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

Abstract

Provided herein are methods of administering an NK cell comprising a polynucleotide comprising a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR).

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 398,834, filed on August 17, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] background

[0004] Targeted therapies, including the use of adoptive cell therapies such as chimeric antigen receptor T cells (CAR-Ts), have revolutionized cancer treatment. These cell therapies can be autologous (CAR-T cells made using the patient's own T cells) or allogeneic (CAR-T cells made using T cells from a healthy donor). Currently, there are no autologous CAR-T therapies approved for the treatment of HER2-specific cancers.

[0005] While their ability to treat targeted hematologic cancers is transformative, challenging clinical hurdles have emerged since 2017, when the US Food and Drug Administration approved the first CAR-T therapy targeting a CD19 B-cell malignancy using an autologous CAR-T cell product. CAR-T cell manufacturing is a resource-intensive process, which can make it impossible to create viable autologous cell therapies for some patients. The average manufacturing time required for autologous CAR-T cell products is three weeks, which may be too long for critically ill patients. Finally, due to the complexity of manufacturing and shipping CAR-T cell products, which require close monitoring at top cancer and medical centers, this treatment option may be out of reach for most patients, both financially and geographically. Importantly, even for those patients who have access to this innovative treatment, CAR-T cell products carry the risk of serious and potentially fatal side effects. These adverse reactions include cytokine release syndrome (CRS) and neurotoxicity, which can be difficult to manage or control.

[0006] Allogeneic CAR-T cell therapies that utilize cells from healthy donors may 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 some issues, including a potential higher risk of graft-versus-host disease (GVHD) and ineffectiveness due to rapid clearance by the patient's immune system.

[0007] Natural killer (NK) cells are lytic cells of the innate immune system with the intrinsic ability to lyse tumor and virus-infected cells. NK cells possess the inherent ability to bridge the gap between innate immunity and the generation of polyclonal adaptive immune responses, thereby generating long-term anti-cancer immune memory. Importantly, NK cells do not require prior antigen exposure to recognize and lyse tumor cells. Cell-to-cell contact of NK cells drives effector functions through degranulation of lytic granules, activation of programmed cell death receptors on target cells, and secretion of immunomodulatory cytokines. The effector functions of NK cells are controlled by a balance of activating and inhibitory receptor signaling. Traditionally, NK cells have been defined as CD56+ and CD3- cells, further subdivided into CD56brightCD16- cytokine-secreting cells and CD56dimCD16+ cytolytic cells. CD16 contact with antibody-activated tumor cells is sufficient to elicit NK cell cytotoxicity and cytokine release. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and macrophage inflammatory protein 1 (MIP1), which 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 act on tumor cells through antibody-dependent cellular cytotoxicity (ADCC). To trigger ADCC, NK cells bind to antibodies through the CD16 receptor on their surface.

[0008] Natural killer (NK) cells also target tumor cells through antibody-dependent cellular cytotoxicity (ADCC), a key component of the innate immune system. Antibody-coated target cells are killed by cells bearing Fc receptors that recognize the constant region of the bound antibody. Contact of CD16 (FCγRIII) with antibody-activated tumor cells is sufficient to elicit NK cell cytotoxicity and cytokine release. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and macrophage inflammatory protein 1 (MIP1), which signal the tumor and recruit T cells. By directly killing tumor cells, NK cells expose tumor antigens for recognition by the adaptive immune system. ADCC is considered a potent mechanism of NK cell action, particularly when bound to antibodies of the immunoglobulin G1 (IgG1) and IgG3 subclasses. To trigger ADCC, NK cells bind to antibodies via their CD16 receptor.

[0009] Similar to T cells, allogeneic NK cells engineered to express CARs with anti-tumor activity may provide an important treatment option for cancer patients. NK cells are not subject to some of the shortcomings of allogeneic CAR-T cells, which, in addition to engineered chimeric antigen receptors, typically retain expression of endogenous T cell receptors. Therefore, allogeneic CAR-NK cell therapy can be safely used in patients without many of the risks associated with allogeneic T cell therapy, including GVHD. However, CAR-NK cells face many of the same challenges as other allogeneic cell therapies, including product sourcing, scalability, durability, and dose-to-dose variability.

[0010] HER2, also known as human epidermal growth factor receptor 2 and ErbB2, is a tyrosine kinase receptor that is highly expressed on the surface of many solid tumors. In normal cells, HER2 plays an important role in cell development. However, mutation or overexpression of HER2 can directly contribute to tumorigenesis and metastasis.

[0011] HER2 amplification is generally considered an important signal for tumorigenesis and is common in several different solid tumor types, including overexpression in 20% to 30% of human breast, ovarian, and gastric cancers.

[0012] Currently, there are eight approved HER2-directed therapies for cancer patients. These FDA-approved agents include monoclonal antibodies, antibody-drug conjugates (ADCs), and small molecule tyrosine kinase inhibitors (TKIs). Under conventional treatments for HER2+ cancers, such as trastuzumab or lapatinib, downstream signaling mutations can lead to resistance to tumor growth inhibition. One mechanism of action of trastuzumab is inhibition of the MAPK and PI3K / Akt pathways, which results in cell cycle arrest. Mutations that confer resistance to trastuzumab treatment include those leading to loss of PTEN (phosphatase and tensin homolog) and activating mutations of PIK3CA (phosphatidylinositol 3-kinase). These mutations can intrinsically activate the PI3K / Akt pathway, driving cell proliferation. PTEN loss has been observed in 36% of HER2-positive primary breast tumor specimens (stage IV disease); these patients have a lower overall response rate to trastuzumab. Furthermore, approximately 25% of patients with trastuzumab resistance harbor PIK3CA mutations. After trastuzumab treatment, patients with PI3KCA mutations experience significantly shorter progression-free survival than those without the mutation. Therefore, while many patients experience meaningful benefit from these therapies, a significant proportion will ultimately experience relapse or disease progression. Once all HER2-directed therapies have been exhausted, patients may be treated with cytotoxic chemotherapy, which offers only limited benefit. The lack of safe and effective treatments for patients who have exhausted HER2-directed therapy options represents a significant and ongoing unmet medical need.

[0013] The present invention addresses these and other deficiencies in the art.

[0014] Summarize

[0015] NK cells are a type of immune cell that can interact with tumor cells through a complex array of receptors on their cell surface and antibody-dependent cellular cytotoxicity (ADCC). NK cells may have advantages over other immune cells, such as T cells used in CAR-T cell therapy and other cell therapies. Autologous CAR-T cells must be engineered from the patient's own cells. This engineering process can take time, during which the patient's disease may significantly worsen. Such patients may require bridging therapy to maintain their condition until their autologous CAR-T cells are ready. Not all patients are suitable for autologous CAR-T therapy. For example, some patients may be too ill or may not have a sufficient number of T cells suitable for engineering purposes. Not all autologous CAR-T cell production batches produce sufficient cell numbers or sufficiently active cells to achieve a therapeutic effect. When such production is successful, patients typically receive only a single dose of autologous CAR-T therapy. Because the risk of acute side effects, such as ICANS and CRS, is greatest immediately after CAR-T cell administration, repeated dosing may be too risky if the patient only derives marginal benefit from a second, third, or subsequent dose. Furthermore, because autologous CAR-T therapy is unique to each patient, the cost of this treatment may make it unaffordable for many patients who could otherwise benefit from it.

[0016] In an exemplary advantage, NK cells can be used in allogeneic therapies, which means that NK cells from one donor can be safely used in one or more patients without the need for HLA matching, gene editing, or other genetic manipulations. Therefore, allogeneic CAR-NK cells can be mass-produced, frozen, shipped around the world, and administered on demand at the point of care. Therefore, the allogeneic cell therapy can be administered to the patient immediately without waiting for the patient's own cells to be modified and administered, and without the need for transitional treatment. Because the allogeneic therapies described herein can be mass-produced using serialized manufacturing methods, the costs associated with manufacturing and shipping in the allogeneic therapies described herein are expected to be significantly lower than those for autologous CAR-T therapies. Serialized manufacturing also reduces batch-to-batch variability and, if conditions permit, allows patients to receive multiple doses of CAR-NK cells from a single batch of a single donor.

[0017] The ability to provide repeated dosing can allow patients to experience or maintain deeper or longer-lasting therapeutic responses. For example, patients can receive response-based dosing, during which they continue to receive CAR-NK cell therapy doses as long as they benefit. The number of doses and cells administered each time can also be determined based on the patient's specific circumstances. In this case, the patient is not limited to the number of cells he or she can provide during the cell collection process associated with autologous CAR-T therapy. Therefore, the CAR-NK cell therapy described herein can be tailored to each patient based on the patient's own response. In some cases, treatment can also be restarted if the patient relapses.

[0018] Allogeneic NK cells may offer an important therapeutic option for cancer patients. A key advantage is that NK cells are well tolerated, with no evidence of graft-versus-host disease, neurotoxicity, or cytokine release syndrome associated with other cell therapies. Another key advantage is that NK cells do not require prior exposure to antigens to recognize and lyse tumor cells. Another key advantage is that NK cells have the inherent ability to bridge the gap between innate immunity and the generation of polyclonal adaptive immune responses, thereby generating long-term anti-cancer immune memory. All of these characteristics contribute to the potential of NK cells as a cancer treatment option.

[0019] 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 (MIP1), which signal tumors and recruit T cells. NK cells directly kill tumor cells, exposing tumor antigens for recognition by the adaptive immune system.

[0020] Furthermore, by utilizing diverse cord blood pools as a source of NK cells, cord blood units can be selected with preferred characteristics (e.g., high-affinity CD16 and killer cell immunoglobulin-like receptor (KIR) B-haplotype) that enhance clinical activity.

[0021] Engineered NK cells (e.g., CAR-NK cells described herein) are superior to autologous cell therapies (e.g., T cells used in CAR-T cell therapies) because NK cells can be used as allogeneic therapies. Therefore, NK cells from one donor can be safely used in one or more patients.

[0022] In conventional treatments for HER2+ cancers, such as trastuzumab or lapatinib, mutations can confer resistance to tumor growth suppression. As described above, some mutations can alter downstream signaling pathways, conferring resistance to trastuzumab or lapatinib. Unlike trastuzumab or lapatinib, HER2-directed CAR-NK cells and therapies described herein, such as AB-201, are activated by binding to HER2 expressed on the surface of target cells. Activated CAR-NK cells then utilize their own cytotoxic pathways to kill target cells. Therefore, this killing is independent of the integrity of signaling within the target cell. Consequently, the CAR-NK cells and cell therapies described herein retain the ability to kill HER2+ tumor cells, even in the presence of some downstream signaling mutations that may confer resistance to approved HER2 therapies. Other mutations may alter the epitopes bound by trastuzumab or lapatinib, rendering these drugs less effective. Since the Her2-directed CAR-NK scFv described herein, including, for example, the scFv of SEQ ID NO: 30, binds to a different domain of HER (domain I) compared to trastuzumab (domain IV) or lapatinib (domain II), the CAR-NK cells described herein may maintain effective binding to HER2+ cells even if other mutations reduce or inhibit the binding ability of trastuzumab or lapatinib.

[0023] In addition, the CAR-NK cells described herein can retain the expression of CD16, including the expression of the 158V / V variant of CD16. Therefore, in some cases, CAR-NK cells can be used in combination with traditional antibody therapy. In a specific example, the antibody therapy can include trastuzumab or lapatinib. As an alternative or in combination, antibody therapy can be directed against alternative or additional targets, including, for example, EGFR. Examples of anti-EGFR antibodies include cetuximab, panitumumab, nimotuzumab, and nexitozumab. These antibodies can cause ADCC reactions of NK cells by binding to CD16 expressed on the surface of NK cells. Therefore, in some cases, the treatment method can include a dual-targeting approach, including the combined use of HER2-targeting CAR-NK cells and anti-EGFR antibody therapy described herein.

[0024] Thus, among other things, the methods provided herein comprise administering 1 million to 100 billion natural killer (NK) cells to a patient in need thereof, wherein the NK cells comprise a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the CAR comprising an extracellular antigen binding domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; b) a nucleic acid encoding IL-15.

[0025] In certain embodiments, the NK cells are administered at a dose comprising 1 to 100 billion NK cells, optionally at a dose comprising 1 million or about 1 million to 20 billion or about 20 billion cells (e.g., 5 million or about 5 million cells, 25 million or about 25 million cells, 50 million or about 75 million cells; 100 million or about 100 million cells, 200 million or about 200 million cells, 300 million or about 300 million cells; 400 million or about 400 million cells, or a range defined by any two of the foregoing values), from 10 million or about 10 million to 20 billion or about 20 billion cells (e.g., 25 million or about 25 million cells, 50 million or about 50 million cells, 75 million or about 75 million cells, 100 million or about 100 million cells, 200 million or about 200 million cells, 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 2 billion cells, 3 billion or about 3 billion cells cells, 4 billion or about 4 billion cells, 5 billion or about 5 billion cells, 6 billion or about 6 billion cells, 7 billion or about 7 billion cells, 8 billion or about 8 billion cells, 9 billion or about 9 billion cells, 10 billion or about 10 billion cells, 20 billion or about 20 billion cells, or a range defined by any two of the foregoing values), and in some cases from 100 million or about 100 million cells to 50 billion or about 50 billion cells (e.g., 150 million or about 150 million cells, 200 million or about 200 million cells , 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 2 billion cells, 3 billion or about 3 billion cells, 4 billion or about 4 billion cells, 5 billion or about 5 billion cells, 6 billion or about 6 billion cells, 7 billion or about 7 billion cells, 8 billion or about 8 billion cells, 9 billion or about 9 billion cells, 10 billion or about 10 billion cells, 20 billion or about 30 billion cells, 40 billion or about 40 billion cells).

[0026] In certain embodiments, the NK cells are administered in a dose of at least 10 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, or at least 1×10 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, or at least 1×10 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, at least 1×10 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 cells, or at least 1×10 10 or 2×10 10 cells.

[0027] In certain embodiments, the NK cells are administered at a dose of about 1×10 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, approximately 1×10 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, approximately 1×10 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 9×107 cells, approximately 1×10 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, approximately 1×10 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 cells, or approximately 1 × 10 per dose 10 or 2×10 10 cells.

[0028] In certain embodiments, the NK cells are administered at a dose of 1 million or about 1 million to 20 billion or about 20 billion CAR-expressing NK cells per dose (e.g., 5 million or about 5 million CAR-expressing NK cells, 25 million or about 25 million CAR-expressing NK cells, 50 million or about 75 million CAR-expressing NK cells; 100 million or about 100 million CAR-expressing NK cells, 200 million or about 200 million CAR-expressing NK cells, 300 million or about 300 million CAR-expressing NK cells; 400 million or about 400 million CAR-expressing NK cells, 500 million or about 500 million CAR-expressing NK cells, 1 billion or about 10 CAR-expressing NK cells, 10 billion or about 10 billion CAR-expressing NK cells, 2 billion or about 2 billion CAR-expressing NK cells, 3 billion or about 3 billion CAR-expressing NK cells, 4 billion or about 4 billion CAR-expressing NK cells, 5 billion or about 5 billion CAR-expressing NK cells, 6 billion or about 6 billion CAR-expressing NK cells, 7 billion or about 7 billion CAR-expressing NK cells, 8 billion or about 8 billion CAR-expressing NK cells, 9 billion or about 9 billion CAR-expressing NK cells, 10 billion or about 10 billion CAR-expressing NK cells, 20 billion or about 20 billion CAR-expressing NK cells, or a range defined by any two of the foregoing values).

[0029] In certain embodiments, the NK cells are administered at a dose of at least 1×10 6 , 2×10 6 , 3×10 6 , 4×106 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 or 9×10 6 CAR-expressing cells, each dose contains or approximately contains 1×10 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 expressing CAR, each dose contains or approximately contains 1×10 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 CAR-expressing cells, each dose contains or approximately contains 1×10 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 each dose contains or contains approximately 1×10 10 or approximately 2×10 10 CAR-expressing cells.

[0030] In certain embodiments, the NK cells are administered at a dose of at least or about 1×10 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, each dose containing at least or at least about 1×10 7 , 2×10 7 , 3×107 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 or 9×10 7 CAR-expressing cells, each dose comprising at least or at least about 1×10 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 CAR-expressing cells, each dose comprising at least or at least about 1×10 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 each dose contains at least or about 1×10 10 CAR-expressing cells.

[0031] Also provided herein is a method comprising administering NK cells to a patient in need thereof, wherein the NK cells comprise a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen binding domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; b) a nucleic acid encoding IL-15. 5 to 10×10 8 cells / kg.

[0032] In certain embodiments, the cell dose comprises 1×10 5 or about 1×10 5 cells / kg and 1×10 8 or about 1×10 8 cells / kg, for example, between 1.5×10 5 or about 1.5×10 5 cells / kg and 1.5×10 7 or about 1.5×10 7 cells / kg, or 4×10 5or about 4×10 5 cells / kg and 4×10^ 6 or about 4×10^ 6 cells / kg.

[0033] In certain embodiments, the NK cells are administered at a dose comprising at or about 1×10 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 9.5×10 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 9.5×10 6 cells / kg, or about 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 1×10 8 , 1.5×10 8 , or 2×10 8 cells / kg.

[0034] In certain embodiments, the NK cells are administered at a dose comprising at least or at least about 1×10 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 9.5×10 5 cells / kg, at least or at least about 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 9.5×106 cells / kg, at least or at least about 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 at least or at least about 1×10 8 or 1.5×10 8 cells / kg.

[0035] Also provided herein are methods comprising administering multiple doses of NK cells to a patient in need thereof, wherein the NK cells comprise a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen binding domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, wherein the dose is administered once every 1 to 16 weeks.

[0036] In certain embodiments, the NK cells are administered once every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, or every 16 weeks.

[0037] Also provided herein are methods comprising administering NK cells to a patient in need thereof, wherein the NK cells comprise a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen binding domain comprising an anti-HER2 antibody or an 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 Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 4 at the time of treatment; (ii) the patient has refractory or relapsed disease after one or more prior systemic therapies; (iii) the patient has an oxygen saturation of at least 92% as measured by a pulse oximeter; (iv) the patient has a left ventricular ejection fraction (LVEF) of at least 50%; and (v) the patient has an absolute neutrophil count (ANC) of at least 1000 / mm3 (1.0×10 9 / L); (vi) the patient's platelet count is at least 75,000 / mm3 (75×10 9 / L); (vii) the patient's hemoglobin level, regardless of whether a blood transfusion is given, is at least 8.0 g / dL; (viii) the estimated glomerular filtration rate (eGFR) is mL / min / 1.73 m using the Cockcraft-Gault equation or the Modification of Diet in Renal Disease (MDRD) equation. 2 , the patient's creatinine clearance is at least 45 mL / min; (ix) the patient's serum total bilirubin is less than 5 mg / dL; (x) the patient's liver transaminase (aspartate transaminase / alanine transaminase / alkaline phosphatase (AST / ALT / ALP)) is less than or equal to 5 times the upper limit of normal (ULN) of the test.

[0038] In certain embodiments, the patient has an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, 2, 3, or 4 at the time of treatment.

[0039] In certain embodiments, the patient is refractory or has relapsed after two or more prior systemic therapies.

[0040] In certain embodiments, the patient's oxygen saturation level as measured by pulse oximetry is at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0041] In certain embodiments, the patient's left ventricular ejection fraction (LVEF) is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%.

[0042] In certain embodiments, the patient has an absolute neutrophil count (ANC) of at least 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 .

[0043] In certain embodiments, the patient's platelet count is at least 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 .

[0044] In certain embodiments, 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, regardless of prior transfusion.

[0045] In certain embodiments, the Cockcraft-Gault equation or the estimated glomerular filtration rate (eGFR) mL / min / 1.73 m2 according to the Modification of Diet in Renal Disease (MDRD) equation is used. 2 , the patient's creatinine clearance 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.

[0046] In certain embodiments, 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.

[0047] In certain embodiments, the patient's liver transaminases (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (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) for the test.

[0048] Also provided herein are methods comprising administering NK cells to a patient in need thereof, wherein the NK cells comprise a polynucleotide comprising: a) encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) R) a nucleic acid comprising an extracellular antigen binding domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; b) a nucleic acid encoding IL-15, wherein the patient meets one or more of the following criteria: (i) the patient does not have active nervous system metastases; (ii) the patient does not have a history of or presence of a clinically relevant central nervous system disease, such as a seizure disorder (e.g., epilepsy), cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, cerebral edema, posterior reversible encephalopathy syndrome (PRES), or any autoimmune disease involving the CNS; (iii) the patient does not have active human immunodeficiency virus (HIV) infection, e.g., as demonstrated by a positive polymerase chain reaction (PCR) test for HIV; (iv) the patient does not have active hepatitis B virus or hepatitis C virus (HCV) infection, e.g., as determined by laboratory testing; (v) the patient's HBV viral load is below the institutional limit of quantification (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 curative antiviral therapy.

[0049] Also provided herein are methods comprising administering NK cells to a patient in need thereof, wherein the NK cells comprise a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the 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 is diagnosed with or has been diagnosed with a HER2+ cancer with an IHC score of 2+; (ii) the patient has a non-HER2 cancer with some degree of HER2 expression; (iii) the patient is diagnosed with or has been diagnosed with a HER2-low cancer; (iv) the patient is diagnosed with or has been diagnosed with a HER2-low cancer with a HER2 expression classification score of 1+ or 2+; (v) the patient is diagnosed with or has been diagnosed with a HER2-low cancer without HER2 amplification; (vi) the patient has a cancer comprising a HER2-activating mutation; or (vii) the patient is diagnosed with a HER2-expressing cancer, i.e., IHC 1+ or IHC 2+. 2+ / ISH- and refractory or relapsed after previous treatment.

[0050] In certain embodiments, the patient is or has been diagnosed with a HER2-low cancer having a HER2 expression classification score of 1+ or 2+ without HER2 amplification.

[0051] In certain embodiments, the HER2-low cancer contains HER2 gain.

[0052] In certain embodiments, the HER2-low cancer lacks HER2 gain.

[0053] In certain embodiments, the patient has been diagnosed with IHC 3+ or IHC 2+ / ISH+ cancer and is refractory or relapsed after prior HER2-targeted therapy.

[0054] In certain embodiments, the patient is diagnosed with a HER2-expressing cancer, ie, IHC 1+ or IHC 2+ / ISH-, and the patient's cancer is unresectable, metastatic, or refractory or relapsed after prior therapy.

[0055] In certain embodiments, in any of the methods described herein, 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.

[0056] In certain embodiments, in any of the methods described herein, 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; CD RH2 encoded by SEQ ID NO:47; and CDRH3 encoded by SEQ ID NO:49.

[0057] In certain embodiments, in any of the methods described herein, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain variable (V) sequence comprising SEQ ID NO: 32. L ) region and a heavy chain variable (V H )district.

[0058] In certain embodiments, in any of the methods described herein, the nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof comprises a V encoding sequence comprising SEQ ID NO: 33. L The nucleic acid of the region and the encoding V containing SEQ ID ID: 37 H nucleic acid in the region.

[0059] In certain embodiments, in any of the methods described herein, the anti-HER2 antibody or antigen-binding fragment thereof comprises a V L Area and V H Area, the V L The V region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32, wherein the V HThe region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:42.

[0060] In certain embodiments, in any of the methods described herein, the anti-HER2 antibody or its antigen-binding fragment is an antigen-binding fragment. In certain embodiments, the antigen-binding fragment comprises a single-chain Fv (scFv). In certain embodiments, the V L District V H In certain embodiments, the V L District V H In certain embodiments, the V L The region is connected to V H region. In certain embodiments, the flexible linker comprises the amino acid sequence set forth in SEQ ID NO:40. In certain embodiments, the flexible linker is encoded by a nucleic acid comprising SEQ ID NO:41. In certain embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO:30. In certain embodiments, the scFv is encoded by a nucleic acid comprising SEQ ID NO:31. In certain embodiments, the scFv comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:30.

[0061] In certain embodiments, in any of the methods described herein, the anti-HER2 CAR specifically binds to a human epidermal growth factor receptor 2 (HER2) protein. In certain embodiments, the HER2 protein comprises the amino acid sequence of SEQ ID NO: 62.

[0062] In certain embodiments, in any of the methods described herein, the CAR comprises a transmembrane domain, optionally a CD28 transmembrane domain. In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 53. In certain embodiments, the CD28 transmembrane domain is encoded by a nucleic acid comprising the nucleic acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 55.

[0063] In certain embodiments, in any of the methods described herein, the CAR further comprises a hinge region located between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, the hinge region comprises at least a portion of the CD8α hinge region. In certain embodiments, the CD8α hinge region comprises the amino acid sequence shown in SEQ ID NO: 50. In certain embodiments, the CD8α hinge region is encoded by a nucleic acid comprising SEQ ID NO: 51 or SEQ ID NO: 52. In certain embodiments, the CD8α hinge region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 50.

[0064] In certain embodiments, in any of the methods described herein, the CAR includes an intracellular signaling domain, optionally wherein the intracellular signaling domain includes a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD3 ζ (CD3 ζ) signaling domain. In certain embodiments, the intracellular signaling domain includes a CD28 intracellular signaling domain and a CD3 ζ signaling domain.

[0065] In certain embodiments, the intracellular signaling domain comprises an OX40L intracellular signaling domain. In certain embodiments, the OX40L intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In certain embodiments, the OX40L intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In certain embodiments, the OX40L intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO: 11 or SEQ ID NO: 12.

[0066] In certain embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain. In certain embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the CD28 intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5. In certain embodiments, the CD28 intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO: 6 or SEQ ID NO: 7.

[0067] In certain embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain. In certain embodiments, the CD3 zeta intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the CD3 zeta intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13. In certain embodiments, the CD3 zeta intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO: 14 or SEQ ID NO: 15.

[0068] In certain embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25.

[0069] In certain embodiments, in any of the methods described herein, the CAR comprises the amino acid sequence shown in SEQ ID NO: 56. In certain embodiments, the CAR is encoded by a nucleic acid comprising SEQ ID NO: 57.

[0070] In certain embodiments, in any of the methods described herein, the IL-15 comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the IL-15 is encoded by a nucleic acid comprising SEQ ID NO: 23 or SEQ ID NO: 24.

