) comprising an anti-HER2 antibody or an extracellular antigen-binding domain containing an antigen-binding fragment thereof; and b) Nucleic acid encoding IL-15, Furthermore, the composition is characterized in that the NK cells are administered to patients who meet one or more of the following criteria: (i) If the patient has been diagnosed with or has been diagnosed with HER2-positive cancer with an IHC score of 2+; (ii) When a patient has cancer that is not HER2-dependent but shows a certain level of HER2 expression; (iii) If the patient has been diagnosed with or has been diagnosed with HER2-low expression cancer; (iv) If the patient has been diagnosed with or has been diagnosed with HER2-low expression cancer with HER2 expression grade 1+ or 2+; (v) If the patient has been diagnosed with or has been diagnosed with HER2-low expression cancer without HER2 amplification; (vi) If the patient has cancer that includes a HER2 activating mutation; or (vii) The patient has been diagnosed with HER2-expressing cancer that is IHC1+ or IHC2+ / ISH- and has been refractory to previous treatment or has relapsed after treatment.

7. The composition according to any one of claims 1 to 6, characterized in that the anti-HER2 antibody or its antigen-binding fragment comprises a light chain complementarity determination region 1 (CDRL1) containing SEQ ID NO: 34, a light chain complementarity determination region 2 (CDRL2) containing SEQ ID NO: 36, a light chain complementarity determination region 3 (CDRL3) containing SEQ ID NO: 38, a heavy chain complementarity determination region 1 (CDRH1) containing SEQ ID NO: 44, a heavy chain complementarity determination region 2 (CDRH2) containing SEQ ID NO: 46, and a heavy chain complementarity determination region 3 (CDRH3) containing SEQ ID NO:

48.

8. The composition according to any one of claims 1 to 6, characterized in that the polynucleotide encodes a polyprotein containing the amino acid sequence of Sequence ID No.

59.

9. The composition according to any one of claims 1 to 6, characterized in that the NK cells are umbilical cord blood NK cells.

10. The NK cells are characterized by containing the KIR-B haplotype (1-6). A composition according to any one of the items.

11. The composition according to any one of claims 1 to 6, characterized in that the NK cells express CD16 having a V / V polymorphism at F158.

12. The composition according to any one of claims 1 to 6, characterized in that the NK cells are administered to a patient who requires the administration of the pharmaceutical composition as part of the composition.

13. The composition according to any one of claims 1 to 6, characterized in that the patient has a disease or condition related to HER2.

14. The composition according to claim 13, characterized in that the disease or condition related to HER2 is or includes breast cancer.

15. The composition according to claim 14, characterized in that the breast cancer is non-invasive, invasive, or metastatic.

16. The composition according to claim 13, characterized in that the disease or condition related to HER2 is or includes gastric cancer.

17. The composition according to claim 13, characterized in that the disease or condition related to HER2 is ovarian cancer or includes ovarian cancer.

18. The composition according to claim 13, characterized in that the disease or condition associated with HER2 is gastroesophageal cancer or includes gastroesophageal cancer.

19. The composition according to claim 13, characterized in that the disease or condition related to HER2 is or includes lung cancer.

20. The composition according to claim 19, characterized in that the lung cancer is non-small cell lung cancer (NSCLC).

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