[0071] In certain embodiments, in any of the methods described herein, the polynucleotide encodes a multimeric protein comprising CAR and IL-15.

[0072] In certain embodiments, in any of the methods described herein, the polynucleotides further include nucleic acids encoding self-cleavage peptides (optionally T2A self-cleavage peptides). In certain embodiments, the CAR is connected to IL-15 via the self-cleavage peptide. In certain embodiments, the self-cleavage peptide can induce ribosome jumping between CAR and IL-15.

[0073] In certain embodiments, in any of the methods described herein, the polynucleotide further comprises a nucleic acid encoding a signal sequence. In certain embodiments, the signal sequence comprises the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the nucleic acid encoding the signal sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 28.

[0074] In certain embodiments, in any of the methods described herein, the polynucleotide encodes a polyprotein comprising the amino acid sequence shown in SEQ ID NO:59.

[0075] In certain embodiments, in any of the methods described herein, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO:60 or SEQ ID NO:61.

[0076] In certain embodiments, in any of the methods described herein, the NK cells are cord blood NK cells.

[0077] In certain embodiments, in any of the methods described herein, the NK cells comprise a KIR-B haplotype.

[0078] In certain embodiments, in any of the methods described herein, the NK cells express CD16 with a V / V polymorphism at F158.

[0079] In certain embodiments, in any of the methods described herein, the NK cells are administered as part of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutically acceptable excipient comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) buffer.

[0080] In certain embodiments, the pharmaceutical composition comprises 30-50 mg / mL human albumin. In certain embodiments, the pharmaceutical composition comprises 40 mg / mL human albumin.

[0081] In certain embodiments, the pharmaceutical composition comprises 20-30 mg / mL of dextran. In certain embodiments, the pharmaceutical composition comprises 25 mg / mL of dextran. In certain embodiments, the dextran is dextran 40.

[0082] In certain embodiments, the pharmaceutical composition comprises 12-15 mg / mL of glucose. In certain embodiments, the pharmaceutical composition comprises 12.5 mg / mL of glucose. In certain embodiments, the pharmaceutical composition comprises less than 27.5 g / L of glucose.

[0083] In certain embodiments, the pharmaceutical composition comprises 50-60 ml / mL of DMSO. In certain embodiments, the pharmaceutical composition comprises 55 mg / mL of DMSO. In certain embodiments, the pharmaceutical composition comprises 40-60% by volume of a buffer. In certain embodiments, the buffer is a phosphate buffer.

[0084] In certain embodiments, 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 buffer.

[0085] In certain embodiments, the pharmaceutical composition further comprises 0.5 mL / mL of water.

[0086] In certain embodiments, in any of the methods described herein, the subject suffers from a HER2-related disease or condition. In certain embodiments, the HER2-related disease or condition is cancer. In certain embodiments, the cancer expresses HER2. In certain embodiments, the cancer is a HER2+ cancer. In certain embodiments, the cancer is a solid tumor or includes a solid tumor. In certain embodiments, the cancer is or includes bladder cancer, breast cancer, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, cervical squamous cell carcinoma, gastric adenocarcinoma, bile duct cancer, ovarian cancer, renal papillary cell carcinoma, and combinations thereof.

[0087] In certain embodiments, the cancer is or comprises breast cancer. In certain embodiments, the breast cancer is non-invasive. In certain embodiments, the breast cancer is non-invasive. In certain embodiments, the breast cancer is metastatic.

[0088] In certain embodiments, the cancer is or comprises gastric cancer.

[0089] In certain embodiments, the cancer is or comprises ovarian cancer.

[0090] In certain embodiments, the cancer is or comprises gastroesophageal cancer.

[0091] In certain embodiments, the cancer is or comprises lung cancer. In certain embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In certain embodiments, the patient has a HER2 activating mutation.

[0092] In certain embodiments, in any of the methods described herein, the method further comprises administering lymphoablative chemotherapy to the subject prior to treatment. In certain embodiments, the lymphoablative chemotherapy is non-myeloablative chemotherapy. In certain embodiments, the lymphoablative chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine. In certain embodiments, the lymphoablative chemotherapy comprises treatment with cyclophosphamide and fludarabine. In certain embodiments, 100 to 500 mg / m2 of cytokine is administered daily. 2 In certain embodiments, 250 mg / m2 of cyclophosphamide is administered daily. 2 In certain embodiments, 500 mg / m2 of cyclophosphamide is administered daily. 2 In certain embodiments, 10 to 50 mg / m2 of cyclophosphamide is administered daily. 2 In certain embodiments, 30 mg / m2 of fludarabine is administered daily. 2 of fludarabine.

[0093] In certain embodiments, in any of the methods described herein, the method further comprises administering IL-2 to the subject. In certain embodiments, the patient is administered 1×10 6 IU / m 2 In certain embodiments, patients are administered 1×10 7 IU of IL-2. In certain embodiments, patients are administered 6×10 7 IU of IL-2. In certain embodiments, the administration of IL-2 occurs within 1-4 hours after the administration of the cells. In certain embodiments, the administration of IL-2 occurs at least 1-4 hours after the administration of the cells.

[0094] In certain embodiments, in any of the methods described herein, the method further comprises administering the cells multiple times. In certain embodiments, the method comprises administering the cells three, four, or eight times. In certain embodiments, the method comprises administering the cells weekly, every two weeks, every three weeks, or every four weeks.

[0095] In certain embodiments, in any of the methods described herein, the method further comprises administering pertuzumab to the subject. In certain embodiments, in any of the methods described herein, the method further comprises administering trastuzumab to the subject. In certain embodiments, in any of the methods described herein, the method further comprises administering nexituzumab to the subject. In certain embodiments, in any of the methods described herein, the method further comprises administering magituximab to the subject. In certain embodiments, in any of the methods described herein, the method further comprises administering a taxane to the subject. In certain embodiments, the taxane is at least one of paclitaxel, docetaxel, and cabazitaxel. In certain embodiments, in any of the methods described herein, the method further comprises administering endocrine therapy to the subject. In certain embodiments, the endocrine therapy comprises at least one of an aromatase inhibitor, fulvestrant, and tamoxifen.

[0096] In certain embodiments, in any of the methods described herein, the method further comprises administering a checkpoint inhibitor to the subject. In certain embodiments, the checkpoint inhibitor inhibits CTLA-4, PD-1, or PD-L1. In certain embodiments, the checkpoint inhibitor is or includes ipilimumab. In certain embodiments, the checkpoint inhibitor is or includes nivolumab. In certain embodiments, the checkpoint inhibitor is or includes pembrolizumab. In certain embodiments, the checkpoint inhibitor is or includes cemiplizumab. In certain embodiments, the checkpoint inhibitor is or includes atezolizumab. In certain embodiments, the checkpoint inhibitor is or includes avelumab. In certain embodiments, the checkpoint inhibitor is or includes durvalumab.

[0097] In certain embodiments, in any of the methods described herein, the method further comprises administering a second therapeutic moiety to the subject. In certain embodiments, the second therapeutic moiety comprises a lympholysis-clearing chemotherapeutic agent. In certain embodiments, the second therapeutic moiety comprises IL-2. In certain embodiments, the second therapeutic moiety comprises at least one of pertuzumab, trastuzumab, nexitozumab, and makituximab. In certain embodiments, the second therapeutic moiety comprises a taxane. In certain embodiments, the second therapeutic moiety comprises a checkpoint inhibitor.

[0098] Also provided herein are polynucleotides comprising a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR) and natural killer cells expressing the polynucleotides.

[0099] Provided herein is a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen binding domain, wherein the extracellular antigen binding domain comprises an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15.

[0100] In certain embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain complementary determining region 1 (CDRL1) comprising SEQ ID NO: 34, a light chain complementary determining region 2 (CDRL2) comprising SEQ ID NO: 36; a light chain complementary determining region 3 (CDRL3) comprising SEQ ID NO: 38, a heavy chain complementary determining region 1 (CDRH1) comprising SEQ ID NO: 44; a heavy chain complementary determining region 2 (CDRH2) comprising SEQ ID NO: 46; and a heavy chain complementary determining region 3 (CDRH3) comprising SEQ ID NO: 48.

[0101] In certain embodiments, 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.

[0102] In certain embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain variable (V L ) region and a heavy chain variable (V H )district.

[0103] In certain embodiments, the nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof comprises a V sequence encoding SEQ ID NO: 33. L The nucleic acid of the region and the encoding V containing SEQ ID ID:42 H nucleic acid in the region.

[0104] In certain embodiments, the anti-HER2 antibody or antigen-binding fragment thereof comprises V L Area and V H Area, the V LThe V region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32, wherein the V H The region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:42.

[0105] In certain embodiments, the anti-HER2 antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0106] In certain embodiments, the antigen-binding fragment comprises a single-chain Fv (scFv).

[0107] In certain embodiments, the V L District is V H The amino terminus of the region.

[0108] In certain embodiments, the V L District is V H The carboxyl terminus of the region.

[0109] In certain embodiments, the V L The region is connected to V H district.

[0110] In certain embodiments, the flexible linker comprises the amino acid sequence shown in SEQ ID NO:40.

[0111] In certain embodiments, the flexible linker is encoded by a nucleic acid comprising SEQ ID NO:41.

[0112] In certain embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO:30.

[0113] In certain embodiments, the scFv is encoded by a nucleic acid comprising SEQ ID NO:31.

[0114] In certain embodiments, the scFv comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:30.

[0115] In certain embodiments, the anti-HER2 CAR specifically binds to human epidermal growth factor receptor 2 (HER2) protein.

[0116] In certain embodiments, the HER2 protein comprises the amino acid sequence of SEQ ID NO:62.

[0117] In certain embodiments, the CAR comprises a transmembrane domain, optionally a CD28 transmembrane domain.

[0118] In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:53.

[0119] In certain embodiments, the CD28 transmembrane domain is encoded by a nucleic acid comprising the nucleic acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 55.

[0120] In certain embodiments, the polynucleotide further comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain.

[0121] In certain embodiments, the hinge region comprises at least a portion of the CD8α hinge region.

[0122] In certain embodiments, the CD8α hinge region comprises the amino acid sequence shown in SEQ ID NO:50.

[0123] In certain embodiments, the CD8α hinge region is encoded by a nucleic acid comprising SEQ ID NO:51 or SEQ ID NO:52.

[0124] In certain embodiments, the CD8α hinge region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:50.

[0125] In certain embodiments, the CAR comprises an intracellular signaling domain, optionally wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD3 zeta (CD3 zeta) signaling domain.

[0126] In certain embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain and a CD3 zeta signaling domain.

[0127] In certain embodiments, the intracellular signaling domain comprises an OX40L intracellular signaling domain.

[0128] In certain embodiments, the OX40L intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0129] In certain embodiments, the OX40L intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0130] In certain embodiments, the OX40L intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO:11 or SEQ ID NO:12.

[0131] In certain embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain.

[0132] In certain embodiments, the CD28 intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:5.

[0133] In certain embodiments, the CD28 intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5.

[0134] In certain embodiments, the CD28 intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO:6 or SEQ ID NO:7.

[0135] In certain embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain.

[0136] In certain embodiments, the CD3ζ intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:13.

[0137] In certain embodiments, the CD3 zeta intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13.

[0138] In certain embodiments, the CD3 zeta intracellular signaling domain is encoded by a nucleic acid comprising SEQ ID NO: 14 or SEQ ID NO: 15.

[0139] In certain embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:25.

[0140] In certain embodiments, the intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25.

[0141] In certain embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:56.

[0142] In certain embodiments, the CAR is encoded by a nucleic acid comprising SEQ ID NO:57.

[0143] In certain embodiments, the IL-15 comprises the amino acid sequence shown in SEQ ID NO:22.

[0144] In certain embodiments, the IL-15 is encoded by a nucleic acid comprising SEQ ID NO:23 or SEQ ID NO:24.

[0145] In certain embodiments, the polynucleotide encodes a multimeric protein comprising CAR and IL-15.

[0146] In certain embodiments, the polynucleotide further comprises a nucleic acid encoding a self-cleaving peptide, optionally a T2A self-cleaving peptide.

[0147] In certain embodiments, the CAR is linked to IL-15 via the self-cleaving peptide.

[0148] In certain embodiments, the self-cleaving peptide is capable of inducing ribosome jumping between CAR and IL-15.

[0149] In certain embodiments, the polynucleotide further comprises a nucleic acid encoding a signal sequence.

[0150] In certain embodiments, the signal sequence comprises the amino acid sequence shown in SEQ ID NO:27.

[0151] In certain embodiments, the nucleic acid encoding the signal sequence comprises the nucleic acid sequence shown in SEQ ID NO:28.

[0152] In certain embodiments, the polynucleotide encodes a polyprotein comprising the amino acid sequence shown in SEQ ID NO:59.

[0153] In certain embodiments, the polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO:60 or SEQ ID NO:61.

[0154] Also provided herein are vectors comprising the polynucleotides described herein.

[0155] In certain embodiments, the vector is a viral vector.

[0156] In certain embodiments, the viral vector is a retroviral vector or a lentiviral vector.

[0157] Also provided herein are cells comprising the polynucleotides and / or vectors described herein.

[0158] Also provided herein are cells expressing a chimeric antigen receptor and IL-15 encoded by a polynucleotide described herein and / or a vector described herein.

[0159] In certain embodiments, the cell is a lymphocyte.

[0160] In certain embodiments, the lymphocytes are natural killer (NK) cells.

[0161] In certain embodiments, the lymphocytes are T cells.

[0162] In certain embodiments, the cell is a human cell.

[0163] In certain embodiments, the cell is a primary cell obtained from a subject.

[0164] In certain embodiments, the cells are primary cells obtained from umbilical cord blood.

[0165] In certain embodiments, the cells comprise a KIR-B haplotype.

[0166] In certain embodiments, the cells express CD16 with the V / V polymorphism at F158.

[0167] Also provided herein are cell populations comprising a plurality of cells described herein.

[0168] In certain embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%; 95%, 96%, 97%, 98% or 99% of the cells comprise a polynucleotide and / or vector described herein.

[0169] In certain embodiments, 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 described herein.

[0170] Also provided herein are pharmaceutical compositions comprising the cell populations described herein.

[0171] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0172] In certain embodiments, the pharmaceutically acceptable excipients include: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) buffer.

[0173] In certain embodiments, the pharmaceutical composition comprises 30-50 mg / mL of human albumin.

[0174] In certain embodiments, the pharmaceutical composition comprises 50 mg / mL human albumin.

[0175] In certain embodiments, the pharmaceutical composition comprises 20-30 mg / mL of dextran.

[0176] In certain embodiments, the pharmaceutical composition comprises 25 mg / mL dextran.

[0177] In certain embodiments, the dextran is dextran 40.

[0178] In certain embodiments, the pharmaceutical composition comprises 12-15 mg / mL of glucose.

[0179] In certain embodiments, the pharmaceutical composition comprises 12.5 mg / mL glucose.

[0180] In certain embodiments, the pharmaceutical composition comprises less than 27.5 g / L of glucose.

[0181] In certain embodiments, the pharmaceutical composition comprises 50-60 ml / mL of DMSO.

[0182] In certain embodiments, the pharmaceutical composition comprises 55 mg / mL of DMSO.

[0183] In certain embodiments, the pharmaceutical composition comprises 40-60% buffer by volume.

[0184] In certain embodiments, the buffer is phosphate buffer.

[0185] In certain embodiments, 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 buffer.

[0186] In certain embodiments, the pharmaceutical composition further comprises 0.5 mL / mL of water.

[0187] Also provided herein are cryovials comprising the compositions described herein.

[0188] Also provided herein are methods of treatment comprising administering a cell described herein, a population of cells described herein, or a composition described herein to a subject having a disease or disorder associated with HER2.

[0189] Also provided herein is a use of a cell described herein, a cell population described herein, or a composition described herein for the preparation of a medicament for treating a disease or condition associated with HER2.

[0190] Also provided herein is a use of a cell described herein, a cell population described herein, or a composition described herein for treating a disease or disorder associated with HER2.

[0191] In certain embodiments, the HER2-related disease or disorder is cancer.

[0192] In certain embodiments, the cancer is HER2 + cancer.

[0193] In certain embodiments, HER2 + The cancer is or comprises a solid tumor that expresses HER2.

[0194] In certain embodiments, the HER2 + is or includes bladder cancer, breast cancer, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, cervical squamous cell carcinoma, gastric adenocarcinoma, bile duct cancer, ovarian cancer, renal papillary cell carcinoma, and combinations thereof.

[0195] In certain embodiments, the HER2 + Of or including breast cancer.

[0196] In certain embodiments, the HER2 + It is or includes stomach cancer.

[0197] In certain embodiments, the HER2 + Of or including ovarian cancer.

[0198] In certain embodiments, the method or use further comprises administering lymphoablative chemotherapy to the subject prior to treatment.

[0199] In certain embodiments, the lymphoablative chemotherapy is non-myeloablative chemotherapy.

[0200] In certain embodiments, the lymphoablative chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.

[0201] In certain embodiments, the lymphoablative chemotherapy comprises cyclophosphamide and fludarabine treatment.

[0202] In certain embodiments, 100 to 500 mg / m2 is administered daily. 2 of cyclophosphamide.

[0203] In certain embodiments, 250 mg / m2 is administered daily. 2 Cyclophosphamide.

[0204] In certain embodiments, 500 mg / m2 is administered daily. 2 Cyclophosphamide.

[0205] In certain embodiments, 10 to 50 mg / m2 is administered daily. 2 of fludarabine.

[0206] In certain embodiments, 30 mg / m2 is administered daily. 2 of fludarabine.

[0207] In certain embodiments, the method or use further comprises administering IL-2 to the subject.

[0208] In certain embodiments, patients are administered 1×10 6 IU / m 2 IL-2.

[0209] In certain embodiments, patients are administered 1×10 7 IU of IL-2.

[0210] In certain embodiments, patients are administered 6×10 7 IU of IL-2.

[0211] In certain embodiments, administration of IL-2 occurs within 1-4 hours after administration of the cells, cell populations, and / or pharmaceutical compositions.

[0212] In certain embodiments, administration of IL-2 occurs at least 1-4 hours after administration of the cells, cell populations, and / or pharmaceutical composition.

[0213] In certain embodiments, the method or use further comprises administering the cell, cell population and / or pharmaceutical composition multiple times.

[0214] In certain embodiments, the method or use comprises administering the cell, cell population and / or pharmaceutical composition three, four or eight times.

[0215] In certain embodiments, the method or use comprises administering the cell, cell population, and / or pharmaceutical composition weekly, biweekly, three-weekly, or four-weekly.

[0216] In certain embodiments, the method or use further comprises administering pertuzumab to the subject.

[0217] In certain embodiments, the method or use further comprises administering trastuzumab to the subject.

[0218] In certain embodiments, the method or use further comprises administering necituzumab to the subject.

[0219] In certain embodiments, the method or use further comprises administering makituximab to the subject.

[0220] In certain embodiments, the method or use further comprises administering a taxane to the subject.

[0221] In certain embodiments, the taxane is at least one of paclitaxel, docetaxel, and cabazitaxel.

[0222] In certain embodiments, the method or use further comprises administering endocrine therapy to the subject.

[0223] In certain embodiments, the endocrine therapy comprises at least one of an aromatase inhibitor, fulvestrant, and tamoxifen.

[0224] In certain embodiments, the method or use further comprises administering a checkpoint inhibitor to the subject.

[0225] In certain embodiments, the checkpoint inhibitor inhibits CTLA-4, PD-1, or PD-L1.

[0226] In certain embodiments, the checkpoint inhibitor is or includes ipilimumab.

[0227] In certain embodiments, the checkpoint inhibitor is or includes nivolumab.

[0228] In certain embodiments, the checkpoint inhibitor is or includes pembrolizumab.

[0229] In certain embodiments, the checkpoint inhibitor is or includes cemiplimab.

[0230] In certain embodiments, the checkpoint inhibitor is or includes atezolizumab.

[0231] In certain embodiments, the checkpoint inhibitor is or includes avelumab.

[0232] In certain embodiments, the checkpoint inhibitor is or includes durvalumab.

[0233] Also provided herein are methods of treatment comprising administering to a subject having a disease or condition associated with HER2 a cell described herein, a cell population described herein, and / or a pharmaceutical composition described herein; and a second therapeutic component.

[0234] In certain embodiments, the second therapeutic portion comprises a lympholytic chemotherapeutic agent.

[0235] In certain embodiments, the second therapeutic moiety comprises IL-2.

[0236] In certain embodiments, the second therapeutic moiety comprises at least one of pertuzumab, trastuzumab, necituzumab, and malituximab.

[0237] In certain embodiments, the second therapeutic moiety comprises a taxane.

[0238] In certain embodiments, the second therapeutic moiety comprises a checkpoint inhibitor.

[0239] 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. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) will prevail.

[0240] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0241] Incorporation by Reference

[0242] 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 specifically and individually indicated to be incorporated by reference.

[0243] BRIEF DESCRIPTION OF THE DRAWINGS

[0244] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be gained by reference to the following detailed description of exemplary embodiments, in which the principles of the present invention are employed, and the accompanying drawings:

[0245] Figure 1 An exemplary embodiment of a method for NK cell expansion and stimulation is shown.

[0246] Figure 2 Examples of different manufacturing scenarios for master cell bank (MCB) and drug product (DP) manufacturing are shown.

[0247] Figure 3 The phenotype of expanded and stimulated NK cell populations is shown.

[0248] Figure 4 It was shown that CAR-NKs containing a costimulatory domain containing OX40L exhibited greater cytotoxic potential than CAR-NKs without OX40L.

[0249] Figure 5 A schematic diagram of the CAR structure is shown.

[0250] Figure 6 Shows Figure 5 Propagation of CAR constructs.

[0251] Figure 7 Shows Figure 5 CAR expression of CAR constructs in .

[0252] Figure 8 Shows Figure 5 CD107a expression of CAR constructs. Bar chart, from left to right: Mock, 2 nd -CA R, 3 rd -CAR.

[0253] Figure 9 Shows Figure 5 IFN-γ expression of CAR constructs. Bar graph, from left to right: Mock, 2 nd -CAR, 3 rd -CAR.

[0254] Figure 10 Shows Figure 5 TNF-α expression of CAR constructs. Bar graph, from left to right: Mock, 2 nd -CA R, 3 rd -CAR.

[0255] Figure 11 Shows Figure 5 Short-term cytotoxicity of the CAR constructs.

[0256] Figure 12 The mechanism of the CAR constructs is shown. From top to bottom: mock NK expressing GFP (mock-NK); CAR without IL-15 (CAR-nk); truncated CAR with IL-15 (CAR(t)-IL-15-NK); CAR with IL-15 (CAR-IL-15-nk, AB-201).

[0257] Figure 13 Shown is the expression of CAR on NK cells in the presence of IL-2.

[0258] Figure 14 Shown is the expression of CAR on NK cells in the absence of IL-2.

[0259] Figure 15 Shown are the proliferation of NK cells in the presence and absence of IL-2.

[0260] Figure 16 NK cell activity in the presence and absence of IL-2 is shown.

[0261] Figure 17 NK cell cytotoxicity was demonstrated.

[0262] Figure 18 The IFNg production of NK cells is shown.

[0263] Figure 19 IL-15 production by NK cells is shown.

[0264] Figure 20 It was shown that IL-15 secretion maintains the survival of bystander NK cells.

[0265] Figure 21 Schematic diagrams of two different CAR constructs are shown.

[0266] Figure 22 Shows Figure 21 CAR expression over time (in days) for the indicated CAR constructs.

[0267] Figure 23 Shows the expression Figure 21 Survival (cell number) of cells expressing the indicated CAR constructs.

[0268] Figure 24 Shows the expression Figure 21 Survival of cells with the indicated CAR constructs (percent survival).

[0269] Figure 25 Shows the expression Figure 21Number of surviving NK cells after 7 days of co-culture of cells with the indicated CAR constructs and target cells without IL-2 support. Bar graph, from left to right: cord blood NK cells (CBNK); day 3 CAR-NK; day 4 CAR-NK.

[0270] Figure 26 AB-201 was shown to have in vitro cytotoxic activity against the breast cancer (HER2-) cell line MDA-MB-468.

[0271] Figure 27 AB-201 demonstrated in vitro cytotoxicity against the ovarian cancer (HER2+) cell line SKOV3.

[0272] Figure 28 AB-201 demonstrated in vitro cytotoxicity against the gastric cancer (HER2+) cell line NCI-N87.

[0273] Figure 29 AB-201 demonstrated in vitro cytotoxicity against the breast cancer (HER2+) cell line HCC1954.

[0274] Figure 30 The in vitro cytotoxic activity of AB-201 against the breast cancer (HER2+) cell line K562 was demonstrated.

[0275] Figure 31 In vitro characterization of AB-201 is shown.

[0276] Figure 32 In vitro characterization of AB-201 is shown.

[0277] Figure 33 In vitro characterization of AB-201 is shown.

[0278] Figure 34 In vitro characterization of AB-201 is shown.

[0279] Figure 35 In vitro characterization of AB-201 is shown.

[0280] Figure 36 In vitro characterization of AB-201 is shown.

[0281] Figure 37 Shown are the results of a long-term killing assay using Incucyte live cell imaging to characterize NCI-N87 gastric cancer cells in culture. Effector to target cell ratio (E:T 0.3:1).

[0282] Figure 38 showed that in the HER2+ HCC1954 breast cancer model, a single dose of 1 million cells of AB-201 achieved a significant survival benefit over trastuzumab.

[0283] Figure 39 It was shown that a single administration of AB-201 on day 4 after establishment of a HER2+ trastuzumab-resistant breast cancer model resulted in tumor regression.

[0284] Figure 40 In vitro characterization of AB-201 is shown.

[0285] Figure 41 In vitro characterization of AB-201 is shown.

[0286] Figure 42 In vitro characterization of AB-201 is shown.

[0287] Figure 43 In vitro characterization of AB-201 is shown.

[0288] Figure 44 Shown are the cytotoxicity of primary cells (non-tumor) following 4-hour coculture of AB-201 or control CB-NK cells with pulmonary artery endothelial cells, keratinocytes, renal epithelial cells, cardiomyocytes, and small airway epithelial cells at effector:target cell (E:T) ratios of 3:1, 1:1, or 0.3:1.

[0289] Figure 45 Shown are tumor volume measurements of NSG mice treated with SKOV3-Luc tumor cells (IP) without AB-201 treatment (open circles, dashed line) or with AB-201 treatment (closed circles, solid line). The vertical line on day 11 indicates the date of AB-201 injection.

[0290] Figure 46 Shown are body weight measurements of NSG mice treated with SKOV3-Luc tumor cells (IP), untreated (open circles, dashed line), or AB-201 treated (closed circles, solid line).

[0291] Figure 47 AB-201 cells were shown to persist at detectable levels in AB-201 treated mice until at least day 52.

[0292] Figure 48 Shown are tumor volumes of irradiated mice inoculated with NCI-N87 cells and given no treatment, cord blood NK cells, or AB-201 cells.

[0293] Figure 49 Shown are tumor volumes of unconditioned mice inoculated with NCI-N87 cells and given no treatment, cord blood NK cells, or AB-201 cells.

[0294] Figure 50 Shows Figure 48 and Figure 49 Body weight measurements of mice as described in .

[0295] Figure 51 Shown are AB-201 infiltration of tumors as shown by immunofluorescence detection of CD56.

[0296] Figure 52 The experimental design is shown: NSG mice were injected intraperitoneally (IP) with 1×10 6 SK-OV-3-Luc tumor cells were randomly divided into groups 4 days later. A single injection of CB-NK (5×10 6 dose) or AB-201 (1×10 6 or 5×10 6 ).

[0297] Figure 53 AB-201 demonstrated efficacy in a SK-OV-3-Luc xenograft tumor model.

[0298] Figure 54 Figure 2 shows the body weight changes of SK-OV-3-Luc tumor-bearing mice. Body weight changes were calculated based on the BW of the mice on the day of NK cell injection.

[0299] Figure 55 Figure 2 shows the presence of AB-201 in peripheral blood lymphoid tissues. At the indicated time points after tumor inoculation (gated on human CD45+CD56+), the levels of AB-201 and CB-NK in peripheral blood and spleen were measured by flow cytometry.

[0300] Figure 56 The expression of cell surface markers on CBNK or AB-201 cells after thawing is shown.

[0301] Figure 57 The specific cytotoxicity of AB-201 and control CB-NK cells against K562, SK-OV-3, HCC1954, and NCI-N87 at different E:T ratios is shown.

[0302] Figure 58 Shown are kinetic analyses of AB-201 and CBNK cytotoxicity against SK-OV-3, HCC1954, and NCI-N87. Long-term cytotoxicity of AB-201 and control CB-NK cells against HER2+ target tumor cell lines representing different solid tumor malignancies was measured for 120 hours using the Incucyte LiveCell Assay System.

[0303] Figure 59Degranulation 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 effector:target (E:T) ratio for 24 hours. After stimulation, cells were harvested and flow cytometry was used to assess cell degranulation (CD107a) and cytokine (IFNγ, TNFα) secretion. Data are presented as the percentage of positive cells in the CD56+ gated population (n=3) (*p<0.05, **p<0.01, compared to CBNK, two-tailed t-test)

[0304] Figure 60 Figure 2 shows IFN-γ secretion by AB-201 following stimulation with K562, SK-OV-3, HCC1954, and NCI-N87 cells. AB-201 or control CB-NK cells were stimulated with various target tumor cells at a 3:1 effector:target (E:T) ratio for 24 hours. Cell-free supernatants were collected, and soluble IFNγ cytokine levels were measured by ELISA (*p < 0.05, **p < 0.01, compared to CBNK cells, two-tailed t-test).

[0305] Figure 61 Figure 2 shows IL-15 secretion by AB-201 following stimulation with K562, SK-OV-3, HCC1954, and NCI-N87 cells. AB-201 or control CB-NK cells were stimulated with various target tumor cells at a 3:1 effector:target (E:T) ratio for 24 hours. Cell-free supernatants were collected, and soluble IL-15 cytokine levels were measured by ELISA. (*p < 0.05, compared with CBNK, two-tailed t-test).

[0306] Details

[0307] Provided herein include, but are not limited to, natural killer (NK) cells, such as CAR-NK cells, methods for producing NK cells, pharmaceutical compositions comprising NK cells, and methods of using NK cells to treat, for example, cancer patients.

[0308] Expansion and stimulation of natural killer cells

[0309] In certain embodiments, natural killer cells are expanded and stimulated, for example, by culturing and stimulation with feeder cells.

[0310] NK cells can be expanded and stimulated as described in, for example, US2020 / 0108096 or WO 2020 / 101361, both of which are incorporated herein by reference in their entirety. Briefly, the source cells can be cultured in a modified HuT-78 ( TIB-161TM ) cells, as described in US2020 / 0108096, HuT-78 ( TIB-161 TM ) cells have been engineered to express 4-1BBL, membrane-bound IL-21, and mutant TNFα.

[0311] Suitable NK cells can also be expanded and stimulated as described herein.

[0312] In certain embodiments, NK cells are expanded and stimulated by: (a) providing NK cells, e.g., a complex comprising NK cells, e.g., CD3(-) cells; and (b) culturing in a medium containing feeder cells and / or stimulatory factors, thereby producing an expanded and stimulated NK cell population.

[0313] Natural killer cell sources

[0314] In certain embodiments, the source of the NK cells is selected from the group consisting of peripheral blood, peripheral blood lymphocytes (PBL), peripheral blood mononuclear cells (PBMCs), bone marrow, umbilical cord blood (umbilical cord blood), isolated NK cells, NK cells derived from induced pluripotent stem cells, NK cells derived from embryonic stem cells, and combinations thereof.

[0315] In certain embodiments, the source of NK cells is one unit of umbilical cord blood.

[0316] In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 7 or about 1×10 7 to 1×10 9 or about 1×10 9 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 8 or about 1×10 8 to 1.5×10 8 or about 1.5×10 8 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 8 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises approximately 1×10 8 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 9 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises approximately 1×10 9 Total nucleated cells.

[0317] In certain embodiments, a source of NK cells, e.g., a unit of umbilical cord blood, comprises from about 20% to about 80% CD16+ cells. In certain embodiments, a source of NK cells, e.g., a unit of umbilical cord blood, comprises from about 20% to 80% or about 80%, from about 20% to 70% or about 70%, from about 20% to 60% or about 60%, from about 20% to 50% or about 50%, from about 20% to 40% or about 40%, from about 20% to 30% or about 30%, from about 30% to 80% or about 80%, from about 30% to 70% or about 70%, from about 30% to 60% or about 60%, from about 30% to 50% or about 50% From about 30% to 40% or about 40%, from about 40% to 80% or about 80%, from about 40% to 70% or about 70%, from about 40% to 60% or about 60%, from about 40% to 50% or about 50%, from about 50% to 80% or about 80%, from about 50% to 70% or about 70%, from about 50% to 60% or about 60%, from about 60% to 80% or about 80%, from about 60% to 70% or about 70%, or from about 70% to 80% or about 80% CD16+ cells. In certain embodiments, a source of NK cells, such as an umbilical cord blood unit, comprises less than or equal to 80% CD16+ cells. In addition, some sources of NK cells may contain a concentration of CD16+ cells greater than 80%.

[0318] In certain embodiments, the source of NK cells, e.g., a cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% ML G2A+ cells.

[0319] In certain embodiments, the source of NK cells, e.g., a cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NK G2C+ cells.

[0320] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% NK G2D+ cells.

[0321] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% NK p46+ cells.

[0322] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% NK p30+ cells.

[0323] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% DN AM-1+ cells.

[0324] In certain embodiments, the source of NK cells, e.g., a cord blood unit, comprises less than or equal to 40%, e.g., less than or equal to 30%, e.g., less than or equal to 20%, e.g., less than or equal to 10%, e.g., less than or equal to 5% NK p44+ cells.

[0325] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% CD25+ cells.

[0326] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% CD62L+ cells.

[0327] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% CD69+ cells.

[0328] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% CXCR3+ cells.

[0329] In certain embodiments, the source of NK cells, such as a cord blood unit, comprises less than or equal to 40%, such as less than or equal to 30%, such as less than or equal to 20%, such as less than or equal to 10%, such as less than or equal to 5% CD57+ cells.

[0330] In certain embodiments, the NK cells of the NK cell source comprise a KIR B allele of the KIR receptor family. See, for example, Hsu et al., "Killer cell immunoglobulin-like receptor (KIR) genomic region: gene order, haplotypes, and allelic polymorphisms," Immunology Reviews, 190:40–52 (2002); and Pyo et al., "Distinct evolutionary patterns of centromeric and telomeric regions of group A and group B haplotypes of the human killer cell immunoglobulin-like receptor locus," PLoS One, 5:e15115 (2010).

[0331] In certain embodiments, the NK cells in the NK cell source comprise the 158V / V variant of CD16 (i.e., homozygous CD16158V polymorphism). See, e.g., Koene et al., "FcγRIIIa-158V / F polymorphism affects natural killer cell FcγRIIIa binding to IgG, independent of the FcγRIIIa-48L / R / H phenotype," Blood, 90: 1109-14 (1997).

[0332] In certain embodiments, the NK cells in the cell source comprise a KIR B allele of the KIR receptor family and the 158V / V variant of CD16.

[0333] In certain embodiments, the NK cells in the cell source are not gene-edited.

[0334] In certain embodiments, the NK cells in the cell source do not include a CD16 transgene.

[0335] In certain embodiments, the NK cells in the cell source do not express exogenous CD16 protein.

[0336] In certain embodiments, the NK cell source is CD3(+)-depleted. In certain embodiments, the method comprises depleting the NK cell source of CD3(+). In certain embodiments, depleting the NK cell source of CD3(+) comprises contacting the NK cell source with a CD3-binding antibody or antigen-binding fragment thereof. In certain embodiments, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1, and HIT3a, and fragments thereof. In certain embodiments, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof is attached to beads, such as magnetic beads. In certain embodiments, depleting the complex of CD3(+) cells comprises contacting the complex with a CD3-targeting antibody or antigen-binding fragment thereof attached to beads, and removing bead-bound CD3(+) cells from the resulting complex. CD3 cells in the complex can be removed by immunomagnetic selection, for example, using a CliniMACS T cell depletion device (LS Depletion Device (162-01) Miltenyi Biotec).

[0337] In certain embodiments, the NK cell source is enriched for CD56+, for example by gating on CD56 expression.

[0338] In certain embodiments, the NK cells are derived, for example, by selecting cells with CD56+CD3- expression for CD56+ enrichment and CD3(+) depletion.

[0339] In certain embodiments, the NK cell source comprises a KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and is enriched for CD56+ and depleted for CD3(+), for example, by selecting cells with CD56+CD3- expression.

[0340] Feeder cells

[0341] Disclosed herein are feeder cells for NK cell expansion. These feeder cells facilitate the expansion of NK cells to a quantity suitable for preparing a pharmaceutical composition described herein. In some cases, the feeder cells allow NK cells to expand without losing CD16 expression, which is typically accompanied by other types of feeder cells or cell expansion using other methods. In some cases, the feeder cells make the expanded NK cells easier to freeze, allowing a higher proportion of NK cells to remain alive after a freeze / thaw cycle, or allowing the cells to remain alive longer when frozen.

[0342] In some cases, the feeder cells allow NK cells to maintain high levels of cytotoxicity, including ADCC, prolong survival, increase persistence, and enhance or maintain high levels of CD 16. In some cases, the feeder cells allow NK cells to expand without causing significant exhaustion or senescence.

[0343] The feeder cells can be used to stimulate NK cells and help them expand more quickly, for example by providing substrates, growth factors and / or cytokines.

[0344] Various types of feeder cells can be used to stimulate NK cells, including but not limited to peripheral blood mononuclear cells (PBMCs), Epstein-Barr virus-transformed B lymphoblasts (such as EBV-LCL), myeloid leukemia cells (such as K562) and CD4(+) T cells (such as HuT), and their derivatives.

[0345] In certain embodiments, the feeder cells are inactivated by, for example, gamma irradiation or mitomycin c treatment.

[0346] Suitable feeder cells for use in the methods described herein are described, for example, in US 2020 / 0108096, which is incorporated herein by reference in its entirety.

[0347] In certain embodiments, the feeder cells are inactivated CD4+ T cells. In certain embodiments, the inactivated CD4(+) T cells are HuT-78 cells ( TIB-161 TM ) or a variant or derivative thereof. In certain embodiments, the derivative of HuT-78 is H9 ( HTB-176 TM ).

[0348] In certain embodiments, the inactivated CD4(+) T cells express OX40L. In certain embodiments, the inactivated CD4(+) T cells are HuT-78 cells expressing OX40L (SEQ ID NO: 4) or a variant thereof, or a variant or derivative thereof.

[0349] In certain embodiments, the feeder cells are HuT-78 cells 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) ("eHut-78 cells"), or variants thereof.

[0350] In certain embodiments, the inactivated CD4(+) T cells are HuT-78 ( TIB-161 TM ) cells or variants or derivatives thereof. In certain embodiments, the feeder 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.

[0351] In certain embodiments, the feeder cells are HuT-78 cells that express OX40L (SEQ ID NO:4) and are engineered to express 4-1BBL (SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and mutant TNFα (SEQ ID NO:3) ("eHut-78 cells"), or variants or derivatives thereof.

[0352] In certain embodiments, the feeder cells are expanded, e.g., from frozen stocks, prior to culture with NK cells, e.g., as described in Example 2.

[0353] Stimulating factors

[0354] In addition to or in place of feeder cells, NK cells can be stimulated using one or more non-feeder cell stimulatory factors, such as signaling factors, in addition to or in place of feeder cells.

[0355] In certain embodiments, the stimulatory factor, such as a signaling factor, as described herein, is a component of the culture medium. In certain embodiments, the stimulatory factor, such as a signaling factor, as described herein, is a supplement to the culture medium.

[0356] In certain embodiments, the stimulatory factor is a cytokine. In certain embodiments, 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, IFNβ, and combinations thereof.

[0357] In certain embodiments, the cytokine is IL-2.

[0358] In certain embodiments, the cytokine is a combination of IL-2 and IL-15.

[0359] In certain embodiments, the cytokine is a combination of IL-2, IL-15, and IL-18.

[0360] In certain embodiments, the cytokine is a combination of IL-2, IL-18, and IL-21.

[0361] nourish

[0362] NK cells can be expanded and stimulated by co-culturing a source of NK cells with feeder cells and / or other stimulatory factors. Suitable sources of NK cells, feeder cells, and stimulatory factors are described herein.

[0363] In some cases, the expanded natural killer cell population is enriched and / or sorted after expansion. In some cases, the expanded natural killer cell population is not enriched and / or sorted after expansion.

[0364] Also described herein are complexes comprising various culture complexes described herein, such as complexes comprising NK cells, for example, a complex comprising an expanded population of umbilical cord blood-derived natural killer cells, wherein the population of natural killer cells comprises a KIR-B haplotype and homozygotes for the CD16 158V polymorphism, and a plurality of engineered HuT78 cells.

[0365] Also described herein are containers, e.g., vials, cryobags, etc., containing the resulting expanded natural killer cell population. In some cases, a plurality of containers, 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, contain a portion of the resulting expanded natural killer cell population.

[0366] Also described herein are bioreactors comprising various culture complexes described herein, such as bioreactors comprising NK cells. For example, a culture of natural killer cells comprising a natural killer cell source (e.g., as described herein) and feeder cells (e.g., as described herein). Also described herein are bioreactors comprising expanded natural killer cell populations.

[0367] culture medium

[0368] Disclosed herein are culture media for expanding NK cells. These culture media facilitate the expansion of the NK cells to a quantity suitable for preparing the pharmaceutical compositions described herein. In some cases, the culture media allow the NK cells to expand without losing CD16 expression, and without the loss of CD16 expression that is typically associated with cell expansion on other helper cells or in other culture media.

[0369] In certain embodiments, the culture medium is a basal medium, optionally supplemented with additional components, eg, as described herein.

[0370] In certain embodiments, the culture medium, such as a basal medium, is a serum-free culture medium. In certain embodiments, the culture medium, such as a basal medium, is a serum-free culture medium supplemented with human plasma and / or serum.

[0371] Suitable basal culture media include, but are not limited to, DMEM, RPMI 1640, MEM, DMEM / F12, SCGM ( Company, 20802-0500 or 20806-0500), LGM-3 TM (Lonza, CC-3211), TexMA CS TM (Miltenyi Biotec, 130-097-196), AlyS TM 505NK-AC (Cell Science Research Institute Co., Ltd., 01600P02), AlyS TM 505NK-EX (Cell Science Research Institute Co., Ltd., 01400P10), CTS TM AIM-V TM S FM (Thermo Fisher Scientific, A3830801), CTS TM OpTmizer TM (Thermo Fisher Scientific, A1048501, AB S-001, StemXxVivo and combinations thereof.

[0372] The culture medium may contain additional components, or supplement additional components, such as growth factors, signaling factors, nutrients, antigen binding agents, etc. The supplementation of the culture medium can be achieved by adding one or more additional components to the culture vessel before, simultaneously with, or after the culture medium is added to the culture vessel. The one or more additional components can be added together or separately. When added separately, multiple additional components do not need to be added simultaneously.

[0373] In certain embodiments, the culture medium comprises plasma, such as human plasma. In certain embodiments, the culture medium is supplemented with plasma, such as human plasma. In certain embodiments, the plasma, such as human plasma, comprises an anticoagulant, such as trisodium citrate.

[0374] In certain embodiments, the culture medium comprises and / or is supplemented with from at or about 0.5% to at or about 10% plasma, such as human plasma, by volume. In certain embodiments, the culture medium is supplemented with from 0.5% or about 0.5% to 9% or about 9%, from 0.5% or about 0.5% to 8% or about 8%, from 0.5% or about 0.5% to 7% or about 7%, from 0.5% or about 0.5% to 6% or about 6%, from 0.5% or about 0.5% to 5% or about 5%, from 0.5% or about 0.5% to 4% or about 4%, from 0.5% or about 0.5% to 3% or about 3%, from 0.5% or about 0.5% to 2% or about 2%, from 0.5% or about 0.5% to 1% or about 1%, from 1% or about 1% to 10% or about 10%, from 1% or about 1% to 9% or about 9 ... from 1% or about 1% to 8% or about 8%, from 1% or about 1% to 7% or about 7%, from 1% or about 1% to 6% or about 6%, from 1% or about 1% to 5% or about 5%, from 1% or about 1% to 4% or about 4%, from 1% or about 1% to 3% or about 3%, from 1% or about 1% to 2% or about 2%, from 2% or about 2% to 10% or about 10%, from 2% or about 2% to 9% or about 9%, from 2% or about 2% to 8% or about 8%, from 2% or about 2% to 7% or about 7%, from 2% or about 2% to 6% or about 6%, from 2% or about 2% to 5% or about 5%, from 2% or about 2% to 4% or about 4%, from 2% or about 2% to 3% or about 3%, from 2% or about 2% to 3% or about 3%, from 2% or about 2% to 4% or about 4%, from 2% or about 2% to 3% or about 3%, from 2% or about 2% to 9% or about 9%, from 2% or about 2% to 8% or about 8%, from 2% or about 2% to 7% or about 7%, from 2% or about 2% to 6% or about 6%, from 2% or about 2% to 5% or about 5%, from 2% or about 2% to 4% or about 4%, from 2% or about 2% to 3% or about 3%, from 2% or about 2% to 3% or about 3%, from 2% or about 2% to 4% or about 4%. 3% or about 3% to 10% or about 10%, from 3% or about 3% to 9% or about 9%, from 3% or about 3% to 8% or about 8%, from 3% or about 3% to 7% or about 7%, from 3% or about 3% to 6% or about 6%, from 3% or about 3% to 5% or about 5%, from 3% or about 3% to 4% or about 4%, from 4% or about 4% to 10% or about 10%, from 4% or about 4% to 9% or about 9%, from 4% or about 4% to 8% or about 8%, from 4% or about 4% to 7% or about 7%, from 4% or about 4% to 6% or about 6%, from 4% or about 4% to 5% or about 5%, from 5% or about 5% to 10% or about 10%, from 5% or about 5% to In some embodiments, the present invention provides plasma, for example, human plasma, which is 9% or about 9%, from 5% or about 5% to 8% or about 8%, from 5% or about 5% to 7% or about 7%, from 5% or about 5% to 6% or about 6%, from 6% or about 6% to 10% or about 10%, from 6% or about 6% to 9% or about 9%, from 6% or about 6% to 8% or about 8%, from 6% or about 6% to 7% or about 7%, from 7% or about 7% to 10% or about 10%, from 7% or about 7% to 9% or about 9%, from 7% or about 7% to 8% or about 8%, from 8% or about 8% to 10% or about 10%, from 8% or about 8% to 9% or about 9%, or from 9% or about 9% to 10% or about 10%, by volume, of plasma, for example, human plasma. In certain embodiments, the culture medium comprises and / or is supplemented with 0.8% to 1.2% by volume human plasma.In certain embodiments, the culture medium comprises and / or is supplemented with 1.0% by volume human plasma. In certain embodiments, the culture medium comprises and / or is supplemented with about 1.0% by volume human plasma.

[0375] In certain embodiments, the culture medium includes serum, such as human serum. In certain embodiments, the culture medium is supplemented with serum, such as human serum. In certain embodiments, the serum is inactivated, such as by heat inactivation. In certain embodiments, the serum is filtered, such as by sterile filtration.

[0376] In certain embodiments, the culture medium includes glutamine. In certain embodiments, the culture medium is supplemented with glutamine. In certain embodiments, the culture medium comprises and / or is supplemented with from 2 mM or about 2 mM to 6 mM or about 6 mM glutamine.In certain embodiments, the culture medium comprises and / or is supplemented with from 2 mM or about 2 mM to 5.5 mM or about 5.5 mM, from 2 mM or about 2 mM to 5.0 mM or about 5.0 mM, from 2 mM or about 2 mM to 5.0 mM or about 5.0 mM, M or about 2 mM to 4.5 mM or about 4.5 mM, from 2 mM or about 2 mM to 4.0 mM or about 4.0 mM, from 2 mM or about 2 mM to 3.5 mM or about 3.5 mM, from 2 mM or about 2 mM to 3.0 mM or about 3.0 mM, from 2 mM or about 2 mM to 2.5 mM or about 2.5 mM, from 2.5 mM or about 2.5 mM to 6.0 mM or about 6.0 mM, from 2.5 mM or about 2.5 mM to 5.5 mM or about 5.5 mM, from 2.5 mM or about 2.5 mM to 5.0 mM or about 5.0 mM, from 2.5 mM or about 2.5 mM to 4.5 mM or about 4.5 mM, from 2.5 mM or about 2.5 mM to 4.0 mM or about 4.0 mM. mM, from 2.5 mM or about 2.5 mM to 3.5 mM or about 3.5 mM, from 2.5 mM or about 2.5 mM to 3.0 mM or about 3.0 mM, from 3.0 mM or about 3.0 mM to 6.0 mM or about 6.0 mM, from 3.0 mM or about 3.0 mM to 5.5 mM or about 5.5 mM, from 3.0 mM or about 3.0 mM to 5.0 mM or about 5.0 mM, from 3.0 mM or about 3.0 mM to 4.5 mM or about 4.5 mM, from 3.0 mM or about 3.0 mM to 4.0 mM or about 4.0 mM, from 3.0 mM or about 3.0 mM to 3.5 mM or about 3.5 mM, from 3.5 mM or about 3.5 mM to 6.0 mM or about 6.0 mM M, from 3.5 mM or about 3.5 mM to 5.5 mM or about 5.5 mM, from 3.5 mM or about 3.5 mM to 5.0 mM or about 5.0 mM, from 3.5 mM or about 3.5 mM to 4.5 mM or about 4.5 mM, from 3.5 mM or about 3.5 mM to 4.0 mM or about 4.0 mM, from 4.0 mM or about 4.0 mM to 6.0 mM or about 6.0 mM, from 4.0 mM or about 4.0 mM to 7.0 mM or about 7.0 mM, from 4.0 mM or about 4.0 mM to 8.0 mM or about 8.0 mM, from 4.0 mM or about 4.0 mM to 9.0 mM or about 9.0 mM, from 4.0 mM or about 4.0 mM to 5.0 mM or about 5 ... In some embodiments, the present invention provides a glutamine supplement of at least 5.5 mM, glutamine at or about 5.5 mM, from 4.0 mM or about 4.0 mM to 5.0 mM or about 5.0 mM, from 4.0 mM or about 4.0 mM to 4.5 mM or about 4.5 mM, from 4.5 mM or about 4.5 mM to 6.0 mM or about 6.0 mM, from 4.5 mM or about 4.5 mM to 5.5 mM or about 5.5 mM, from 4.5 mM or about 4.5 mM to 5.0 mM or about 5.0 mM, from 5.0 mM or about 5.0 mM to 6.0 mM or about 6.0 mM, from 5.0 mM or about 5.0 mM to 5.5 mM or about 5.5 mM, or from 5.5 mM or about 5.5 mM to 6.0 mM or about 6.0 mM. In certain embodiments, the culture medium comprises and / or is supplemented with 3.2 mM to 4.8 mM glutamine.In certain embodiments, the culture medium comprises and / or is supplemented with 4.0 mM glutamine. In certain embodiments, the culture medium comprises and / or is supplemented with about 4.0 mM glutamine.

[0377] In certain embodiments, the culture medium comprises one or more cytokines. In certain embodiments, the culture medium is supplemented with one or more cytokines.

[0378] In certain embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, and combinations thereof.

[0379] In certain embodiments, the culture medium comprises and / or is supplemented with IL-2. In certain embodiments, the culture medium comprises and / or is supplemented with from 150 IU / mL or about 150 IU / mL to 2,500 IU / mL or about 2,500 IU / mL of IL-2. In certain embodiments, the culture medium comprises and / or is supplemented with from 200 IU / mL or about 200 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, from 200 IU / mL or about 200 IU / mL to 2,000 IU / mL or about 2,000 IU / mL. L, from 200 IU / mL or about 200 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 200 IU / mL or about 200 IU / mL to 1,500 IU / mL or about 1,500 IU / mL, from 200 IU / mL or about 200 IU / mL to 1,250 IU / mL or about 1,250 IU / mL, from 200 IU / mL or about 200 IU / mL to 1,000 IU / mL or about 1,000 IU / mL, from 200 IU / mL or about 200 IU / mL to 1,000 IU / mL or about 1,000 IU / mL, from 200 IU / mL or about 200 IU / mL to 1,000 IU / mL or about 1,000 IU / mL, mL to 750 IU / mL or about 750 IU / mL, from 200 IU / mL or about 200 IU / mL to 500 IU / mL or about 500 IU / mL, from 200 IU / mL or about 200 IU / mL to 250 IU / mL or about 250 IU / mL, from 250 IU / mL or about 250 IU / mL to 2,500 IU / mL or about 2,500 IU / mL, from 250 IU / mL or about 250 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, from 250 IU / mL or about 250 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, U / mL or about 250 IU / mL to 2,000 IU / mL or about 2,000 IU / mL, from 250 IU / mL or about 250 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 250 IU / mL or about 250 IU / mL to 1,500 IU / mL or about 1,500 IU / mL, from 250 IU / mL or about 250 IU / mL to 1,250 IU / mL or about 1,250 IU / mL, from 250 IU / mL or about 250 IU / mL to 1,00 1,000 IU / mL, from 250 IU / mL or about 250 IU / mL to 750 IU / mL or about 750 IU / mL, from 250 IU / mL or about 250 IU / mL to 500 IU / mL or about 500 IU / mL, from 500 IU / mL or about 500 IU / mL to 2,500 IU / mL or about 2,500 IU / mL, from 500 IU / mL or about 500 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, from 500 IU / mL or about 500 IU / mL to 2,000 IU / mL or about 2,000 IU / mL.000 IU / mL, from 500 IU / mL or about 500 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 500 IU / mL or about 500 IU / mL to 1,500 IU / mL or about 1,500 IU / mL L, from 500 IU / mL or about 500 IU / mL to 1,250 IU / mL or about 1,250 IU / mL, from 500 IU / mL or about 500 IU / mL to 1,000 IU / mL or about 1,000 IU / mL, from 500 IU / mL or about 500 IU / mL to 750 IU / mL or about 750 IU / mL, from 750 IU / mL or about 750 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, from 750 IU / mL or about 750 IU / mL to 2,000 IU / mL or about 2,000 IU / mL, from 750 IU / mL or about 750 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, 0 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 750 IU / mL or about 750 IU / mL to 1,500 IU / mL or about 1,500 IU / mL, from 750 IU / mL or about 750 IU / mL to 1,250 IU / mL or about 1,250 IU / mL, from 750 IU / mL or about 750 IU / mL to 1,000 IU / mL or about 1,000 IU / mL, from 1000 IU / mL or about 1000 IU / mL to 2,500 IU / mL or about 2,500 IU / mL, from 1000 IU / mL or about 1000 IU / mL to 2 1,250 IU / mL or about 1,250 IU / mL, from 1,000 IU / mL or about 1,000 IU / mL to 2,000 IU / mL or about 2,000 IU / mL, from 1,000 IU / mL or about 1,000 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 1,000 IU / mL or about 1,000 IU / mL to 1,500 IU / mL or about 1,500 IU / mL, from 1,000 IU / mL or about 1,000 IU / mL to 1,250 IU / mL or about 1,250 IU / mL, from 1,250 IU / mL or about 1,250 IU / mL to 2, 1,500 IU / mL or about 1,500 IU / mL, from 1,250 IU / mL or about 1,250 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, from 1,250 IU / mL or about 1,250 IU / mL to 2,000 IU / mL or about 2,000 IU / mL, from 1,250 IU / mL or about 1,250 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 1,250 IU / mL or about 1,250 IU / mL to 1,500 IU / mL or about 1,500 IU / mL, from 1,500 IU / mL or about 1,500 IU / mL to 2,2,500 IU / mL or about 2,500 IU / mL, from 1,500 IU / mL or about 1,500 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, from 1,500 IU / mL or about 1,500 IU / mL to 2,000 IU / mL or about 2,000 IU / mL, from 1,500 IU / mL or about 1,500 IU / mL to 1,750 IU / mL or about 1,750 IU / mL, from 1,750 IU / mL or about 1,750 IU / mL to 2,500 IU / mL or about 2,500 IU / mL, from 1,750 IU / mL or about 1,750 IU / mL to 2,500 IU / mL or about 2,500 IU / mL, 2,250 IU / mL or about 2,250 IU / mL, from 1,750 IU / mL or about 1,750 IU / mL to 2,000 IU / mL or about 2,000 IU / mL, from 2,000 IU / mL or about 2,000 IU / mL to 2,500 IU / mL or about 2,500 IU / mL, from 2,000 IU / mL or about 2,000 IU / mL to 2,250 IU / mL or about 2,250 IU / mL, or from 2,250 IU / mL or about 2,250 IU / mL to 2,500 IU / mL or about 2,500 IU / mL.

[0380] In certain embodiments, the culture medium comprises and / or is supplemented with 64 μg / L to 96 μg / L of IL-2. In certain embodiments, the culture medium comprises and / or is supplemented with 80 μg / L of IL-2 (approximately 1,333 IU / mL). In certain embodiments, the culture medium comprises and / or is supplemented with about 80 μg / L of IL-2.

[0381] In certain embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2 and IL-15.

[0382] In certain embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2, IL-15, and IL-18.

[0383] In certain embodiments, the culture medium comprises and / or is supplemented with a combination of IL-2, IL-18, and IL-21.

[0384] In certain embodiments, the culture medium comprises and / or is supplemented with glucose. In certain embodiments, the culture medium comprises and / or is supplemented with glucose from 0.5 g / L or about 0.5 g / L to 3.5 g / L or about 3.5 g / L. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.5 g / L or about 0.5 g / L to 3.0 g / L or about 3.0 g / L, from 0.5 g / L or about 0.5 g / L to 2.5 g / L or about 2.5 g / L, from 0.5 g / L or about 0.5 g / L to 2.0 g / L or about 2.0 g / L, from 0.5 g / L or about 0.5 g / L to 1.5 g / L or about 1.5 g / L, from 0.5 g / L or about 0.5 g / L to 1.0 g / L or about 1.0 g / L, from 1.0 g / L or about 1.0 g / L to 3.0 g / L or about 3.0 g / L, from 1.0 g / L or about 1.0 g / L to 2.5 g / L or about 2.5 g / L, from 1.0 g / L or about 1.0 g / L to 3.0 g / L or about 3.0 g / L or about 2.5 g / L, from 1.5 g / L or about 1.5 g / L to 3.0 g / L or about 3.0 g / L, from 1.5 g / L or about 1.5 g / L to 2.5 g / L or about 2.5 g / L, from 1.5 g / L or about 1.5 g / L to 2.0 g / L or about 2.0 g / L, from 2.0 g / L or about 2.0 g / L to 3.0 g / L or about 3.0 g / L, from 2.0 g / L or about 2.0 g / L to 2.5 g / L or about 2.5 g / L, or from 2.5 g / L or about 2.5 g / L to 3.0 g / L or about 3.0 g / L of glucose. In certain embodiments, the culture medium comprises and / or is supplemented with 1.6 g / L to 2.4 g / L of glucose. In certain embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of glucose. In certain embodiments, the culture medium comprises and / or is supplemented with about 2.0 g / L of glucose.

[0385] In certain embodiments, the culture medium comprises and / or is supplemented with sodium pyruvate. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.1 mM or about 0.1 mM to 2.0 mM or about 2.0 mM sodium pyruvate. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.1 mM or about 0.1 mM to 1.8 mM or about 1.8 mM, from 0.1 mM or about 0.1 mM to 1.6 mM or about 1.6 mM, from 0.1 mM or about 0.1 mM to 1.4 mM or about 1.4 mM, from 0.1 mM or about 0.1 mM to 1.2 mM or about 1.2 mM, from 0.1 mM or about 0.1 mM to 1.0 mM or about 1.0 mM, from 0.1 mM or about 0.1 mM to 0.8 mM or about 0.8 mM, from 0.1 mM or about 0.1 mM to 0.6 mM or about 0.6 mM, from 0.1 mM or about 0.1 mM to 0.4 mM or about 0.4 mM, from 0.1 mM or about 0.1 mM to 0.4 mM or about 0.4 mM, M or about 0.1 mM to 0.2 mM or about 0.2 mM, from 0.2 mM or about 0.2 mM to 2.0 mM or about 2.0 mM, from 0.2 mM or about 0.2 mM to 1.8 mM or about 1.8 mM, from 0.2 mM or about 0.2 mM to 1.6 mM or about 1.6 mM, from 0.2 mM or about 0.2 mM to 1.4 mM or about 1.4 mM, from 0.2 mM or about 0.2 mM to 1.4 mM or about 1.4 mM, from 0.2 mM or about 0.2 mM to 1.8 mM or about 1.8 mM, M or about 0.2 mM to 1.2 mM or about 1.2 mM, from 0.2 mM or about 0.2 mM to 1.0 mM or about 1.0 mM, from 0.2 mM or about 0.2 mM to 0.8 mM or about 0.8 mM, from 0.2 mM or about 0.2 mM to 0.6 mM or about 0.6 mM, from 0.2 mM or about 0.2 mM to 0.4 mM or about 0.4 mM, from 0.4 mM or about 0.4 ...4 mM or about 0.4 mM, from 0.2 mM or about 0.2 mM to 0.8 mM or about 0.8 mM, from 0.2 mM or about 0.2 mM to 0.6 mM or about 0.6 mM, from 0.2 mM or about 0.2 mM to 0.4 mM or about 0.4 mM, from 0.2 mM or about 0.2 mM to 0.4 mM or about 0.4 mM, from 0.2 mM or about 0.2 mM to 0.4 mM or about 0.4 mM, from 0.2 mM or about 0.2 mM to 0.8 mM or about 0.8 mM, M or about 0.4 mM to 2.0 mM or about 2.0 mM, from 0.4 mM or about 0.4 mM to 1.8 mM or about 1.8 mM, from 0.4 mM or about 0.4 mM to 1.6 mM or about 1.6 mM, from 0.4 mM or about 0.4 mM to 1.4 mM or about 1.4 mM, from 0.4 mM or about 0.4 mM to 1.2 mM or about 1.2 mM, from 0.4 mM or about 0.4 mM to 1.6 mM or about 1.6 mM, M or about 0.4 mM to 1.0 mM or about 1.0 mM, from 0.4 mM or about 0.4 mM to 0.8 mM or about 0.8 mM, from 0.4 mM or about 0.4 mM to 0.6 mM or about 0.6 mM, from 0.6 mM or about 0.6 mM to 2.0 mM or about 2.0 mM, from 0.6 mM or about 0.6 mM to 1.8 mM or about 1.8 ... M or about 0.6 mM to 1.6 mM or about 1.6 mM, from 0.6 mM or about 0.6 mM to 1.4 mM or about 1.4 mM, from 0.6 mM or about 0.6 mM to 1.2 mM or about 1.2 mM, from 0.6 mM or about 0.6 mM to 1.0 mM or about 1.0 mM, from 0.6 mM or about 0.6 mM to 0.8 mM or about 0.8 mM, from 0.6 mM or about 0.6 mM to 0.8 mM or about 0.8 mM, from 0.6 mM or about 0.6 mM to 1.2 mM or about 1.2 mM,8 mM or about 0.8 mM to 2.0 mM or about 2.0 mM, from 0.8 mM or about 0.8 mM to 1.8 mM or about 1.8 mM, from 0.8 mM or about 0.8 mM to 1.6 mM or about 1.6 mM, from 0.8 mM or about 0.8 mM to 1.4 mM or about 1.4 mM, from 0.8 mM or about 0.8 mM to 1.2 mM or about 1.2 mM, from 0.8 mM or about 0.8 mM to 1.0 mM or about 1.0 mM, from 1.0 mM or about 1.0 mM to 2.0 mM or about 2.0 mM, from 1.0 mM or about 1.0 mM to 1.8 mM or about 1.8 mM, mM or about 1.8 mM, from 1.0 mM or about 1.0 mM to 1.6 mM or about 1.6 mM, from 1.0 mM or about 1.0 mM to 1.4 mM or about 1.4 mM, from 1.0 mM or about 1.0 mM to 1.2 mM or about 1.2 mM, from 1.2 mM or about 1.2 mM to 2.0 mM or about 2.0 mM, from 1.2 mM or about 1.2 mM to 1.8 mM or about 1.8 mM, from 1.2 mM or about 1.2 mM to 1.6 mM or about 1.6 mM, from 1.2 mM or about 1.2 mM to 1.4 mM or about 1.4 mM, from 1.4 mM or about 1.4 mM to 1.2 mM or about 1.2 mM. In some embodiments, the culture medium comprises and / or is supplemented with 0.8 mM to 1.2 mM sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with 1.0 mM sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with about 1.0 mM sodium pyruvate. In some embodiments, the culture medium comprises and / or is supplemented with about 1.0 mM sodium pyruvate.

[0386] In certain embodiments, the culture medium comprises and / or is supplemented with sodium bicarbonate. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.5 g / L or about 0.5 g / L to 3.5 g / L or about 3.5 g / L of sodium bicarbonate. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.5 g / L or about 0.5 g / L to 3.0 g / L or about 3.0 g / L, from 0.5 g / L or about 0.5 g / L to 2.5 g / L or about 2.5 g / L, from 0.5 g / L or about 0.5 g / L to 2.0 g / L or about 2.0 g / L, from 0.5 g / L or about 0.5 g / L to 1.5 g / L or about 1.5 g / L, from 0.5 g / L or about 0.5 g / L to 1.0 g / L or about 1.0 g / L, from 1.0 g / L or about 1.0 g / L to 3.0 g / L or about 3.0 g / L, from 1.0 g / L or about 1.0 g / L to 2.5 g / L or about 2.5 g / L, from 1.0 g / L or about 1.0 g / L to 3.0 g / L or about 3.0 g / L about 1.0 g / L to 2.0 g / L or about 2.0 g / L, from 1.0 g / L or about 1.0 g / L to 1.5 g / L or about 1.5 g / L, from 1.5 g / L or about 1.5 g / L to 3.0 g / L or about 3.0 g / L, from 1.5 g / L or about 1.5 g / L to 2.5 g / L or about 2.5 g / L, from 1.5 g / L or about 1.5 g / L to 3.0 g / L or about 3.0 g / L, In certain embodiments, the culture medium comprises and / or is supplemented with 1.6 g / L to 2.4 g / L of sodium bicarbonate. In certain embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of sodium bicarbonate. In certain embodiments, the culture medium comprises and / or is supplemented with 2.0 g / L of sodium bicarbonate. In certain embodiments, the culture medium comprises and / or is supplemented with about 2.0 g / L of sodium bicarbonate.

[0387] In certain embodiments, the culture medium comprises and / or is supplemented with albumin, e.g., human albumin, e.g., a human albumin solution described herein. In certain embodiments, the culture medium comprises and / or is supplemented with from at or about 0.5% to at or about 3.0% by volume of a 20% albumin solution, e.g., a 20% human albumin solution. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.5% or about 0.5% to 3.0% or about 3.0%, from 0.5% or about 0.5% to 2.5% or about 2.5%, from 0.5% or about 0.5% to 2.0% or about 2.0%, from 0.5% or about 0.5% to 1.5% or about 1.5%, from 0.5% or about 0.5% to 1.0% or about 1.0%, from 1.0% or about 1.0% to 3.0% or about 3.0%, from 1.0% or about 1.0% to 2.5% or about 2.5%, from 1.0% or about 1.0% to 2.0% or about 2.0%. In some embodiments, the culture medium comprises and / or is supplemented with 1.6% to 2.4% by volume of a 20% albumin solution, for example, a 20% human albumin solution. In some embodiments, the culture medium comprises and / or is supplemented with 1.6% to 2.4% by volume of a 20% albumin solution, for example, a 20% human albumin solution. In certain embodiments, the culture medium comprises and / or is supplemented with 2.0% by volume of a 20% albumin solution, e.g., a 20% human albumin solution. In certain embodiments, the culture medium comprises and / or is supplemented with about 2.0% by volume of a 20% albumin solution, e.g., a 20% human albumin solution.

[0388] In certain embodiments, the culture medium comprises and / or is supplemented with from 2 g / L or about 2 g / L to 6 g / L or about 6 g / L albumin, e.g., human albumin. In certain embodiments, the culture medium comprises and / or is supplemented with from 2 g / L or about 2 g / L to 5.5 g / L or about 5.5 g / L, from 2 g / L or about 2 g / L to 5.0 g / L or about 5.0 g / L, from 2 g / L or about 2 g / L to 4.5 g / L or about 4.5 g / L, from 2 g / L or about 2 g / L to 4.0 g / L or about 4.0 g / L, from 2 g / L or about 2 g / L to 3.5 g / L or about 3.5 g / L, from 2 g / L or about 2 g / L to 3.0 g / L or about 3.0 g / L, from 2 g / L or about 2 g / L to 2.5 g / L or about 2.5 g / L, from 2 g / L or about 2 g / L to 3.0 g / L or about 3.0 g / L, g / L, from 2.5 g / L or about 2.5 g / L to 5.5 g / L or about 5.5 g / L, from 2.5 g / L or about 2.5 g / L to 5.0 g / L or about 5.0 g / L, from 2.5 g / L or about 2.5 g / L to 4.5 g / L or about 4.5 g / L, from 2.5 g / L or about 2.5 g / L to 4.0 g / L or about 4.0 g / L, from 2.5 g / L or about 2.5 g / L to 3.5 g / L or about 3.5 g / L, from 2.5 g / L or about 2.5 g / L to 3.0 g / L or about 3.0 g / L, from 3.0 g / L or about 3.0 g / L g / L or about 4.0 g / L, from 3.0 g / L or about 3.0 g / L to 3.5 g / L or about 3.5 g / L, from 3.5 g / L or about 3.5 g / L to 6 g / L or about 6 g / L, from 3.0 g / L or about 3.0 g / L to 5.5 g / L or about 5.5 g / L, from 3.0 g / L or about 3.0 g / L to 5.0 g / L or about 5.0 g / L, from 3.0 g / L or about 3.0 g / L to 4.5 g / L or about 4.5 g / L, from 3.0 g / L or about 3.0 g / L to 4.0 g / L or about 4.0 g / L, from 3.0 g / L or about 3.0 g / L to 3.5 g / L or about 3.5 g / L, from 3.5 g / L or about 3.5 g / L to 6 g / L or about 6 g / L, and from 3.5 g / L or about 3.5 g / L to 5.5 g / L or about 5 g / L or about 4.0 g / L, from 3.5 g / L or about 3.5 g / L to 4.0 g / L or about 4.0 g / L, from 4.0 g / L or about 4.0 g / L to 6 g / L or about 6 g / L, from 4.0 g / L or about 4.0 g / L to 5.5 g / L or about 5.5 g / L, from 4.0 g / L or about 4.0 g / L to 5.0 g / L or about 5.0 g / L, from 4.0 g / L or about 4.0 g / L to 4.5 g / L or about 4.5 g / L, from 3.5 g / L or about 3.5 g / L to 4.0 g / L or about 4.0 g / L,

[0389] In some embodiments, the culture medium comprises and / or is supplemented with 3.2 g / L to 4.8 g / L of albumin, for example, human albumin. In some embodiments, the culture medium comprises and / or is supplemented with 4 g / L to 5.5 g / L, for example, human albumin. In some embodiments, the culture medium comprises and / or is supplemented with 4 g / L of albumin, for example, human albumin. In certain embodiments, the culture medium comprises and / or is supplemented with about 4 g / L of albumin, e.g., human albumin.

[0390] In certain embodiments, the culture medium comprises and / or is supplemented with Poloxamer 188. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.1 g / L or about 0.1 g / L to 2.0 g / L or about 2.0 g / L of Poloxamer 188. In certain embodiments, the culture medium comprises and / or is supplemented with from 0.1 g / L or about 0.1 g / L to 1.8 g / L or about 1.8 g / L, from 0.1 g / L or about 0.1 g / L to 1.6 g / L or about 1.6 g / L, from 0.1 g / L or about 0.1 g / L to 1.4 g / L or about 1.4 g / L, from 0.1 g / L or about 0.1 g / L to 1.2 g / L or about 1.2 g / L, from 0.1 g / L or about 0.1 g / L to 1.0 g / L or about 1.0 g / L, from 0.1 g / L or about 0.1 g / L to 0.8 g / L or about 0.8 g / L, from 0.1 g / L or about 0.1 g / L to 0.1 g / L or about 0.1 g / L. / L to 0.6g / L or about 0.6g / L, from 0.1g / L or about 0.1g / L to 0.4g / L or about 0.4g / L, from 0.1g / L or about 0.1g / L to 0.2g / L or about 0.2g / L, from 0.2g / L or about 0.2g / L to 2.0g / L or about 2.0g / L, from 0.2g / L or about 0.2g / L to 1.8g / L or about 1.8g / L, from 0.2g / L or about 0.2g / L to 1.6g / L or about 1.6g / L, from 0.2g / L or about 0.2g / L to 1.4g / L or about 1.4g / L, and from 0.2g / L or about 0.2g / L to 1. In some embodiments, the present invention relates to a method for the treatment of a disease in which the patient's body is damaged or missing, and the patient's body is damaged or missing. In some embodiments, the patient's body is damaged or missing, and the patient's body is damaged or missing. In some embodiments, the patient's body is damaged or missing, and the patient's body is damaged or missing. In some embodiments, the patient's body is damaged or missing, and the patient's body is damaged or missing. In some embodiments, the patient's body is damaged or missing, and the patient's body is damaged or missing. In some embodiments, the patient's body is damaged or missing, and the patient's body is damaged or missing. In some embodiments, the patient's body is damaged or missing, and the patient's body is damaged or missing. L or about 1.6 g / L, from 0.4 g / L or about 0.4 g / L to 1.4 g / L or about 1.4 g / L, from 0.4 g / L or about 0.4 g / L to 1.2 g / L or about 1.2 g / L, from 0.4 g / L or about 0.4 g / L to 1.0 g / L or about 1.0 g / L, from 0.4 g / L or about 0.4 g / L to 0.8 g / L or about 0.8 g / L, from 0.4 g / L or about 0.4 g / L to 0.6 g / L or about 0.6 g / L, from 0.6 g / L or about 0.6 g / L to 2.0 g / L or about 2.0 g / L, and from 0.6 g / L or about 0.6 g / L to 1.8 g / L or about 1.8g / L, from 0.6g / L or about 0.6g / L to 1.6g / L or about 1.6g / L, from 0.6g / L or about 0.6g / L to 1.4g / L or about 1.4g / L, from 0.6g / L or about 0.6g / L to 1.2g / L or about 1.2g / L, from 0.6g / L or about 0.6g / L to 1.0g / L or about 1.0g / L, from 0.6g / L or about 0.6g / L to 0.8g / L or about 0.8g / L, from 0.8g / L or about 0.8g / L to 2.0g / L or about 2.0g / L, from 0.8g / L or about 0.8g / L to 1.2g / L or about 1.2g / L, L to 1.8 g / L or about 1.8 g / L, from 0.8 g / L or about 0.8 g / L to 1.6 g / L or about 1.6 g / L, from 0.8 g / L or about 0.8 g / L to 1.4 g / L or about 1.4 g / L, from 0.8 g / L or about 0.8 g / L to 1.2 g / L or about 1.2 g / L, from 0.8 g / L or about 0.8 g / L to 1.0 g / L or about 1.0 g / L, from 1.0 g / L or about 1.0 g / L to 2.0 g / L or about 2.0 g / L, from 1.0 g / L or about 1.0 g / L to 1.8 g / L or about 1.8 g / L, from 1.0 g / L or about 1.0 g / L to 2.0 g / L or about 2.0 g / L, g / L, from 1.2 g / L or about 1.2 g / L to 2.0 g / L or about 2.0 g / L, from 1.2 g / L or about 1.2 g / L to 1.8 g / L or about 1.8 g / L, from 1.2 g / L or about 1.2 g / L to 1.6 g / L or about 1.6 g / L, from 1.2 g / L or about 1.2 g / L to 1.4 g / L or about 1.4 g / L, In some embodiments, the culture medium comprises and / or is supplemented with 0.8 g / L to 1.2 g / L of Poloxamer 188. In some embodiments, the culture medium comprises and / or is supplemented with 0.8 g / L to 1.2 g / L of Poloxamer 188. In certain embodiments, the culture medium comprises and / or is supplemented with 1.0 g / L of Poloxamer 188. In certain embodiments, the culture medium comprises and / or is supplemented with about 1.0 g / L of Poloxamer 188.

[0391] In certain embodiments, the culture medium contains and / or is supplemented with one or more antibiotics.

[0392] Table 1 lists a first exemplary culture medium.

[0393] Table 1 Exemplary Culture Medium #1

[0394] Components Exemplary concentration ranges Exemplary concentrations CellgroSCGM liquid culture medium Undiluted Undiluted human plasma 0.8–1.2% (volume ratio) 1.0% volume ratio Glutamine 3.2–4.8 mM 4.0mM IL-2 64–96 μg / L 80 μg / L

[0395] Table 2 lists a second exemplary culture medium.

[0396] Table 2 Exemplary Medium #2

[0397]

[0398]

[0399] CD3-binding antibodies

[0400] In certain embodiments, the culture medium comprises and / or is supplemented with a CD3-binding antibody or antigen-binding fragment thereof. In certain embodiments, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1, and HIT3a, or variants thereof. In certain embodiments, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof.

[0401] In certain embodiments, the CD3 binding antibody or antigen-binding fragment thereof and feeder cells are added to the culture vessel prior to adding NK cells and / or cell culture medium.

[0402] In certain embodiments, the culture medium comprises and / or is supplemented with from 5 ng / mL or about 5 ng / mL to 15 ng / mL or about 15 ng / mL of OKT3. In certain embodiments, the culture medium comprises and / or is supplemented with from 5 ng / mL or about 5 ng / mL to 12.5 ng / mL or about 12.5 ng / mL, from 5 ng / mL or about 5 ng / mL to 10 ng / mL or about 10 ng / mL, from 5 ng / mL or about 5 ng / mL to 7.5 ng / mL or about 7.5 ng / mL, from 7.5 ng / mL or about 7.5 ng / mL to 15 ng / mL or about 15 ng / mL, from 7.5 ng / mL or about 7.5 ng / mL to 12.5 ng / mL or about 12.5 ng / mL. In some embodiments, the culture medium comprises and / or is supplemented with 10 ng / mL of OKT3. In some embodiments, the culture medium comprises and / or is supplemented with about 10 ng / mL of OKT3. In some embodiments, the culture medium comprises and / or is supplemented with about 10 ng / mL of OKT3.

[0403] Culture container

[0404] Many containers are consistent with the present disclosure.In certain embodiments, the culture vessel is selected from the group consisting of a flask, a bottle, a petri dish, a multi-walled plate, a roller bottle, a bag, and a bioreactor.

[0405] In certain embodiments, the culture vessel is treated to render it hydrophilic. In certain embodiments, the culture vessel is treated to promote attachment and / or proliferation. In certain embodiments, the culture vessel is coated with serum, collagen, laminin, gelatin, poly-L-lysine, fibronectin, extracellular matrix proteins, or combinations thereof.

[0406] In certain embodiments, different types of culture vessels are used for different culture stages.

[0407] In certain embodiments, the culture vessel has a volume of between 100 mL or about 100 mL and 1,000 L or about 1,000 L. In certain embodiments, the culture vessel has a volume of at or about 125 mL, at or about 250 mL, at or about 500 mL, at or about 1 L, at or about 5 L, at or about 10 L, or at or about 20 L.

[0408] In certain embodiments, the culture vessel is a bioreactor.

[0409] In certain embodiments, the bioreactor is a shaking (wave) bioreactor. In certain embodiments, the bioreactor is a stirred bioreactor. In certain embodiments, the bioreactor is a rotating wall container. In certain embodiments, the bioreactor is a perfusion bioreactor. In certain embodiments, the bioreactor is an isolation / expansion automation system. In certain embodiments, the bioreactor is an automated or semi-automatic bioreactor. In certain embodiments, the bioreactor is a disposable bag bioreactor.

[0410] In certain embodiments, the bioreactor has a volume of about 100 mL to about 1,000 L. In certain embodiments, the bioreactor has a volume of about 10 L to about 1,000 L. In certain embodiments, the bioreactor has a volume of about 100 L to about 1,000 L. In certain embodiments, the bioreactor has a volume of about 10 L to about 800 L. In certain embodiments, the bioreactor has a volume of 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 1,000 L, about 20 L to about 900 L, about 20 L to about 800 L, about 20 L to about 100 L, about 20 L to about 800 L, about 20 L to about 900 L, about 20 L to about 800 L, about 20 L to about 100 L, about 20 L to about 100 L, about 20 L to about 100 L, about 20 L to about 8 ...800 L, about 20 L to about 0L to about 700L, about 20L to about 600L, about 20L to about 500L, about 20L to about 400L, about 20L to about 300L, about 20L to about 200L, about 20L to about 100L, about 20L to about 90L, about 20L to about 80L, about 20L to about 70L, about 20L to about 60L, about 20L to about 50L, about 20L to about 40L, about 20L to about 30L, about 30L to about 1,000L, about 30L to about 900L, about 30L to about 800L, about 30L to about 700L, about 30L to about 600L, about 30L to about 500L, from about 30L to about 400L, from about 30L to about 300L, from about 30L to about 200L, from about 30L to about 100L, from about 30L to about 90L, from about 30L to about 80L, from about 30L to about 70L, from about 30L to about 60L, from about 30L to about 50L, from about 30L to about 40L, from about 40L to about 1,000L, from about 40L to about 900L, from about 40L to about 800L, from about 40L to about 700L, from about 40L to about 600L, from about 40L to about 500L, from about 40L to about 400L, from about 40L to about 300L, from about 40L to about 200L, from about 40L to about 10 0L, about 40L to about 90L, about 40L to about 80L, about 40L to about 70L, about 40L to about 60L, about 40L to about 50L, about 50L to about 1,000L, about 50L to about 900L, about 50L to about 800L, about 50L to about 700L, about 50L to about 600L, about 50L to about 500L, about 50L to about 400L, about 50L to about 300L, about 50L to about 200L, about 50L to about 100L, about 50L to about 90L, about 50L to about 80L, about 50L to about 70L, about 50L to about 60L, about 60L to about 1,000L, 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 L, 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 80L 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 a bout 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 30 0L 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 500L, 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 1,000 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 certain embodiments, the volume of the bioreactor is about 50 L.

[0411] In certain embodiments, the bioreactor has a volume of 100 mL to 1,000 L. In certain embodiments, the bioreactor has a volume of 10 L to 1,000 L. In certain embodiments, the bioreactor has a volume of 100 L to 1,000 L. In certain embodiments, the bioreactor has a volume of 10 L to 800 L. In certain embodiments, the volume of the bioreactor is from 10 L to 700 L, from 10 L to 600 L, from 10 L to 500 L, from 10 L to 400 L, from 10 L to 300 L, from 10 L to 200 L, from 10 L to 100 L, from 10 L to 90 L, from 10 L to 80 L, from 10 L to 70 L, from 10 L to 60 L, from 10 L to 50 L, from 10 L to 40 L, from 10 L to 30 L, from 10 L to 20, from 20 L to 1,000 L, from 20 L to 900 L, from 20 L to 800 L, from 20 L to 700 L, from 20 L to 600 L, from 20 L to 500 L, from 20 L to 500 L, from 20 L to 600 L 0L to 400L, 20L to 300L, 20L to 200L, 20L to 100L, 20L to 90L, 20L to 80L, 20L to 70L, 20L to 60L, 20L to 50L, 20L to 40L, 20L to 30L, 30L to 1,000L, 30L to 900L, 30L to 800L, 30L to 700L, 30L to 600L, 30L to 500L, 30L to 400L, 30L to 300L, 30L to 200L, 30L to 100L, 30L to 90L, 30L to 80L, 30L to 70L, 30L to 60L, 30L to 50L, 30L to 40L, 40L to 1,000L, 40L to 900L, 40L to 800L, 40L to 700L, 40L to 600L, 40L to 500L, 40L to 400L, 40L to 300L, 40L to 200L, 40L to 100L, 40L to 90L, 40L to 80L, 40L to 70L, 40L to 60L, 40L to 50L, 50L to 1,000L, 50L to 900L, 50L to 800L, 50L to 700L, 50L to 6 00L, 50L to 500L, 50L to 400L, 50L to 300L, 50L to 200L, 50L to 100L, 50L to 90L, 50L to 80L, 50L to 70L, 50L to 60L, 60L to 1,000L, 60L to 900L, 60L to 800L, 60L to 700L, 60L to 600L, 60L to 500L, 60L to 400L, 60L to 300L, 60L to 200L, 60L to 100L, 60L to 90L, 60L to 80L, 60L to 70L, 70L to 1,000L, 70L to 900L, 70L to 800L, 70L to 700L, 70L to 600L, 70L to 500L, 70L to 400L, 70L to 300L, 70L to 200L, 70L to 100L, 70L to 90L, 70L to 80L, 80L to 1,000L, 80L to 900L, 80L to 800L, 80L to 700L, 80L to 600L, 80L to 500L, 80L to 400L, 80L to 300L, 80L to 200L, 80L to 100L, 80L to 90L, 90L to 1,000L, 90L to 900L, 90L to 800L, 90L to 700L, 90L to 600L, 90L to 500L, 90L to 400L, 90L to 300L, 90L to 200L, 90L to 100L, 100L to 1,000L, 100L to 900L, 100L to 800L, 100L to 700L, 100L to a bout 600L, 100L to 500L, 100L to 400L, 100L to 300L, 100L to 200L, 200L to 1,000L, 200L to 900L, 200L to 800L, 200L to 700L, 200L to 600L, 200L to 500L, 200L to 400L, 200L to 300L, 300L to 1,000L, 300L to 900L, 300L to 800L, 300L to 700L, 300L to 600L, 300L to 500L, 300L to 400L, 400L to 1,000L, 400L to 900L, 400L to 800L, 400L to 700L, 400L to 600L, 400L to 500L, 500L to 1,000L, 500L to 900L, 500L to 800L, 500L to 700L, 500L to 600L, 600L to 1,000L, 600L to 900L, 600L to 800L, 600L to 700L, 700L to 1,000L, 700L to 900L, 700L to 800L, 800L to 1,000L, 800L to 900L, or 900L to 1,000L. In certain embodiments, the bioreactor has a volume of 50L.

[0412] Cell expansion and stimulation

[0413] In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, is co-cultured with feeder cells to produce expanded and stimulated NK cells.

[0414] In certain embodiments, the co-cultivation is performed in a culture medium described herein, such as Exemplary Medium #1 (Table 1) or Exemplary Medium #2 (Table 2).

[0415] In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 7 or about 1×10 7 to 1×10 9 or about 1×10 9 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 8 or about 1×10 8 to 1.5×10 8 or about 1.5×10 8 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 8 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises approximately 1×10 8 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises 1×10 9 In certain embodiments, a source of natural killer cells, such as a single unit of umbilical cord blood, comprises approximately 1×10 9 Total nucleated cells.

[0416] In certain embodiments, cells co-cultured with a natural killer cell source, such as a single unit of umbilical cord blood and feeder cells, are collected and frozen, for example in a cryopreservation complex as described herein. In certain embodiments, the frozen cells from the co-culture are a ready-to-inject drug product. In certain embodiments, the frozen cells from the co-culture are used as a master cell bank (MCB), from which a ready-to-inject drug product is produced by, for example, one or more additional co-culture steps as described herein. Thus, for example, a natural killer cell source can be expanded and stimulated as described herein to produce NK cells suitable for expansion and stimulation of a ready-to-inject drug product without producing any intermediate products. A natural killer cell source can also be expanded and stimulated as described herein to produce an intermediate product, such as a first master cell bank (MCB). The first MCB can be used to produce NK cells suitable for expansion and stimulation of a ready-to-inject drug product, or, alternatively, can be used to produce another intermediate product, such as a second MCB. The second MCB can be used to produce NK cells suitable for expansion and stimulation of a ready-to-inject drug product, or can be used to produce another intermediate product, such as a third MCB, and so on.

[0417] In certain embodiments, the ratio of feeder cells to natural killer cell-derived cells or MC B cells inoculated into the co-culture is from 1:1 or about 1:1 to 4:1 or about 4:1. In certain embodiments, the ratio of feeder cells to natural killer cell-derived cells or MCB cells is from 1:1 or about 1:1 to 3.5:1 or about 3.5:1, from 1:1 or about 1:1 to 3:1 or about 3:1, from 1:1 or about 1:1 to 2.5:1 or about 2.5:1, from 1:1 or about 1:1 to 2:1 or about 2:1, from 1:1 or about 1:1 to 1.5:1 or about 1.5:1, from 1.5:1 or about 1.5:1 to 4:1 or about 4:1, from 1.5:1 or about 1.5:1 to 3.5:1 or about 3.5:1, from 1.5:1 or about 1.5:1 to 3:1 or about 3:1, from 1.5:1 or about 1.5:1 to 2.5:1 or about 2.5:1, from 1 From the foregoing, the foregoing invention relates to a pharmaceutical composition comprising: ... In certain embodiments, the ratio of feeder cells to natural killer cell-derived cells or MCB cells seeded into the co-culture is 2.5: 1. In certain embodiments, the ratio of feeder cells to natural killer cell-derived cells or MCB cells seeded into the co-culture is about 2.5:1.

[0418] In certain embodiments, the co-cultivation is carried out in a disposable culture bag, such as a 1 L disposable culture bag. In certain embodiments, the co-cultivation is carried out in a disposable culture bag, such as a 50 L disposable culture bag. In certain embodiments, culture medium is added to the co-culture after the initial inoculation.

[0419] In certain embodiments, 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 days or longer. In certain embodiments, the co-cultivation is carried out for up to 16 days.

[0420] In certain embodiments, the co-cultivation is performed at or about 37°C.

[0421] In certain embodiments, the co-cultivation is performed at or about pH 7.9.

[0422] In certain embodiments, the co-cultivation is performed at a dissolved oxygen (DO) level of 50% or higher.

[0423] In certain embodiments, Exemplary Medium #1 (Table 1) is used to produce MCB and Exemplary Medium #2 (Table 2) is used to produce cells suitable for a ready-to-use drug product.

[0424] In certain embodiments, the source of natural killer cells (e.g., a single unit of umbilical cord blood) is co-cultured with feeder cells to produce cells from 50×10 8 or about 50×10 8 to 50×10 12 or about 50×10 12 In some embodiments, the expansion produces cells from 50×10 8 or about 50×10 8 to 25×10 10 or about 25×10 10 pcs, from 10×10 8 or about 10×10 8 to 1×10 10 or about 1×10 10 pcs, from 50×10 8 or about 50×10 8 to 75×10 9 or about 75×10 9 pcs, from 50×10 8 or about 50×10 8 to 50×10 9 or about 50×10 9 pcs, from 50×10 8 or about 50×10 8 to 25×10 9 or about 25×10 9 pcs, from 50×10 8 or about 50×10 8 to 1×10 9 or about 1×10 9 pcs, from 50×10 8 or about 50×10 8 to 75×10 8 or about 75×10 8 pcs, from 75×10 8 or about 75×10 8 to 50×10 10 or about 50×10 10pcs, from 75×10 8 or about 75×10 8 to 25×10 10 or about 25×10 10 pcs, from 75×10 8 or about 75×10 8 to 1×10 10 or about 1×10 10 pcs, from 75×10 8 or about 75×10 8 to 75×10 9 or about 75×10 9 pcs, from 75×10 8 or about 75×10 8 to 50×10 9 or about 50×10 9 pcs, from 75×10 8 or about 75×10 8 to 25×10 9 or about 25×10 9 pcs, from 75×10 8 or about 75×10 8 to 1×10 9 or about 1×10 9 pcs, from 1×10 9 or about 1×10 9 to 50×10 10 or about 50×10 10 pcs, from 1×10 9 or about 1×10 9 to 25×10 10 or about 25×10 10 pcs, from 1×10 9 or about 1×10 9 to 1×10 10 or about 1×10 10 pcs, from 1×10 9 or about 1×10 9 to 75×10 9 or about 75×10 9 pcs, from 1×10 9 or about 1×10 9 to 50×10 9 or about 50×10 9 pcs, from 1×10 9 or about 1×10 9 to 25×10 9 or about 25×10 9 pcs, from 25×10 9 or about 25×10 9 to 50×10 10or about 50×10 10 pcs, from 25×10 9 or about 25×10 9 to 25×10 10 or about 25×10 10 pcs, from 25×10 9 or about 25×10 9 to 1×10 10 or about 1×10 10 pcs, from 25×10 9 or about 25×10 9 to 75×10 9 or about 75×10 9 pcs, from 25×10 9 or about 25×10 9 to 50×10 9 or about 50×10 9 pcs, from 50×10 9 or about 50×10 9 to 50×10 10 or about 50×10 10 pcs, from 50×10 9 or about 50×10 9 to 25×10 10 or about 25×10 10 pcs, from 50×10 9 or about 50×10 9 to 1×10 10 or about 1×10 10 pcs, from 50×10 9 or about 50×10 9 to 75×10 9 or about 75×10 9 indivual,

[0425] From 75×10 9 or about 75×10 9 to 50×10 10 or about 50×10 10 pcs, from 75×10 9 or about 75×10 9 to 25×10 10 or about 25×10 10 pcs, from 75×10 9 or about 75×10 9 to 1×10 10 or about 1×10 10 pcs, from 1×10 10 or about 1×10 10 to 50×10 10 or about 50×10 10pcs, from 1×10 10 or about 1×10 10 to 25×10 10 or about 25×10 10 or from 25×10 10 or about 25×10 10 to 50×10 10 or about 50×10 10 Individual cells, such as, for example, MCB cells or cells of a ready-to-inject pharmaceutical product.

[0426] In certain embodiments, expansion results in from 60 or about 60 vials to 100 or about 100 vials, each vial containing from 600 million or about 600 million to 1 billion or about 1 billion cells, e.g., MCB cells or ready-to-inject drug product cells. In certain embodiments, expansion results in from 80 or about 80 vials, each vial containing or consisting of 800 million or about 800 million cells, e.g., MCB cells or ready-to-inject drug product cells.

[0427] In certain embodiments, the expansion increases the number of cells in the source of natural killer cells by from 100 or about 100 to 500 or about 500-fold, such as the number of MCB NK cells relative to the number of cells, such as NK cells. In some embodiments, the expansion increases the number of cells in the source of natural killer cells by from 100 or about 100 to 500 or about 500 fold, from 100 or about 100 to 400 or about 400 fold, from 100 or about 100 to 300 or about 300 fold, from 100 or about 100 to 200 or about 200 fold, from 200 or about 200 to 450 or about 450 fold, from 200 or about 200 to 400 or about 400 fold, from 100 or about 100 to 350 or about 350 fold, from 200 or about 200 to 300 or about 300 fold, from 200 or about 200 to 250 or about 250 fold, from 250 or about 250 to 500 or about 500 fold. 450 or about 450 times, from 300 or about 300 to 500 or about 500 times, from 300 or about 300 to 450 or about 450 times, from 300 or about 300 to 400 or about 400 times, from 300 or about 300 to 500 or about 500 times, from 300 or about 300 to 400 or about 400 times, from 300 or about 300 to 350 or about 350 times, from 350 or about 350 to 500 or about 500 times, from 350 or about 350 to 450 or about 450 times, or from 350 or about 350 to 400 or about 400 times, e.g., the number of MCB NK cells relative to the number of cells, e.g., NK cells.

[0428] In some embodiments, the expansion increases the number of cells (e.g., the number of MCB NK cells) relative to the number of cells (e.g., NK cells) in a source of natural killer cells by 100 or about 100 to 70,000 or about 70,000-fold. In some embodiments, the expansion increases the number of cells (e.g., the number of MCB NK cells) relative to the number of cells (e.g., NK cells) in a source of natural killer cells by at least 1,0000-fold, such as 15,000-fold, 2,0000-fold, 25,000-fold, 3,0000-fold, 35,000-fold, 4,0000-fold, 45,000-fold, 5,0000-fold, 55,000-fold, 6,0000-fold, 65,000-fold, or 7,0000-fold.

[0429] In certain embodiments, co-culture of MCB cells and feeder cells produces from at or about 500 million to at or about 1.5 billion cells, eg, NK cells suitable for use in MCB and / or ready-to-inject pharmaceutical products. In certain embodiments, co-culture of MCB cells and feeder cells produces from 500 million or about 500 million to 1.5 billion or about 1.5 billion, from 500 million or about 500 million to 1.25 billion or about 1.25 billion, from 500 million or about 500 million to 1 billion or about 1 billion, from 500 million or about 500 million to 750 million or about 750 million, from 750 million or about 750 million to 1.5 billion or about 1.5 billion, from 500 million or about 500 million to 1.25 billion or about 1.25 billion, from 750 million or about 750 million to 1 billion or about 1 billion, from 1 billion or about 1 billion to 1.5 billion or about 1.5 billion, from 1 billion or about 1 billion to 1.25 billion or about 1.25 billion, or from 1.25 billion or about 1.25 billion to 1.5 billion or about 1.5 billion cells, e.g., NK cells suitable for use in MCB and / or ready-to-inject pharmaceutical products.

[0430] In certain embodiments, the co-culture of MCB cells and feeder cells produces from 50 or about 50 flasks to 150 or about 150 flasks of cells, e.g., cells for a ready-to-inject drug product, each flask comprising from 750 million or about 750 million to 1.25 billion or about 1.25 billion cells, e.g., NK cells suitable for MCB and / or ready-to-inject drug products. In certain embodiments, the co-culture of MCB cells and feeder cells produces 100 or about 100 flasks of cells, e.g., cells for a ready-to-inject drug product, each flask comprising 1 billion or about 1 billion cells, e.g., NK cells suitable for MCB and / or ready-to-inject drug products.

[0431] In certain embodiments, the expansion produces an increase in the number of cells from 100 or about 100 to 500 or about 500-fold, for example, the number of NK cells suitable for use in MCB and / or ready-to-use pharmaceutical preparations relative to the number of starting MCB cells. In some embodiments, the expansion produces an increase in the number of cells of from 100 or about 100 to 500 or about 500-fold, from 100 or about 100 to 400 or about 400-fold, from 100 or about 100 to 300 or about 300-fold, from 100 or about 100 to 200 or about 200-fold, from 200 or about 200 to 450 or about 450-fold, from 200 or about 200 to 400 or about 400-fold, from 100 or about 100 to 350 or about 350-fold, from 200 or about 200 to 300 or about 300-fold, from 200 or about 200 to 250 or about 250-fold, from 250 or about 250 to 500 or about 500-fold, from 250 or about 25 ... For example, the number of NK cells suitable for use in MCB and / or ready-to-inject pharmaceutical products relative to the number of starting MCB cells can be from 200 or about 200 to 400 or about 400-fold, from 250 or about 250 to 350 or about 350-fold, from 250 or about 250 to 300 or about 300-fold, from 300 or about 300 to 500 or about 500-fold, from 300 or about 300 to 450 or about 450-fold, from 300 or about 300 to 400 or about 400-fold, from 300 or about 300 to 350 or about 350-fold, from 350 or about 350 to 500 or about 500-fold, from 350 or about 350 to 450 or about 450-fold, or from 350 or about 350 to 400 or about 400-fold, for example.

[0432] In certain embodiments, the expansion produces an increase in the number of cells from at or about 100 to at or about 70,000,000-fold, e.g., the number of NK cells suitable for use in MCB and / or ready-to-inject pharmaceutical products relative to the number of starting MCB NK cells. In certain embodiments, the expansion produces an increase in the number of cells by at least 1,0000-fold, e.g., 15,000-fold, 2,0000-fold, 25,000-fold, 3,0000-fold, 35,000-fold, 4,0000-fold, 45,000-fold, 5,0000-fold, 55,000-fold, 6,0000-fold, 65,000-fold, or 7,0000-fold, e.g., the number of NK cells suitable for use in MCB and / or ready-to-inject pharmaceutical products relative to the number of starting MCB NK cells.

[0433] As described herein, in embodiments where cells are engineered during expansion and stimulation, not all expanded and stimulated cells are necessarily successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising CAR and / or IL-15 as described herein. Thus, the methods described herein may further comprise sorting engineered cells, e.g., engineered cells as described herein, from non-engineered cells.

[0434] In certain embodiments, using an engineered cell antigen-specific reagent, for example, an antibody directed against an antigen of an engineered cell rather than an antigen of a non-engineered cell, an engineered cell, such as a transduced cell, is sorted from a non-engineered cell, such as a non-transduced cell. In certain embodiments, the antigen of the engineered cell is a component of a CAR, for example, a CAR as described herein.

[0435] Antigen-based cell separation systems are commercially available, e.g. Sorting system (Miltenyi Biotec).

[0436] In certain embodiments, flow cytometry is used to sort engineered cells, eg, transduced cells, from non-engineered cells, eg, non-transduced cells.

[0437] In certain embodiments, the sorted engineered cells are used as MCBs. In certain embodiments, the sorted engineered cells are used as a component in a ready-to-use drug product.

[0438] In certain embodiments, engineered cells (e.g., transduced cells) are sorted from non-engineered cells (e.g., non-transduced cells) using microfluidic cell sorting methods. Microfluidic cell sorting methods are described, for example, in Dalili et al., "A Review of Sorting, Separation, and Isolation of Cells and Microbeads for Biomedical Applications: Microfluidic Methods," Analyst 144:87 (2019).

[0439] In certain embodiments, from 1% or about 1% to 99% or about 99% of the expanded and stimulated cells are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15 as described herein. In certain embodiments, from 1% or about 1% to 90% or about 90%, from 1% or about 1% to 80% or about 80%, from 1% or about 1% to 70% or about 70%, from 1% or about 1% to 60% or about 60%, from 1% or about 1% to 50% or about 50%, from 1% or about 1% to 40% or about 40%, from 1% or about 1% to 30% or about 30%, from 1% or about 1% to 20% or about 20%, from 1% or about 1% to 10% or about 10%, from 1% or about 1% to 5% or about 5%, from 5% or about 5% to 99% or about 99%, from 5% or about 5% to 90% or about 90%, from 5% or about 5% to 80% or about 80%. 80%, from 5% or about 5% to 70% or about 70%, from 5% or about 5% to 60% or about 60%, from 5% or about 5% to 50% or about 50%, from 5% or about 5% to 40% or about 40%, from 5% or about 5% to 30% or about 30%, from 5% or about 5% to 20% or about 20%, from 5% or about 5% to 10% or about 10%, from 10% or about 10% to 99% or about 99%, from 10% or about 10% to 90% or about 90%, from 10% or about 10% to 80% or about 80%, from 10% or about 10% to 70% or about 70%, from 10% or about 10% to 60% or about 60%, from 10% or about 10% to 10% or about 1 ... 99%, from 20% or about 20% to 90% or about 90%, from 20% or about 20% to 80% or about 80%, from 20% or about 20% to 70% or about 70%, from 20% or about 20% to 60% or about 60%, from 20% or about 20% to 50% or about 50%, from 20% or about 20% to 40% or about 40%, from 20% or about 20% to 30% or about 30%, from 30% or about 3 ...20% or about 20% to 30% or about 30%, from %, from 30% or about 30% to 99% or about 99%, from 30% or about 30% to 90% or about 90%, from 30% or about 30% to 80% or about 80%, from 30% or about 30% to 70% or about 70%, from 30% or about 30% to 60% or about 60%, from 30% or about 30% to 50% or about 50%, from 30% or about 30% to 40% or about 40%, from 40% or about 40% to 99% or about 99%, from 40% or about 40% to 90% or about 90%, from 40% or about 40% to 80% or about 80%, from 40% or about 40% to 70% or about 70%, from 40% or about 40% to 60% or about 60%,From 40% or about 40% to 50% or about 50%, from 50% or about 50% to 99% or about 99%, from 50% or about 50% to 90% or about 90%, from 50% or about 50% to 80% or about 80%, from 50% or about 50% to 70% or about 70%, from 50% or about 50% to 60% or about 60%, from 60% or about 60% to 99% or about 99%, from 60% or about 60% to 90% or about 90%, from 60% or about 60% to 80% or about 80%, from 60% or about 60% to 70% or about 70%, from 60% or about 60% to 90% or about 90%, from 60% or about 60% to 80% or about 80%, from 60% or about 60% to 70% or about 70%, from 70% or about 70% to 99% or about 99%, from 70% or about 70% to 90% or about 90%, from 70% or about 70% to 80% or about 80%, from 80% or about 80% to 99% or about 99%, from 80% or about 80% to 90% or about 90%, or from 90% or about 90% to 99% or about 99% of the expanded and stimulated cells are successfully engineered, e.g., successfully transduced, e.g., with a vector comprising a heterologous protein, e.g., a heterologous protein comprising a CAR and / or IL-15 as described herein.

[0440] In certain embodiments, frozen cells from the first or second MCB are thawed and cultured. In certain embodiments, a single vial of frozen cells from the first or second MCB is thawed and cultured, e.g., the single vial containing 800 million or about 800 million cells, e.g., cells from the first or second MCB. In certain embodiments, the frozen first or second MCB cells are cultured with additional feeder cells to produce cells suitable for use as a second or third MCB or for a ready-to-inject drug product. In certain embodiments, cells co-cultured from the first or second MCB are harvested and frozen.

[0441] In certain embodiments, cells from the co-culture of a natural killer cell source, a first MCB, or a second MCB are collected and frozen in a cryopreservation compound, e.g., a cryopreservation compound described herein. In certain embodiments, the cells are washed after collection. Thus, provided herein are pharmaceutical compositions comprising activated and activated NK cells, e.g., activated and stimulated NK cells prepared by the methods described herein, e.g., collected and washed activated and stimulated NK cells prepared by the methods described herein, and a cryopreservation compound, e.g., a cryopreservation compound described herein.

[0442] In certain embodiments, prior to freezing, the cells are mixed with a cryopreservative, e.g., as described herein. In certain embodiments, the cells are frozen in a freezing bag. In certain embodiments, the cells are frozen in a freezing vial.

[0443] In certain embodiments, the method further comprises isolating NK cells from the expanded and stimulated NK cell population.

[0444] Figure 1 An exemplary process for expanding and stimulating NK cells is shown in .

[0445] Engineering

[0446] In certain embodiments, the method further comprises engineering the NK cells, e.g., to express a heterologous protein, e.g., a heterologous protein described herein, e.g., a heterologous protein comprising CAR and / or IL-15.

[0447] In certain embodiments, engineering NK cells to express a heterologous protein described herein comprises transducing, e.g., stably transducing, NK cells with a vector comprising a polynucleotide encoding a heterologous protein described herein. Suitable vectors are described herein.

[0448] In certain embodiments, engineering NK cells to express heterologous proteins described herein comprises introducing heterologous proteins by gene editing (e.g., zinc finger nuclease (ZFN) gene editing, ARCUS gene editing, CRISPR-Cas9 gene editing, or mega TAL gene editing) combined with adeno-associated virus (AAV) technology.

[0449] In certain embodiments, engineered NK cells are to express heterologous proteins as described herein, for example, during or after culturing the complex in a culture medium comprising feeder cells. For example, in some cases, engineering (such as transduction) occurs during expansion and stimulation as described herein, for example, during co-cultivation of NK cell sources and feeder cells as described herein, for example, on the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th or 18th day of co-cultivation.

[0450] In certain embodiments, the method further comprises engineering the NK cells, eg, expressing, overexpressing, knocking out, or knocking out a gene or gene product.

[0451] In certain embodiments, the natural killer cells are not genetically engineered.

[0452] In certain embodiments, NK cells are engineered (e.g., transduced) in a culture medium supplemented with a stimulating factor (e.g., as described herein). These cytokines can be used to provide growth or survival signals to NK cells during the engineering process, or to improve transduction efficiency. In certain embodiments, the stimulating factor is a cytokine. In certain embodiments, 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.

[0453] In certain embodiments, the cytokine is IL-21. The IL-21 can be used at a final concentration of 10 to 100 ng / mL, including, for example, or about 10, 15, 20, 25, 30, 34, 40, 45, 50, 55, 60, 70, 80, 90 or 100 ng / mL. In certain embodiments, the cytokine is IL-2. In certain embodiments, the cytokine is a combination of IL-2 and IL-21. In certain embodiments, the cytokine is a combination of IL-2, IL-18 and IL-21.

[0454] In certain embodiments, the stimulating factor is added to the culture medium during through engineering approaches (e.g., transduction). In certain embodiments, after through engineering approaches (e.g., transduction), for example, 1 to 48 hours after through engineering approaches, 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 engineering, the stimulating factor is added to the culture medium. In certain embodiments, the stimulating factor is added to the culture medium during transduction and after through engineering approaches (e.g., 1 to 48 hours after transduction).

[0455] In certain embodiments, after cultivating in the culture medium containing feeder cells, supplement stimulating factors in the culture medium. Therefore, in some cases, when through engineering approaches (for example, transduction), the culture medium will comprise feeder cells. In some cases, before supplementing stimulating factors or through engineering approaches, the feeder cells in the culture are removed. In some cases, before supplementing stimulating factors or through engineering approaches, the feeder cells in the culture are not removed. In some cases, in the through engineering approaches process, feeder cells are not additionally added to the culture, no matter whether any remaining feeder cells are removed. In some cases, in the through engineering approaches process, feeder cells and stimulating factors are additionally added in the culture medium. In some cases, in the through engineering approaches process, feeder cells are not additionally added to the culture medium, but stimulating factors are added in the through engineering approaches process.

[0456] Characterization of expanded and stimulated NK cells

[0457] The expanded and stimulated NK cell populations, e.g., after in vitro expansion and stimulation as described herein, not only have numbers / densities that do not naturally occur in the human body (e.g., as described above), but also have phenotypic characteristics (e.g., gene expression and / or surface protein expression) that are different from the starting source material or other naturally occurring NK cell populations.

[0458] In some cases, the starting NK cell source is a sample from a single individual, for example, a single umbilical cord blood unit that has not been expanded in vitro. Thus, in some cases, the expanded and stimulated NK cells share a common lineage, i.e., they are all derived from the expansion of the starting NK cell source and, therefore, share a genotype through clonal expansion of a cell population from a single organism. However, they cannot occur naturally at the density achieved by in vitro expansion and are also different from the starting NK cell source in terms of phenotypic characteristics.

[0459] In some cases, the expanded and stimulated NK cell population comprises at least 100 million expanded natural killer cells, for example, 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 9 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, 10 trillion expanded natural killer cells.

[0460] In certain embodiments, the expanded and stimulated NK cells comprise at least 80%, e.g., at least 90%, at least 95%, at least 99%, or 100% CD56+CD3- cells.

[0461] In certain embodiments, the expanded and stimulated NK cells are not genetically engineered.

[0462] In certain embodiments, the expanded and stimulated NK cells do not include a CD16 transgene.

[0463] In certain embodiments, the expanded and stimulated NK cells do not express exogenous CD16 protein.

[0464] The expanded and stimulated NK cells can be characterized, for example, by surface expression, such as one or more of CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44.

[0465] In some cases, the surface protein expression levels described herein are achieved without positive selection for the specific surface protein referenced. For example, in some cases, a source of NK cells, such as a single umbilical cord unit, includes both KIR B alleles of the KIR receptor family and the 158V / V variant of CD16 and is CD56+ enriched and CD3(+) depleted, for example by gating for CD56+CD3- expression, but no other surface protein expression selection is performed during expansion and stimulation.

[0466] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., 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.

[0467] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., 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.

[0468] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., 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.

[0469] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., 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.

[0470] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., 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.

[0471] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., 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% CD94+ (KLRD1) cells.

[0472] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, comprise less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD3+ cells.

[0473] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, comprise less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD14+ cells.

[0474] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, comprise less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD19+ cells.

[0475] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, comprise less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CXCR+ cells.

[0476] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, comprise less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD122+(IL2RB) cells.

[0477] As described herein, the inventors have demonstrated that, surprisingly, NK cells expanded and stimulated by the methods described herein express CD16 at high levels throughout the expansion and stimulation process, resulting in a cell population with high CD16 expression. The high expression of CD 16 avoids the need to engineer the expanded cells to express CD16, which is very important for starting ADCCs, and therefore, this is an unexpected and non-obvious effect of the expansion and stimulation methods described herein. Therefore, in certain embodiments, the expanded and stimulated NK cells, for example, expansion and stimulation from a single umbilical cord blood unit, for example, as described above, include 50% or more, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells.

[0478] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, include KIR B alleles of the KIR receptor family and the 158V / V variant of CD16, and comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ cells.

[0479] In certain embodiments, the percentage of expanded and stimulated NK cells expressing CD16, e.g., from a single cord blood unit expanded and stimulated, e.g., as described above, is the same as or greater than the percentage of natural killer cells in seed cells from cord blood.

[0480] In certain embodiments, the percentage of the expanded and stimulated NK cells that express NKG2D, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, is the same as or greater than the percentage of natural killer cells in seed cells from cord blood.

[0481] In certain embodiments, the percentage of the expanded and stimulated NK cells that express NKp30, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, is the same as or greater than the percentage of natural killer cells in seed cells from cord blood.

[0482] In certain embodiments, the percentage of the expanded and stimulated NK cells expressing DNAM-1, e.g., from a single cord blood unit expanded and stimulated, e.g., as described above, is the same as or greater than the percentage of natural killer cells in seed cells from cord blood.

[0483] In certain embodiments, the percentage of the expanded and stimulated NK cells that express NKp44, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, is the same as or greater than the percentage of natural killer cells in seed cells from cord blood.

[0484] In certain embodiments, the percentage of the expanded and stimulated NK cells that express NKp46, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, is the same as or greater than the percentage of natural killer cells in seed cells from cord blood.

[0485] As described herein, the inventors also demonstrated that, surprisingly, NK cells expanded and stimulated by the methods described herein express CD38 at low levels. CD38 is a valid target for the treatment of certain cancers, such as multiple myeloma and acute myeloid leukemia. See, for example, Jiao et al., "CD38: Targeted Therapy for Multiple Myeloma and Therapeutic Potential for Solid Cancers," Expert Opinion on Investigational Drugs, 29(11): 1295-1308 (2020).

[0486] Thus, in certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, comprise less than or equal to 80% CD38+ cells, e.g., less than or equal to 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells.

[0487] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, comprise a KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and comprise less than or equal to 80% CD38+ cells, e.g., less than or equal to 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD38+ cells.

[0488] In certain embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated from a single umbilical cord blood unit, e.g., as described above, comprise a KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and comprise less than or equal to 80% CD38+ cells, e.g., less than or equal to 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.

[0489] In certain embodiments, the percentage of expanded and stimulated NK cells expressing NKp46, e.g., expanded and stimulated from a single cord blood unit, e.g., as described above, comprises a KIR B allele of the KIR receptor family and the 158V / V variant of CD16, and comprises 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., less than or equal to 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%, at least 80%, at least 90%, at least 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% 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%, at least 95%, at least 99%, or 100% DNAM-1+ cells. and / or vii) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% NKp44+ cells; and / or viii) 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; and / or ix) less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD3+ cells; and / or x) less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD3+ cells; less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD14+ cells; and / or xi) less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD19+ cells; and / or xii) less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CXCR+ cells; and / or xiii) less than or equal to 20%, e.g., less than or equal to 10%, less than or equal to 5%, less than or equal to 1% or 0% CD122+ (IL2RB) cells.

[0490] In certain embodiments, feeder cells do not persist in the expanded and stimulated NK cells, however, residual characteristics of the feeder cells can be detected, for example, by the presence of residual cells (e.g., by detecting cells with expression of a particular surface protein) or residual nucleic acids and / or proteins expressed by the feeder cells.

[0491] For example, in some cases, the methods described herein include expanding and stimulating natural killer cells using engineered feeder cells (e.g., eHuT-78 feeder cells described above) that are engineered to express sequences that are not expressed by cells from a natural killer cell source (including natural killer cells). For example, the engineered feeder 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) ("eHut-78 cells"), or variants thereof.

[0492] Although these feeder cells may not persist in the expanded and stimulated NK cells, the expanded and stimulated NK cells may retain a measurable amount of residual cells, proteins and / or nucleic acids from the feeder cells. Therefore, their presence in the expanded and stimulated NK cells can be detected, for example, by detecting the cells themselves (e.g., flow cytometry), the proteins they express, and / or the nucleic acids they express.

[0493] Thus, also described herein are expanded and stimulated NK cell populations that contain residual feeder cells (live or dead) 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). In some cases, the expanded and stimulated NK cells contain more than 0% but 0.3% or less residual feeder cells, such as eHuT-78 feeder cells.

[0494] In some cases, the expanded and stimulated NK cells include residual feeder cell nucleic acids, e.g., encoding residual 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and / or mutated TNFα (SEQ ID ID: 3) or portions thereof. In some cases, membrane-bound IL-21 includes a CD8 transmembrane domain.

[0495] In some cases, the expanded and stimulated NK cells contain a % of residual feeder cells greater than 0% and less than or equal to 0.2%, as measured, for example, by the relative proportion of feeder cell-specific proteins or nucleic acid sequences (i.e., proteins or nucleic acid sequences not expressed by natural killer cells) in the sample, e.g., by qPCR, as described herein.

[0496] In certain embodiments, the residual feeder cells are CD4 (+) T cells. In certain embodiments, the residual feeder cells are engineered CD4 (+) T cells. In certain embodiments, the residual feeder cells are 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) ("eHut-78 cells"), or variants thereof. Thus, in some cases, the feeder cell-specific protein is 4-1BBL (UniProtKB P41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and / or mutant TNFα (SEQ ID ID: 3). Thus, the cellular nucleic acid is a nucleic acid encoding 4-1BBL (UniProtKBP41273, SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2), and / or mutant TNFα (SEQ ID ID: 3), or a portion thereof. In certain instances, the membrane-bound IL-21 comprises a CD8 transmembrane domain.

[0497] A variety of different methods can be used to analyze and detect the presence of nucleic acids or protein gene products in biological samples. As used herein, "detection" can refer to a method for discovering, determining, or confirming the presence or existence of a compound and / or substance (e.g., a cell, protein, and / or nucleic acid). In certain embodiments, the detection method can be used to detect a protein. In certain embodiments, the detection can include chemiluminescence or fluorescence techniques. In certain embodiments, the detection can include immunological methods (e.g., quantitative enzyme-linked immunosorbent assay (ELISA), immunoblotting, or dot blot), in which antibodies are used to specifically react with the entire protein or a specific epitope of the protein. In certain embodiments, the detection can include protein immunoprecipitation (Jungblut et al., J Biotechnol., 31; 41(2-3): 111-20 (1995); Franco et al., Eur J Morphol., 39(1): 3-25 (2001)). In certain embodiments, the detection method can be used to detect a nucleic acid (e.g., DNA and / or RNA). In certain embodiments, detection can include 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 CE nviron Carcinog Ecotoxicol Rev. 20(2):77-116 (2002)).

[0498] Thus, also described herein are methods for detecting expanded and stimulated NK cell populations, e.g., using the methods described herein to expand and stimulate NK cells that have been co-cultured with engineered feeder cells (e.g., eHuT-78 feeder cells described herein).

[0499] Anti-HER2 CAR-NK

[0500] Provided herein are engineered cells, e.g., engineered natural killer cells, e.g., CAR-NK cells, e.g., anti-HER2 CAR-NK cells. In certain embodiments, the CAR-NK cells are engineered to express IL-15.

[0501] In certain embodiments, the natural killer cells are engineered, such as by transduction, during expansion and stimulation, such as during expansion and stimulation as described herein. In certain embodiments, the natural killer cells are engineered, such as during the preparation of a master cell bank (MCB), as described herein. In certain embodiments, the natural killer cells are engineered, such as during expansion and stimulation, such as during the preparation of a master cell bank (MCB), as described above. Thus, in certain embodiments, the NK cells are host cells, and provided herein are natural killer host cells expressing heterologous proteins, such as heterologous proteins as described herein.

[0502] In certain embodiments, the natural killer cells are engineered prior to expansion and stimulation. In certain embodiments, the natural killer cells are engineered prior to expansion and stimulation.

[0503] In certain embodiments, the NK cells are engineered by transducing them with a vector. Suitable vectors are described herein, such as lentiviral vectors, such as lentiviral vectors of heterologous proteins, such as heterologous proteins as described herein. In certain embodiments, as described herein, NK cells are transduced during the preparation of the first master cell bank (MCB).

[0504] In certain embodiments, NK cells are transduced at a multiplicity of infection of 1 or about 1 to 40 or about 40 viral particles per cell. In certain embodiments, NK cells are transduced at a multiplicity of infection of 1, 5, 10, 15, 20, 25, 30, 35, or 40 viral particles per cell.

[0505] Chimeric antigen receptor

[0506] In certain embodiments, the heterologous protein is a fusion protein, for example, a fusion protein comprising a chimeric antigen receptor (CAR), which is introduced into NK cells, for example, during expansion and stimulation.

[0507] In certain embodiments, the CAR comprises one or more of the following components: a signal sequence, an extracellular domain, a hinge region, a transmembrane domain, and one or more intracellular signal sequences. In certain embodiments, the CAR further comprises a spacer sequence.

[0508] In certain embodiments, the CAR comprises (from N-terminus to C-terminus): a signal sequence, an extracellular domain, a hinge region, a spacer region, a transmembrane domain, a first signal sequence, a second signal sequence, and a third signal sequence.

[0509] In certain embodiments, the CAR comprises (from N-terminus to C-terminus): a signal sequence, an extracellular domain, a hinge region, a transmembrane domain, a first signal sequence, a second signal sequence, and a third signal sequence.

[0510] The signal sequence can be cut from the mature CAR protein. This cleavage can be mediated by a signal peptidase and can occur during or after transport to generate a mature protein. Therefore, in certain embodiments, the CAR (from N-terminus to C-terminus) comprises: an extracellular domain, a hinge region, a spacer, a transmembrane domain, a first signal sequence, a second signal sequence, and a third signal sequence.

[0511] In certain embodiments, the CAR comprises (from N-terminus to C-terminus): an extracellular domain, a hinge region, a transmembrane domain, a first signal sequence, a second signal sequence, and a third signal sequence.

[0512] In certain embodiments, the extracellular domain comprises an antibody or an antigen-binding portion thereof.

[0513] In certain embodiments, the one or more intracellular signal sequences are CD28 intracellular signal sequences. In certain embodiments, the CD28 intracellular signal sequence comprises or consists of SEQ ID NO: 5.

[0514] In certain embodiments, the one or more intracellular signal sequences are OX40L intracellular signal sequences. See, for example, Matsumura et al., "OX40 ligand gp34 intracellular signaling: Induction of c-jun and c-fos mRNA expression by binding of gp34 to its receptor OX40," J. Immunol, 163:3007-11 (1999), which is incorporated herein by reference in its entirety. In certain embodiments, the OX40L signal sequence comprises or consists of SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0515] In certain embodiments, the one or more intracellular signal sequences are CD3ζ intracellular signal sequences. In certain embodiments, the CD3ζ intracellular signal sequence comprises SEQ ID NO: 13, or consists of it.

[0516] In certain embodiments, the CAR comprises a CD28 intracellular signal sequence (SEQ ID NO: 5), an OX40L intracellular signal sequence (SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10), or a CD3 zeta intracellular signal sequence (SEQ ID NO: 13).

[0517] In certain embodiments, the CAR comprises an intracellular signaling domain comprising, or consisting of, SEQ ID NO: 25.

[0518] In certain embodiments, the CAR does not comprise an OX40L intracellular signaling domain sequence.

[0519] In certain embodiments, the CAR comprises a CD28 intracellular signal sequence (SEQ ID NO: 5) and a CD3ζ intracellular signal sequence (SEQ ID NO: 13), but does not include an OX40L intracellular signal sequence.

[0520] In certain embodiments, the signal sequence is a CD8α signal sequence. In certain embodiments, the signal sequence comprises or consists of SEQ ID NO: 27.

[0521] In certain embodiments, the extracellular domain comprises a single-chain variable fragment (scFv). In certain embodiments, the extracellular domain comprises an anti-HER2 antibody or an antigen-binding fragment thereof. In certain embodiments, the extracellular domain comprises an anti-HER2 scFv.

[0522] In certain embodiments, 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 CDRH1 domain comprising or consisting of SEQ ID NO: 46, and a CDRH1 domain comprising or consisting of SEQ ID NO: 48.

[0523] In certain embodiments, 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.

[0524] In certain embodiments, 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.

[0525] In certain embodiments, the anti-HER2 scFv comprises or consists of SEQ ID NO:30.

[0526] In certain embodiments, the hinge region comprises or consists of a CD8α hinge region. In certain embodiments, the CD8α hinge region comprises or consists of SEQ ID NO: 50.

[0527] In certain embodiments, the transmembrane domain is a CD28 transmembrane domain. In certain embodiments, the CD28 transmembrane domain comprises or consists of SEQ ID NO: 53.

[0528] In certain embodiments, the fusion protein comprises or consists of SEQ ID NO: 56.

[0529] IL-15

[0530] In certain embodiments, the NK cells are engineered to express IL-15, such as human IL-15 (UniProtKB#P40933; NCBI gene number #3600), such as soluble human IL-15 or an ortholog thereof, or a variant of any of the foregoing. In certain embodiments, the IL-15 is expressed as part of a fusion protein that further comprises a cleavage site. In certain embodiments, the IL-15 is expressed as part of a polyprotein that comprises a self-cleavage peptide, such as a T2A ribosomal skipping sequence site (sometimes also referred to as a self-cleavage site). See, e.g., Radcliffe and Mitrophanous, “Generation of Multiple Gene Products from a Single Vector: Self-cleaving 2A Peptides,” Gene Therapy, vol. 11: pp. 1673-1674 (2004); also see Liu et al., “Systematic Comparison of 2A Peptides for Cloning Multiple Genes in Polycistronic Vectors,” Scientific Reports, vol. 7, no. 1: 2193 (2017).

[0531] In certain embodiments, the IL-15 comprises or consists of SEQ ID NO:22.

[0532] In certain embodiments, the self-cleaving peptide is a 2A self-cleaving peptide. In certain embodiments, the self-cleaving peptide is a T2A, P2A, E2A or F2A self-cleaving peptide. In certain embodiments, the self-cleaving peptide comprises the sequence of SEQ ID NO: 16. In certain embodiments, the self-cleaving peptide comprises the sequence of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, or consists thereof.

[0533] In certain embodiments, the T2A cleavage site comprises or consists of SEQ ID NO: 17.

[0534] In certain embodiments, IL-15 is expressed as part of a fusion protein comprising a CAR, such as a CAR described herein.

[0535] In certain embodiments, the fusion protein comprises (oriented from N-terminus to C-terminus): a CAR comprising a cleavage site and IL-15.

[0536] In certain embodiments, the fusion protein comprises SEQ ID NO: 26. SEQ ID NO: 26.

[0537] In certain embodiments, the fusion protein comprises or consists of SEQ ID NO:59.

[0538] Inhibitory receptors

[0539] In certain embodiments, NK cells are engineered to alter (eg, reduce) the expression of one or more inhibitory receptor genes.

[0540] In certain embodiments, the inhibitory receptor gene is an HLA-specific inhibitory receptor gene. In certain embodiments, the inhibitory receptor gene is a non-HLA-specific inhibitory receptor gene.

[0541] In certain embodiments, the inhibitor receptor gene is selected from the group consisting of KIR, CD94 / NKG2A, LILRB1, PD-1, Irp60, Siglec-7, LAIR-1, and combinations thereof.

[0542] Polynucleic acids, vectors and host cells

[0543] Also provided herein are polynucleic acids encoding fusion proteins or portions thereof, eg, polynucleotide sequences encoding the polypeptides described herein, as shown in the sequence listing provided herein.

[0544] Also provided herein are vectors comprising the polynucleic acids and cells comprising the vectors, such as NK cells.

[0545] In certain embodiments, the vector is a lentiviral vector. See, for example, Milone et al., "Clinical Applications of Lentiviral Vectors," Leukemia, 32:1529–41 (2018). In certain embodiments, the vector is a retroviral vector. In certain embodiments, the vector is a retroviral vector. In certain embodiments, the vector is a non-viral vector, such as a piggyback non-viral vector (PB transposon, see Wu et al., "Piggyback is a flexible and highly active transposon compared to Sleeping Beauty, Tol2, and Mos1," PNAS, 103(41):15008-13 (2006)), a Sleeping Beauty non-viral vector (SB transposon, see Hudecek et al., "Towards non-viral: the Sleeping Beauty transposon system breaks through clinical application," Reviews in Biochemistry and Molecular Biology, 52(4):355-380 (2017)), or an mRNA vector.

[0546] Cryopreservation

[0547] Cryopreservation composition

[0548] Provided herein are cryopreservation compositions, e.g., cryopreservation compositions suitable for intravenous administration, e.g., intravenous administration of NK cells, e.g., intravenous administration of NK cells described herein. In certain embodiments, a pharmaceutical composition comprises a cryopreservation composition and cells, e.g., NK cells described herein.

[0549] albumin

[0550] In certain embodiments, the cryopreservation composition comprises albumin, e.g., human albumin (UniProtKB accession number P0278, SEQ ID NO: 63) or a variant thereof. In certain embodiments, the cryopreservation composition comprises a homologue of albumin, e.g., human albumin, or a variant thereof. In certain embodiments, the cryopreservation composition comprises a biologically active portion of albumin, e.g., human albumin, or a variant thereof.

[0551] In certain embodiments, the albumin, such as human albumin, is provided as a solution, also referred to herein as an albumin solution or a human albumin solution. Thus, in certain embodiments, the cryopreservation composition is or comprises an albumin solution, such as a human albumin solution. In certain embodiments, the albumin solution is a serum-free protein solution.

[0552] In certain embodiments, the albumin solution is suitable for intravenous use.

[0553] In certain embodiments, the albumin solution comprises from 40 or about 40 g / L to 200 or about 200 g / L of albumin. In certain embodiments, the albumin solution comprises from 40 or about 40 g / L to 50 or about 50 g / L of albumin, e.g., human albumin. In certain embodiments, the albumin solution comprises about 200 g / L of albumin, e.g., human albumin. In certain embodiments, the albumin solution comprises 200 g / L of albumin, e.g., human albumin.

[0554] In certain embodiments, the albumin solution comprises a protein composition wherein 95% or more is albumin, e.g., human albumin. In certain embodiments, 96%, 97%, 98%, or 99% or more of the protein is albumin, e.g., human albumin.

[0555] In certain embodiments, the albumin solution further contains sodium. In certain embodiments, the albumin solution comprises from 100 or about 100 mmol to 200 or about 200 mmol sodium. In certain embodiments, the albumin solution comprises from 130 or about 130 mmol to 160 or about 160 mmol sodium.

[0556] In certain embodiments, the albumin solution further comprises potassium. In certain embodiments, the albumin solution comprises 3 mmol or less of potassium. In certain embodiments, the albumin solution further comprises 2 mmol or less of potassium.

[0557] In certain embodiments, the albumin solution further comprises one or more stabilizers. In certain embodiments, the stabilizer is selected from the group consisting of sodium octanoate, octanoic acid, (2S)-2-acetamido-3-(1H-indole-3-yl) propionic acid (also known as acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-theanine), 2-acetamido-3-(1H-indole-3-yl) propionic acid (also known as N-acetyltryptophan, DL-acetyltryptophan, and N-acetyl-DL-tryptophan). In certain embodiments, the one or more stabilizers are present in a solution at a concentration of less than 0.1 mmol per gram of protein. In certain embodiments, the solution comprises, for example, from 0.05 or about 0.05 mmol to 0.1 or about 0.1 mmol per gram of protein, for example, from 0.064 or about 0.064 mmol to 0.096 or about 0.096 mmol per gram of protein. In certain embodiments, the total stabilizer content is less than 0.1 mmol per gram of protein in the solution. In certain embodiments, the solution includes, for example, from 0.05 or about 0.05 mmol to 0.1 or about 0.1 mmol, such as from 0.064 or about 0.064 mmol to 0.096 or about 0.096 mmol, of total stabilizer per gram of protein in the solution.

[0558] In certain embodiments, the albumin solution is composed of a protein composition of which greater than 95% is albumin, sodium, potassium, and an aqueous stabilizer solution of one or more stabilizers selected from the group consisting of sodium octanoate, octanoic acid, (2S)-2-acetylamino-3-(1H-indol-3-yl) propionic acid (also known as acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-theanine), 2-acetylamino-3-(1H-indol-3-yl) propionic acid (also known as N-acetyltryptophan, DL-acetyltryptophan, and N-acetyl-DL-tryptophan).

[0559] In certain embodiments, the cryopreservation composition comprises from 10% or about 10% to 50% or about 50% by volume of an albumin solution, such as an albumin solution described herein. In certain embodiments, the cryopreservation composition comprises from 10% or about 10% to 50% or about 50%, from 10% or about 10% to 45% or about 45%, from 10% or about 10% to 40% or about 40%, from 10% or about 10% to 35% or about 35%, from 10% or about 10% to 30% or about 30%, from 10% or about 10% to 25% or about 25%, from 10% or about 10% to 20% or about 20%, from 10% or about 10% to 15% or about 15%, from 15% or about 15% to 5 0% or about 50%, from 15% or about 15% to 45% or about 45%, from 15% or about 15% to 40% or about 40%, from 15% or about 15% to 35% or about 35%, from 15% or about 15% to 30% or about 30%, from 15% or about 15% to 25% or about 25%, from 15% or about 15% to 20% or about 20%, from 20% or about 20% to 50% or about 50%, from 20% or about 20% to 45% or about 45%, from 20% or about 20% to 40% or about 40 ... %, from 20% or about 20% to 35% or about 35%, from 20% or about 20% to 30% or about 30%, from 20% or about 20% to 25% or about 25%, from 25% or about 25% to 50% or about 50%, from 25% or about 25% to 45% or about 45%, from 25% or about 25% to 40% or about 40%, from 25% or about 25% to 35% or about 35%, from 25% or about 25% to 30% or about 30%, from 30% or about 30% to 50% or about 50%, from 30% or about 30% to 45% In some embodiments, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by volume of an albumin solution, such as an albumin solution described herein. In certain embodiments, the cryopreservation composition comprises a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% albumin solution by volume, such as the albumin solution described herein.

[0560] In certain embodiments, the cryopreservation composition comprises from 20 or about 20 g / L to 100 or about 100 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises from 20 or about 20 g / L to 100 or about 100 g / L, from 20 or about 20 g / L to 90 or about 90 g / L, from 20 or about 20 g / L to 80 or about 80 g / L, from 20 or about 20 g / L to 70 or about 70 g / L, from 20 or about 20 g / L to 60 or about 60 g / L, from 20 or about 20 g / L to 50 or about 50 g / L, from 20 or about 20 g / L to 40 or about 40 g / L, from 20 or about 20 g / L to 30 or about 30 g / L, from 30 or about 30 g / L to 1 g / L, from 30 or about 30 g / L to 90 or about 90 g / L, from 30 or about 30 g / L to 80 or about 80 g / L, from 30 or about 30 g / L to 70 or about 70 g / L, from 30 or about 30 g / L to 60 or about 60 g / L, from 30 or about 30 g / L to 50 or about 50 g / L, from 30 or about 30 g / L to 40 or about 40 g / L, from 40 or about 40 g / L to 100 or about 100 g / L, from 40 or about 40 g / L to 90 or about 90 g / L, and from 40 or about 40 g / L to 80 or about 80 g / L. g / L, from 40 or about 40 g / L to 70 or about 70 g / L, from 40 or about 40 g / L to 60 or about 60 g / L, from 40 or about 40 g / L to 50 or about 50 g / L, from 50 or about 50 g / L to 100 or about 100 g / L, from 50 or about 50 g / L to 90 or about 90 g / L, from 50 or about 50 g / L to 80 or about 80 g / L, from 50 or about 50 g / L to 70 or about 70 g / L, from 50 or about 50 g / L to 60 or about 60 g / L, from 60 or about 60 g / L to 100 or about 100 g / L, from 60 or about 60 g / L to 100 or about 100 g / L, from 50 or about 50 g / L to 90 or about 90 g / L, from 50 or about 50 g / L to 80 or about 80 g / L, In some embodiments, the present invention provides an albumin, such as human albumin, between 60 or about 60 g / L and 90 or about 90 g / L, from 60 or about 60 g / L to 80 or about 80 g / L, from 60 or about 60 g / L to 70 or about 70 g / L, from 70 or about 70 g / L to 100 or about 100 g / L, from 70 or about 70 g / L to 90 or about 90 g / L, from 70 or about 70 g / L to 80 or about 80 g / L, from 80 or about 80 g / L to 100 or about 100 g / L, from 80 or about 80 g / L to 90 or about 90 g / L, or from 90 or about 90 g / L to 100 or about 100 g / L.

[0561] In certain embodiments, the cryopreservation composition comprises 20 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises 40 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises 70 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises 100 g / L of albumin, such as human albumin.

[0562] In certain embodiments, the cryopreservation composition comprises about 20 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises about 40 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises about 70 g / L of albumin, such as human albumin. In certain embodiments, the cryopreservation composition comprises about 100 g / L of albumin, such as human albumin.

[0563] In certain embodiments, the cryopreservation composition further comprises a stabilizer, such as an albumin stabilizer. In certain embodiments, the stabilizer is selected from the group consisting of sodium octanoate, octanoic acid, (2S)-2-acetamido-3-(1H-indole-3-yl) propionic acid (also known as acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-theanine), 2-acetamido-3-(1H-indole-3-yl) propionic acid (also known as N-acetyltryptophan, DL-acetyltryptophan, and N-acetyl-DL-tryptophan). In certain embodiments, the cryopreservation composition comprises one or more stabilizers per gram of protein in the composition, each less than 0.1 mmol. In certain embodiments, the cryopreservation composition comprises, for example, from 0.05 or about 0.05 mmol to 0.1 or about 0.1 mmol, for example, from 0.064 or about 0.064 mmol to 0.096 or about 0.096 mmol, per gram of protein in the composition. In certain embodiments, the cryopreservation composition comprises less than 0.1 mmol of total stabilizer per gram of protein in the cryopreservation composition. In certain embodiments, the cryopreservation composition comprises, for example, from 0.05 or about 0.05 mmol to 0.1 or about 0.1 mmol, such as from 0.064 or about 0.064 mmol to 0.096 or about 0.096 mmol, of total stabilizer per gram of albumin in the cryopreservation composition.

[0564] Glucan

[0565] In certain embodiments, the cryopreservation composition comprises dextran or a derivative thereof.

[0566] Glucan is a polysaccharide composed of approximately 95% α-D-(1-6) bonds (designated as (C6H 10 O5) n). Dextran fractions are provided with molecular weights ranging from about 1000 daltons to about 2,000,000 daltons. They are represented by numbers (Dextran X), such as Dextran 1, Dextran 10, Dextran 40, Dextran 70, etc., where X corresponds to the average molecular weight divided by 1000 daltons. Thus, for example, Dextran 40 has an average molecular weight of or about 40,000 daltons.

[0567] In certain embodiments, the dextran has an average molecular weight of from 1,000 or about 1,000 daltons to 2,000,000 or about 2,000,000 daltons. In certain embodiments, the dextran has an average molecular weight of at or about 40,000 daltons. In certain embodiments, the dextran has an average molecular weight of at or about 70,000 daltons.

[0568] In certain embodiments, the dextran is selected from the group consisting of dextran 40, dextran 70, and combinations thereof. In certain embodiments, the dextran is dextran 40.

[0569] In certain embodiments, the dextran, such as dextran 40, is provided in the form of a solution, also referred to herein as a dextran solution or a dextran 40 solution. Thus, in certain embodiments, the complex comprises a dextran solution, such as a dextran 40 solution.

[0570] In certain embodiments, the dextran solution is suitable for intravenous use.

[0571] In some embodiments, the dextran solution comprises about 5% to about 50% w / w dextran, such as dextran 40. In some embodiments, the dextran solution comprises from 5% or about 5% to 50% or about 50%, from 5% or about 5% to 45% or about 45%, from 5% or about 5% to 40% or about 40%, from 5% or about 5% to 35% or about 35%, from 5% or about 5% to 30% or about 30%, from 5% or about 5% to 25% or about 25%, from 5% or about 5% to 20% or about 20%, from 5% or about 5% to 15% or about 15%, from 5% or about 5% to 10% or about 10%, from 10% or about 10% to 50% or about 50%, from 10% or about 10% to 45% or about 45%, from 10% or about 10% to 50% or about 50%, to 40% or about 40%, from 10% or about 10% to 35% or about 35%, from 10% or about 10% to 30% or about 30%, from 10% or about 10% to 25% or about 25%, from 10% or about 10% to 20% or about 20%, from 10% or about 10% to 15% or about 15%, from 15% or about 15% to 50% or about 50%, from 15% or about 15% to 45% or about 45%, from 15% or about 15% to 40% or about 40%, from 15% or about 15% to 35% or about 35%, from 15% or about 15% to 30% or about 30%, and from 15% or about 15% to 25% or about 25% , from 15% or about 15% to 20% or about 20%, from 20% or about 20% to 50% or about 50%, from 20% or about 20% to 45% or about 45%, from 20% or about 20% to 40% or about 40%, from 20% or about 20% to 35% or about 35%, from 20% or about 20% to 30% or about 30%, from 20% or about 20% to 25% or about 25%, from 25% or about 25% to 50% or about 50%, from 25% or about 25% to 45% or about 45%, from 25% or about 25% to 40% or about 40%, from 25% or about 25% to 35% or about 35%, from 25% or about 25% to 35% or about 35%, from 25% or about 25% to 40% or about 40%, In some embodiments, the present invention provides a dextran, for example, dextran 40, from 30% to 30% or about 30%, from 30% or about 30% to 50% or about 50%, from 30% or about 30% to 45% or about 45%, from 30% or about 30% to 40% or about 40%, from 30% or about 30% to 35% or about 35%, from 35% or about 35% to 50% or about 50%, from 35% or about 35% to 45% or about 45%, from 35% or about 35% to 40% or about 40%, from 40% or about 40% to 50% or about 50%, from 40% or about 40% to 45% or about 45%, or from 45% or about 45% to 50% or about 50% w / w dextran, for example, dextran 40. In certain embodiments, 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, such as dextran 40.

[0572] In some embodiments, the dextran solution comprises from 25 or about 25 g / L to 200 or about 200 g / L of dextran, for example, dextran 40. In some embodiments, the dextran solution comprises from 35 or about 35 g / L to 200 or about 200 g / L, from 25 or about 25 g / L to 175 or about 175 g / L, from 25 or about 25 g / L to 150 or about 150 g / L, from 25 or about 25 g / L to 125 or about 125 g / L, from 25 or about 25 g / L to 100 or about 100 g / L, from 25 or about 25 g / L to 75 or about 75 g / L, from 25 or about 25 g / L to 50 or about 50 g / L, g / L, from 50 or about 50 g / L to 100 or about 100 g / L, from 50 or about 50 g / L to 75 or about 75 g / L, from 75 or about 75 g / L to 200 or about 200 g / L, from 50 or about 50 g / L to 175 or about 175 g / L, from 50 or about 50 g / L to 150 or about 150 g / L, from 50 or about 50 g / L to 125 or about 125 g / L, from 50 or about 50 g / L to 100 or about 100 g / L, from 50 or about 50 g / L to 75 or about 75 g / L, from 75 or about 75 g / L to 200 or about 200 g / L, from 75 or about 75 g / L to 100 or about 100 g / L, g / L, from 75 or about 75 g / L to 150 or about 150 g / L, from 75 or about 75 g / L to 125 or about 125 g / L, from 75 or about 75 g / L to 100 or about 100 g / L, from 100 or about 100 g / L to 200 or about 200 g / L, from 100 or about 100 g / L to 175 or about 175 g / L, from 100 or about 100 g / L to 150 or about 150 g / L, from 100 or about 100 g / L to 125 or about 125 g / L g / L, or from 175 or about 175 g / L to 200 or about 200 g / L, or from 175 or about 175 g / L to 200 or about 200 g / L. In certain embodiments, the dextran solution comprises 25 g / L, 50 g / L, 75 g / L, 100 g / L, 125 g / L, 150 g / L, 175 g / L, or 200 g / L of dextran, e.g., dextran 40. In certain embodiments, the dextran solution comprises 100 g / L of dextran, e.g., dextran 40. In certain embodiments, the dextran solution comprises about 25 g / L, about 50 g / L, about 75 g / L, about 100 g / L, about 125 g / L, about 150 g / L, about 175 g / L, or about 200 g / L of dextran, e.g., dextran 40. In certain embodiments, the dextran solution comprises about 100 g / L of dextran, e.g., dextran 40.

[0573] In some embodiments, the dextran solution further comprises glucose (also known as dextrose). In some embodiments, the dextran solution comprises from 10 or about 10 g / L to 100 or about 100 g / L of glucose.In some embodiments, the dextran solution comprises from 10 or about 10 g / L to 100 or about 100 g / L, from 10 or about 10 g / L to 90 or about 90 g / L, from 10 or about 10 g / L to 80 or about 80 g / L, from 10 or about 10 g / L to 70 or about 70 g / L, from 10 or about 10 g / L to 60 or about 60 g / L, from 10 or about 10 g / L to 50 or about 50 g / L, from 10 or about 10 g / L to 40 or about 40 g / L, from 10 or about 10 g / L to 30 or about 30 g / L, from 10 or about 10 g / L to 20 or about 20 g / L, from 20 or about 20 g / L to 100 or about 100 g / L, from 20 or about 20 g / L to 90 or about 100 g / L, g / L, from 20 or about 20 g / L to 40 or about 40 g / L, from 20 or about 20 g / L to 50 or about 50 g / L, from 20 or about 20 g / L to 60 or about 60 g / L, from 20 or about 20 g / L to 70 or about 70 g / L, from 20 or about 20 g / L to 80 or about 80 g / L, from 20 or about 20 g / L to 90 or about 90 g / L, from 20 or about 20 g / L to 80 or about 80 g / L, from 20 or about 20 g / L to 70 or about 70 g / L, from 20 or about 20 g / L to 60 or about 60 g / L, from 20 or about 20 g / L to 50 or about 50 g / L, from g / L, from 30 or about 30 g / L to 100 or about 100 g / L, from 30 or about 30 g / L to 90 or about 90 g / L, from 30 or about 30 g / L to 80 or about 80 g / L, from 30 or about 30 g / L to 70 or about 70 g / L, from 30 or about 30 g / L to 60 or about 60 g / L, from 30 or about 30 g / L to 70 or about 70 g / L, from 30 or about 30 g / L to 80 or about 80 g / L, g / L to 50 or about 50 g / L, from 30 or about 30 g / L to 40 or about 40 g / L, from 40 or about 40 g / L to 100 or about 100 g / L, from 40 or about 40 g / L to 90 or about 90 g / L, from 40 or about 40 g / L to 80 or about 80 g / L, from 40 or about 40 g / L to 70 or about 70 g / L, from 40 or about 40 g / L to 80 or about 80 g / L, from 40 or about 40 g / L to 80 or about 80 g / L, from 40 or about 40 g / L to 90 or about 90 g / L, g / L to 80 or about 80 g / L, from 50 or about 50 g / L to 70 or about 70 g / L, from 50 or about 50 g / L to 90 or about 90 g / L, from 50 or about 50 g / L to 80 or about 80 g / L, from 50 or about 50 g / L to 90 or about 90 g / L, from 50 or about 50 g / L to 100 or about 10 ... In some embodiments, the present invention provides an amount of glucose in the range of 50 or about 50 g / L to 60 or about 60 g / L, from 60 or about 60 g / L to 100 or about 100 g / L, from 60 or about 60 g / L to 90 or about 90 g / L, from 60 or about 60 g / L to 80 or about 80 g / L, from 60 or about 60 g / L to 70 or about 70 g / L, from 70 or about 70 g / L to 100 or about 100 g / L, from 70 or about 70 g / L to 90 or about 90 g / L, from 70 or about 70 g / L to 80 or about 80 g / L, from 80 or about 80 g / L to 100 or about 100 g / L, from 80 or about 80 g / L to 90 or about 90 g / L, or from 90 or about 90 g / L to 100 or about 100 g / L glucose.In certain embodiments, the glucan solution comprises 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, or 100 g / L of glucose. In certain embodiments, the glucan solution comprises 50 g / L of glucose. In certain embodiments, the glucan solution comprises 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 certain embodiments, the glucan solution comprises 50 g / L of glucose.

[0574] In certain embodiments, the dextran solution consists of dextran (eg, dextran 40) and an aqueous solution of glucose.

[0575] In certain embodiments, the cryopreservation composition comprises from 10% or about 10% to 50% or about 50% by volume of a dextran solution, such as a dextran solution described herein. In certain embodiments, the cryopreservation composition comprises from 10% or about 10% to 50% or about 50%, from 10% or about 10% to 45% or about 45%, from 10% or about 10% to 40% or about 40%, from 10% or about 10% to 35% or about 35%, from 10% or about 10% to 30% or about 30%, from 10% or about 10% to 25% or about 25%, from 10% or about 10% to 20% or about 20%, from 10% or about 10% to 15% or about 15%, from 15% or about 15% to 5 0% or about 50%, from 15% or about 15% to 45% or about 45%, from 15% or about 15% to 40% or about 40%, from 15% or about 15% to 35% or about 35%, from 15% or about 15% to 30% or about 30%, from 15% or about 15% to 25% or about 25%, from 15% or about 15% to 20% or about 20%, from 20% or about 20% to 50% or about 50%, from 20% or about 20% to 45% or about 45%, from 20% or about 20% to 40% or about 40 ... %, from 20% or about 20% to 35% or about 35%, from 20% or about 20% to 30% or about 30%, from 20% or about 20% to 25% or about 25%, from 25% or about 25% to 50% or about 50%, from 25% or about 25% to 45% or about 45%, from 25% or about 25% to 40% or about 40%, from 25% or about 25% to 35% or about 35%, from 25% or about 25% to 30% or about 30%, from 30% or about 30% to 50% or about 50%, from 30% or about 30% to 45% In some embodiments, the cryopreservation composition comprises 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by volume of a dextran solution, such as a dextran solution described herein. In certain embodiments, the cryopreservation composition comprises about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by volume of a dextran solution, such as the dextran solution described herein.

[0576] In certain embodiments, the cryopreservation composition comprises from 10 or about 10 g / L to 50 or about 50 g / L of dextran, such as dextran 40. In certain embodiments, the cryopreservation composition comprises from 10 or about 10 g / L to 50 or about 50 g / L, from 10 or about 10 g / L to 45 or about 45 g / L, from 10 or about 10 g / L to 40 or about 40 g / L, from 10 or about 10 g / L to 35 or about 35 g / L, from 10 or about 10 g / L to 30 or about 30 g / L, from 10 or about 10 g / L to 25 or about 25 g / L, from 10 or about 10 g / L to 20 or about 20 g / L, from 10 or about 10 g / L to 15 or about 15 g / L, from 15 or about 15 g / L to 50 or about 50 g / L. g / L, from 15 or about 15 g / L to 45 or about 45 g / L, from 15 or about 15 g / L to 40 or about 40 g / L, from 15 or about 15 g / L to 35 or about 35 g / L, from 15 or about 15 g / L to 30 or about 30 g / L, from 15 or about 15 g / L to 25 or about 25 g / L, from 15 or about 15 g / L to 20 or about 20 g / L, from 20 or about 20 g / L to 50 or about 50 g / L, from 20 or about 20 g / L to 45 or about 45 g / L, from 20 or about 20 g / L to 40 or about 40 g / L, from 20 or about 20 g / L to 45 or about 45 g / L, g / L, from 25 or about 25 g / L to 40 or about 40 g / L, from 25 or about 25 g / L to 35 or about 35 g / L, from 25 or about 25 g / L to 30 or about 30 g / L, from 20 or about 20 g / L to 25 or about 25 g / L, from 25 or about 25 g / L to 50 or about 50 g / L, from 25 or about 25 g / L to 45 or about 45 g / L, from 25 or about 25 g / L to 40 or about 40 g / L, from 25 or about 25 g / L to 35 or about 35 g / L, from 25 or about 25 g / L to 30 or about 30 g / L, from 30 or about 30 g / L to 50 or about 50 g / L, from 30 or about 30 g / L to 45 or about 45 g / L In some embodiments, the present invention can include a dextran solution, such as a dextran solution described herein, in an amount of 30 or about 30 g / L to 40 or about 40 g / L, from 30 or about 30 g / L to 35 or about 35 g / L, from 35 or about 35 g / L to 50 or about 50 g / L, from 35 or about 35 g / L to 45 or about 45 g / L, from 35 or about 35 g / L to 40 or about 40 g / L, from 40 or about 40 g / L to 50 or about 50 g / L, from 40 or about 40 g / L to 45 or about 45 g / L, or from 45 or about 45 g / L to 50 or about 50 g / L, by volume. In certain embodiments, the cryopreservation composition comprises 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, or 50 g / L of dextran, such as dextran 40.In certain embodiments, the cryopreservation composition comprises about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L, about 35 g / L, about 40 g / L, about 45 g / L, or about 50 g / L of dextran, such as dextran 40.

[0577] glucose

[0578] In certain embodiments, the cryopreservation composition comprises glucose (D-glucose and / or L-glucose).

[0579] In certain embodiments, as described herein, the cryopreservation composition comprises a dextran solution comprising glucose.

[0580] In certain embodiments, the cryopreservation composition comprises a dextran solution that does not comprise glucose. In certain embodiments, for example, when the dextran solution does not comprise glucose, glucose is added separately to the cryopreservation composition.

[0581] In certain embodiments, the cryopreservation composition comprises from 5 or 5 g / L to 25 or about 25 g / L of glucose. In certain embodiments, the cryopreservation composition comprises from 5 or 5 g / L to 25 or about 25 g / L, from 5 or 5 g / L to 20 or about 20 g / L, from 5 or 5 g / L to 15 or about 15 g / L, from 5 or 5 g / L to 10 or about 10 g / L, from 10 or 10 g / L to 25 or about 25 g / L, from 10 or 10 g / L to 20 or about 20 g / L, from 10 or 10 g / L to 15 or about 15 g / L, from 15 or 15 g / L to 25 or about 25 g / L, from 15 or 15 g / L to 20 or about 20 g / L, or from 20 or 20 g / L to 25 or about 25 g / L of glucose. In certain embodiments, the cryopreservation composition comprises 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, 20 g / L, 22.5 g / L, or 25 g / L glucose. In certain embodiments, the cryopreservation composition comprises 12.5 g / L glucose. In certain embodiments, the cryopreservation composition comprises about 5 g / L, about 7.5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 22.5 g / L, about 25 g / L glucose. In certain embodiments, the cryopreservation composition comprises about 12.5 g / L glucose.

[0582] In certain embodiments, the cryopreservation composition comprises less than 2.75% w / v glucose. In certain embodiments, the cryopreservation composition comprises less than 27.5 g / L glucose. In certain embodiments, the cryopreservation composition comprises less than 2% w / v glucose. In certain embodiments, the cryopreservation composition comprises less than 1.5% w / v glucose. In certain embodiments, the cryopreservation composition comprises about 1.25% w / v or less glucose.

[0583] Dimethyl sulfoxide

[0584] In certain embodiments, the cryopreservation composition comprises dimethyl sulfoxide (DMSO, also known as methyl sulfoxide and methylsulfoxylmethane).

[0585] In certain embodiments, DMSO is provided in the form of a solution, also referred to herein as a DMSO solution. Thus, in certain embodiments, the cryopreservation composition comprises a DMSO solution.

[0586] In certain embodiments, the DMSO solution is suitable for intravenous use.

[0587] In certain embodiments, the DMSO solution contains 1.1 g / mL DMSO. In certain embodiments, the DMSO solution contains about 1.1 g / mL DMSO.

[0588] In certain embodiments, the cryopreservation compound comprises from 1% or about 1% to 10% or 10% DMSO solution. In certain embodiments, the cryopreservation compound comprises from 1% or about 1% to 10% or 10%, from 1% or about 1% to 9% or 9%, from 1% or about 1% to 8% or 8%, from 1% or about 1% to 7% or 7%, from 1% or about 1% to 6% or 6%, from 1% or about 1% to 5% or 5%, from 1% or about 1% to 4% or 4%, from 1% or about 1% to 3% or 3%, from 1% or about 1% to 2% or 2%, from 2% or about 2% to 10% or 10%, from 2% or about 2% to 10% or 10%, from 2% or about 2% to 10% or 10%. to 9% or 9%, from 2% or about 2% to 8% or 8%, from 2% or about 2% to 7% or 7%, from 2% or about 2% to 6% or 6%, from 2% or about 2% to 5% or 5%, from 2% or about 2% to 4% or 4%, from 2% or about 2% to 3% or 3%, from 3% or about 3% to 10% or 10%, from 3% or about 3% to 9% or 9%, from 3% or about 3% to 8% or 8%, from 3% or about 3% to 7% or 7%, from 3% or about 3% to 6% or 6%, from 3% or about 3% to 4% or 4%, to 5% or 5%, from 3% or about 3% to 4% or 4%, from 4% or about 4% to 10% or 10%, from 4% or about 4% to 9% or 9%, from 4% or about 4% to 8% or 8%, from 4% or about 4% to 7% or 7%, from 4% or about 4% to 6% or 6%, from 4% or about 4% to 5% or 5%, from 5% or about 5% to 10% or 10%, from 5% or about 5% to 9% or 9%, from 5% or about 5% to 8% or 8%, from 5% or about 5% to 7% or 7%, from 5% or about 5% to In some embodiments, the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by volume of a DMSO solution. In some embodiments, the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% by volume of a DMSO solution. In some embodiments, the cryopreservation composition comprises 5% DMSO solution. In certain embodiments, 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% by volume of a DMSO solution. In certain embodiments, the cryopreservation composition comprises about 5% DMSO solution.

[0589] In certain embodiments, the cryopreservation composition includes from at or about 11 g / L to at or about 110 g / L of DMSO solution. In certain embodiments, the cryopreservation composition comprises from 11 or about 11 g / L to 110 or 110 g / L, from 11 or about 11 g / L to 99 or 99 g / L, from 11 or about 11 g / L to 88 or 88 g / L, from 11 or about 11 g / L to 77 or 77 g / L, from 11 or about 11 g / L to 66 or 66 g / L, from 11 or about 11 g / L to 55 or 55 g / L, from 11 or about 11 g / L to 44 or 44 g / L, from 11 or about 11 g / L to 33 or 33 g / L, from 11 or about 11 g / L to 22 or 22 g / L, from 22 or about 22 g / L to 110 or 110 ... g / L, from 22 or about 22 g / L to 88 or 88 g / L, from 22 or about 22 g / L to 77 or 77 g / L, from 22 or about 22 g / L to 66 or 66 g / L, from 22 or about 22 g / L to 55 or 55 g / L, from 22 or about 22 g / L to 44 or 44 g / L, from 22 or about 22 g / L to 33 or 33 g / L, from 33 or about 33 g / L to 110 or 110 g / L, from 33 or about 33 g / L to 99 or 99 g / L, from 33 or about 33 g / L to 88 or 88 g / L, from 33 or about 33 g / L to 77 or 77 g / L, from 22 or about 22 g / L to 66 or 66 g / L, from 22 or about 22 g / L to 55 or 55 g / L, from 22 or about 22 g / L to 44 or 44 g / L, g / L, from 44 or about 44 g / L to 88 or 88 g / L, from 44 or about 44 g / L to 77 or 77 g / L, from 44 or about 44 g / L to 66 or 66 g / L, from 44 or about 44 g / L to 55 or 55 g / L, from 55 or about 55 g / L to 110 or 110 g / L, from 55 or about 55 g / L to 99 or 99 g / L, from 55 or about 55 g / L to 88 or 88 g / L, from 44 or about 44 g / L to 77 or 77 g / L, from 44 or about 44 g / L to 66 or 66 g / L, from 44 or about 44 g / L to 55 or 55 g / L, from 55 or about 55 g / L to 110 or 110 g / L, from 55 or about 55 g / L to 99 or 99 g / L, from 55 or about 55 g / L to 88 or 88 g / L, from 55 or about 55 g / L to 77 or 77 g / L In some embodiments, the present invention relates to an agent that is selected from the group consisting of: a) DMSO at or about 55 g / L to 66 or 66 g / L, b) DMSO at or about 55 g / L to 66 or 66 g / L, c) DMSO at or about 66 g / L to 110 or 110 g / L, d) DMSO at or about 55 g / L to 66 or 66 g / L, d) DMSO at or about 66 g / L to 99 or 99 g / L, d) DMSO at or about 66 g / L to 88 or 88 g / L, d) DMSO at or about 66 g / L to 99 or 99 g / L, d) DMSO at or about 66 g / L to 88 or 88 g / L, d) DMSO at or about 66 g / L to 77 or 77 g / L, d) DMSO at or about 77 g / L to 110 or 110 g / L, d) DMSO at or about 99 or 99 g / LIn certain embodiments, the cryopreservation composition comprises 11 g / L, 22 g / L, 33 g / L, 44 g / L, 55 g / L, 66 g / L, 77 g / L, 88 g / L, 99 g / L, or 110 g / L DMSO. In certain embodiments, the cryopreservation composition comprises 55 g / L DMSO. In certain embodiments, the cryopreservation composition comprises about 11 g / L, about 22 g / L, about 33 g / L, about 44 g / L, about 55 g / L, about 66 g / L, about 77 g / L, about 88 g / L, about 99 g / L, or about 110 g / L DMSO. In certain embodiments, the cryopreservation composition comprises about 55 g / L DMSO.

[0590] buffer

[0591] In certain embodiments, the cryopreservation composition comprises a buffer, such as a buffer suitable for intravenous administration.

[0592] The buffer solution includes, but is not limited to, phosphate buffered saline (PBS), Ringer's solution, Tyrode's buffer, Hank's balanced salt solution, Earle's balanced salt solution, and Tris.

[0593] In certain embodiments, the buffer is phosphate buffered saline (PBS).

[0594] Exemplary Cryopreservation Compositions

[0595] In certain embodiments, the cryopreservation composition comprises or consists of: (1) albumin, such as human albumin; (2) dextran, such as dextran 40; (3) DMSO; and (4) a buffer solution. In certain embodiments, the cryopreservation composition further comprises glucose. In certain embodiments, the cryopreservation composition consists of (1) albumin, such as human albumin; (2) dextran, such as dextran 40; (3) glucose; 4) DMSO; and (5) a buffer solution.

[0596] In certain embodiments, the cryopreservation composition comprises (1) an albumin solution as described herein, (2) a dextran solution as described herein, (3) a DMSO solution as described herein, and (4) a buffer.

[0597] In certain embodiments, the cryopreservation composition consists of (1) an albumin solution as described herein, (2) a dextran solution as described herein, (3) a DMSO solution as described herein, and (4) a buffer.

[0598] In certain embodiments, the cryopreservation composition does not include cell culture medium.

[0599] In specific embodiments, the cryopreservation composition includes or comprises about 40 mg / mL human albumin, 25 mg / mL Dextran 40, 12.5 mg / mL glucose, and 55 mg / mL DMSO.

[0600] In specific embodiments, the cryopreservation composition comprises or 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% phosphate buffered saline (PBS) in water, or consists of or consists of 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% phosphate buffered saline (PBS) in water.

[0601] In a specific embodiment, the cryopreservation composition includes or comprises about 32 mg / mL human albumin, 25 mg / mL Dextran 40, 12.5 mg / mL glucose, and 55 mg / mL dimethyl sulfoxide.

[0602] In specific embodiments, the cryopreservation composition comprises or consists of or consists of approximately 32 mg / mL human albumin, 25 mg / mL dextran 40, 12.5 mg / mL glucose, and 55 mg / mL dimethyl sulfoxide, and 0.54 mL / mL 100% phosphate buffered saline (PBS) in water.

[0603] Table 3, Table 4, Table 5, and Table 6 show exemplary cryopreservation compositions.

[0604] Table 3 Exemplary cryopreservation compositions

[0605]

[0606] Table 4 Exemplary Cryopreservation Composition #1

[0607]

[0608] Table 5 Exemplary Cryopreservation Composition #2

[0609]

[0610] Table 6 Exemplary Cryopreservation Composition #3

[0611]

[0612] Cryopreservation method

[0613] The cryopreservation compositions described herein can be used to cryopreserve cells, such as therapeutic cells, such as natural killer (NK) cells, such as the NK cells described herein.

[0614] In certain embodiments, the cell is an animal cell. In certain embodiments, the cell is a human cell.

[0615] In certain embodiments, the cell is an immune cell. In certain embodiments, the immune cell is selected from the group consisting of basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.

[0616] In certain embodiments, the immune cells are natural killer (NK) cells. In certain embodiments, natural killer cells are amplified and stimulated by the methods described herein. In certain embodiments, the NK cells are CAR-NK cells, such as CAR-NK cells described herein.

[0617] In certain embodiments, freezing the cells comprises: mixing the cells with a cryopreservation composition described herein, or a component thereof, to produce a composition, such as a pharmaceutical composition; and freezing the mixture.

[0618] In certain embodiments, freezing cells comprises: mixing a composition comprising cells with a freezing composition described herein or a component thereof to produce a composition, such as a pharmaceutical composition; and then freezing the mixture. In certain embodiments, the composition comprising cells comprises: cells and a buffer. Suitable buffers are described herein.

[0619] In certain embodiments, freezing cells comprises: mixing a complex comprising cells and a buffer (eg, PBS) with a composition comprising albumin, dextran, and DMSO, as described herein; and freezing the mixture.

[0620] In certain embodiments, freezing cells comprises mixing a composition comprising cells and a buffer (e.g., PBS 1:1) with a composition comprising 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.

[0621] In certain embodiments, the composition comprising cells and a buffer (e.g., PBS) comprises from 2×10 7 or about 2×10 7 to 2×10 9 or about 2×10 9In certain embodiments, the composition comprising cells and a buffer (e.g., PBS) comprises 2×10 8 In some embodiments, the composition comprising cells and a buffer (e.g., PBS) comprises approximately 2×10 8 cells / mL.

[0622] In certain embodiments, freezing cells comprises: mixing cells, a buffer (eg, PBS), an albumin (eg, human albumin), a dextran (eg, dextran 40), and DMSO; and freezing the mixture.

[0623] In certain embodiments, the mixture comprises from 1×10 7 or about 1×10 7 to 1×10 9 or about 1×10 9 In some embodiments, the mixture comprises 1×10 8 In some embodiments, the mixture comprises about 1×10 8 cells / mL.

[0624] Suitable ranges for albumin, dextran and DMSO are as described above.

[0625] In certain embodiments, the composition is frozen at -135°C or below.

[0626] In certain embodiments, the composition is frozen at a controlled rate.

[0627] Pharmaceutical composition

[0628] Provided herein are pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein.

[0629] In some cases, the dosage unit includes 100 million to 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.

[0630] The pharmaceutical composition generally includes a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration.

[0631] In certain embodiments, the pharmaceutical composition comprises: a) natural killer cells described herein; and b) a cryopreservation composition.

[0632] Suitable cryopreservation compositions are described herein.

[0633] In certain embodiments, the composition is frozen. In certain embodiments, the composition has been frozen for at least three months, e.g., at least six months, at least nine months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

[0634] In certain embodiments, at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the natural killer cells are viable after thawing.

[0635] In certain embodiments, the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cells.

[0636] In certain embodiments, the therapeutic cells are animal cells. In certain embodiments, the therapeutic cells are human cells.

[0637] In certain embodiments, the therapeutic cells are immune cells. In certain embodiments, the immune cells are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof.

[0638] In certain embodiments, the immune cells are natural killer (NK) cells. In certain embodiments, natural killer cells are expanded and stimulated by the methods described herein, such as the CAR-NK described herein.

[0639] In certain embodiments, the pharmaceutical composition further comprises: c) a buffer. Suitable buffer solutions are described herein, for example, for use in freezing compositions.

[0640] In certain embodiments, the pharmaceutical composition comprises from 1×10 7 or about 1×10 7 to 1×10 9 or about 1×10 9 In certain embodiments, the pharmaceutical composition comprises 1×10 8 In certain embodiments, the pharmaceutical composition comprises about 1×10 8 cells / mL.

[0641] In certain embodiments, the pharmaceutical composition further comprises an antibody or antigen-binding fragment thereof, such as an antibody described herein.

[0642] The pharmaceutical composition is typically formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal.

[0643] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: the Science and Practice of Pharmacy, 21st ed., 2005; and books in the series Drugs and Pharmaceutical Sciences: A Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following ingredients: a sterile diluent, such as water for injection, physiological saline, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; 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 tonic, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0644] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that is easy to inject. It should be stable under manufacturing and storage conditions and must prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, and the solute or dispersion medium comprises such as water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by using a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is best to include isotonic agents in the composition, such as polyols such as sugar, mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0645] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into an appropriate solvent with one or more of the above-mentioned ingredients as required, followed by filtration sterilization. Generally, dispersions can be prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0646] Treatment

[0647] The NK cells described herein, such as the CAR-NK cells described herein, can be used to treat cancer or other proliferative diseases.

[0648] Thus, also provided herein are methods of treating a patient having a disease, e.g., a disease associated with cancer, e.g., a HER2+ cancer, comprising administering NK cells, e.g., the NK cells described herein, e.g., the CAR-NK cells described herein.

[0649] Also provided herein are methods for preventing, reducing and / or inhibiting the recurrence, growth, proliferation, migration and / or metastasis of cancer cells or cancer cell populations in a subject in need thereof, comprising administering NK cells, such as the NK cells described herein, such as the CAR-NK cells described herein.

[0650] Also provided herein are methods of enhancing, improving and / or increasing a response to an anti-cancer therapy in a subject in need thereof, comprising administering NK cells, e.g., the NK cells described herein, e.g., the CAR-NK cells described herein.

[0651] Also provided herein are methods of inducing the immune system in a subject in need thereof, comprising administering NK cells, e.g., the NK cells described herein, e.g., the CAR-NK cells described herein.

[0652] The methods described herein include methods for treating diseases associated with abnormal apoptosis or differentiation processes, such as cell proliferative diseases or cell differentiation diseases, such as cancer, including solid tumors and hematopoietic cancers. Typically, the method includes administering a therapeutically effective amount of a treatment as described herein to a subject in need or determined to be in need of such treatment. In some embodiments, the method includes administering a therapeutically effective amount of a treatment comprising NK cells, such as CAR-NK cells as described herein.

[0653] As used herein, the terms "treatment method", "treatment" and "treatment mode" refer to reversing, alleviating, delaying the onset of a disease associated with an abnormal apoptosis or differentiation process, or inhibiting its progression. For example, treatment can reduce tumor size or growth rate. Administration of a therapeutically effective amount of a compound described herein for the treatment of a disease associated with an abnormal apoptosis or differentiation process will result in a reduction in tumor size or a reduction in growth rate, a reduction in the risk or frequency of recurrence, a delay in recurrence, a reduction in metastasis, an increase in survival rate and / or a reduction in morbidity and mortality, etc. In some embodiments, treatment can be administered after the onset of one or more symptoms. In other embodiments, treatment can be administered in the absence of symptoms. For example, susceptible individuals can be treated before the onset of symptoms (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment can also be continued after symptom relief, for example to prevent or delay its recurrence.

[0654] As used herein, the term "inhibit" refers to inhibiting the growth, division, maturation or viability of cancer cells, either alone or in combination with other cancer cells, and / or causing the death of cancer cells, by cytotoxicity, nutrient depletion or induction of apoptosis.

[0655] As used herein, "delaying" the development of a disease or disorder or one or more symptoms thereof refers to postponing, hindering, slowing down, slowing down, stabilizing and / or delaying the development of a disease, disorder or its symptoms. The length of time for such delay may vary, depending on the history of the disease and / or the subject being treated. As known to those skilled in the art, sufficient or significant delay can actually include prevention, in that the subject does not develop the disease, disorder or its symptoms. For example, a method of "delaying" the development of cancer is a method of reducing the probability of disease development and / or reducing the extent of the disease within a given time frame, compared to not using the method. Such comparisons may be based on clinical studies using a statistically significant number of subjects.

[0656] As used herein, "preventive method" or "prophylaxis" refers to a regimen that prevents the onset of a disease or disorder so that clinical symptoms of the disease do not develop. Thus, a "preventive method" involves administering treatment (e.g., administering a therapeutic substance) to a subject before signs of the disease are detected in the subject and / or before a certain stage of the disease (e.g., administering a therapeutic substance to a subject with cancer that has not yet metastasized). The subject can be an individual at risk of developing the disease or disorder, or an individual at risk of disease progression (e.g., cancer metastasis). For example, an individual has one or more risk factors known to be associated with the development or onset of a disease or disorder. For example, an individual may have a mutation associated with the development or progression of cancer. Furthermore, it should be understood that prevention may not completely inhibit the occurrence of a disease or disorder. In some cases, prevention includes reducing the risk of developing a disease or disorder. The reduction in risk may not result in the complete elimination of the risk of developing a disease or disorder.

[0657] By "increase" or "enhance" the amount (e.g., with respect to anti-tumor response, cancer cell metastasis) is meant 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 or more times (e.g., 100, 500, 1000 times) the amounts or levels described herein (including all integers and decimal points between and above 1, 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.

[0658] By "lower" or "decrease" or a "lesser" amount (e.g., with respect to tumor size, cancer cell proliferation or growth) is meant a decrease (including all integers and decimal points between and above 1, e.g., 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 times (e.g., 100, 500, 1000 times) of the amounts or levels recited herein. It may also include a reduction 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%, or 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.

[0659] disease

[0660] The methods and compositions disclosed herein can be used to target a wide range of diseases, such as cell proliferative disorders. One benefit of this approach is the use of allogeneic cells to target specific cells. Unlike previous therapies, such as chemotherapy or radiation therapy, using the methods and pharmaceutical compositions herein, one can specifically target potential cells exhibiting deleterious proliferative activity without the need to administer systemic drugs or toxins that randomly affect proliferating cells.

[0661] Examples of cell proliferative and / or differentiation diseases include cancers, such as tumors, sarcomas, metastatic diseases, or hematopoietic neoplastic diseases, such as leukemias. Metastatic tumors can arise from a variety of primary tumor types, including but not limited to tumors of prostate, colon, lung, breast, and liver origin.

[0662] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to an abnormal state or condition characterized by rapid proliferation and growth of cells that have the capacity for autonomous growth. Hyperproliferative and neoplastic disease states can be classified as pathological, that is, characterizing or constituting a disease state, or as non-pathological, that is, deviations from normal but not associated with a disease state. The term is intended to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of their histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair.

[0663] The term "cancer" or "tumor" includes malignancies of various organ systems, such as those affecting the lung, breast, thyroid, lymphoid, gastrointestinal, and genitourinary tracts; as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, carcinomas and / or testicular tumors, non-small cell lung cancer, carcinomas, and carcinomas of the ovary.

[0664] The term "carcinoma" is generally recognized and refers to a malignant tumor of epithelial or endocrine tissue, including respiratory cancer, gastrointestinal cancer, genitourinary cancer, testicular cancer, breast cancer, prostate cancer, endocrine cancer, and melanoma. In some embodiments, the disease is renal cancer or melanoma. Exemplary cancers include cancers formed from cervical, lung, prostate, breast, head and neck, colon, and ovarian tissue. The term also includes carcinosarcoma, for example, including malignant tumors composed of carcinoma tissue and sarcoma tissue. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which tumor cells form a recognizable glandular structure.

[0665] The term "sarcoma" is generally accepted to refer to malignant tumors of mesenchymal derivation.

[0666] Other examples of proliferative diseases include hematopoietic neoplastic diseases. As used herein, the term "hematopoietic neoplastic disease" includes diseases involving hyperplasia / tumor cells of hematopoietic origin, for example, diseases caused by myeloid, lymphoid or erythroid lineages or their precursor cells. Preferably, these diseases originate from poorly differentiated acute leukemias, such as erythroid leukemia and acute megakaryocytic leukemia. Other exemplary myeloid diseases include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia / hemoglobin. 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). Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), and classical Hodgkin lymphoma.

[0667] In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, classic teratoma / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, cardiac tumor, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CM), L), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesia neuroblastoma, Ewing sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer (such as intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart cancer, cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma pancreatic islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, Langerhans cell histiocytosis, cancer, leukemia, lip and mouth cancer, cancer, lung cancer (such as non-small cell lung cancer, small cell lung cancer, pleuropulmonary blastoma and tracheobronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer, midline tract cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasms, myeloid leukemia, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, myeloproliferative neoplasms, nasal cavity and nasal Sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacytoma, multiple myeloma, thoracic pulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma and rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g.,Childhood rhabdomyosarcoma, childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sézary syndrome, skin cancer, small bowel cancer, soft tissue osteosarcoma, squamous cell carcinoma, squamous neck cancer, stomach cancer, T-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thyroid cancer, tracheobronchial cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, and Wilms' tumor.

[0668] In some embodiments, the cancer is a solid tumor.

[0669] In some embodiments, the cancer is a metastatic tumor.

[0670] In some embodiments, the cancer is a HER2+ cancer.

[0671] In some embodiments, the HER2+ cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, cervical squamous cell carcinoma, gastric adenocarcinoma, bile duct cancer, ovarian cancer, renal papillary cell carcinoma, and combinations thereof.

[0672] In some embodiments, the HER2+ cancer is selected from the group consisting of breast cancer, gastric cancer, and ovarian cancer.

[0673] In some embodiments, the HER2+ cancer is breast cancer. In some embodiments, the HER2+ cancer is gastric cancer. In some embodiments, the HER2+ cancer is ovarian cancer.

[0674] patient

[0675] Patients suitable for the compositions and methods of the present invention include those suffering from, diagnosed with, or suspected of having a cell proliferative and / or differentiation disorder, such as cancer. Patients treated with the present technology generally respond better to the methods and compositions of the present invention, in part because the pharmaceutical compositions are allogeneic and target cells identified by the antigen-binding domain, rather than general proliferating cells. Consequently, off-target effects are minimized, and patients are more likely to complete their treatment regimen without experiencing substantial, deleterious off-target effects.

[0676] In some embodiments, the methods of treatment 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 differentiation disease (e.g., cancer). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0677] As used herein, a subject refers to a mammal, including, for example, a human.

[0678] In some embodiments, the mammal is selected from the group consisting of an armadillo, a donkey, a bat, a bear, a beaver, a cat, a chimpanzee, a cow, a coyote, a deer, a dog, a dolphin, an elephant, a fox, a panda, a gibbon, a giraffe, a goat, a gopher, a hedgehog, a hippopotamus, a horse, a humpback whale, a jaguar, a kangaroo, a koala, a leopard, a lion, a llama, a lynx, a mole, a monkey, a mouse, a narwhal, an orangutan, an orca, an otter, a cow, a pig, a polar bear, a porcupine, a cougar, a rabbit, a raccoon, a mouse, a rhino, a sheep, a squirrel, a tiger, a walrus, a weasel, a wolf, a zebra, a goat, a horse, and combinations thereof.

[0679] In some embodiments, the mammal is a human.

[0680] The subject, e.g., a human subject, can be a child, e.g., from about 0 to about 14 years old. The subject can be an adolescent, e.g., from about 15 to about 24 years old. The subject can be an adult, e.g., from about 25 to about 64 years old. The subject can be an elderly person, e.g., over 65 years old.

[0681] In some embodiments, the subject can be a person who exhibits one or more symptoms associated with a cell proliferative and / or differentiated disease, such as a cancer, such as a tumor. Any of the methods of treatment provided herein can be used to treat cancers at different stages. For example, cancer stages include, but are not limited to, early, late, locally advanced, remission, refractory, relapse after remission, and progressive. In some embodiments, the subject is in the early stages of cancer. In other embodiments, the subject is in the late stages of cancer. In various embodiments, the subject is in Phase I, Phase II, Phase III, or Phase IV of cancer. The methods of treatment described herein can promote the reduction or retraction of tumors, reduce or inhibit tumor growth or cancer cell proliferation, and / or induce, increase, or promote tumor cell killing. In some embodiments, the subject is in remission of cancer. The methods of treatment described herein can prevent or delay the metastasis or recurrence of cancer.

[0682] In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status at the time of treatment of 0, 1, 2, 3, or 4. In some embodiments, the subject has an ECOG performance status of 0 to 4, 0 to 3, 0 to 2, 0 to 1, 1 to 4, 1 to 3, 1 to 2, 2 to 4, 2 to 3, or 3 to 4 at the time of treatment.

[0683] In some embodiments, the subject is at risk for, or has a genetic or other predisposition (eg, risk factor) for, developing a diagnosed or undiagnosed cell proliferation and / or differentiation disorder, such as cancer.

[0684] As used herein, "at-risk" individuals refer to individuals at risk of developing a disease to be treated, such as cell proliferative and / or differentiation diseases, such as cancer. In general, "at-risk" subjects may or may not have detectable disease, and may or may not have shown detectable disease before the methods of treatment described herein. "Risk" refers to that an individual has one or more so-called risk factors, which are measurable parameters associated with the development of a disease or condition, and are known in the art. Subjects with one or more of these risk factors have a higher probability of developing cancer than individuals without these risk factors. In general, risk factors may include, for example, age, sex, race, diet, past medical history, the presence of a precursor disease, genetic (such as hereditary) factors, and environmental exposure. In some embodiments, cancer risk subjects include, for example, subjects with relatives who have experienced the disease, and subjects whose risk is determined by analyzing genetic or biochemical markers.

[0685] In addition, the subject may be receiving one or more standard therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or a combination thereof. Thus, one or more kinase inhibitors may be administered before, during, or after chemotherapy, radiotherapy, immunotherapy, surgery, or a combination thereof.

[0686] In certain embodiments, the subject can be a human who is: (i) substantially refractory to at least one chemotherapy, or (ii) relapsed after chemotherapy treatment, or both (i) and (ii). In some embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapies (including standard or experimental chemotherapies).

[0687] In some embodiments, the subject has a cancer that expresses HER2.

[0688] In some embodiments, the patient is diagnosed with or has been diagnosed with a HER2+ cancer.

[0689] In some embodiments, the patient is diagnosed with or has been diagnosed with HER2+ cancer by immunohistochemical staining of a biopsy or surgical specimen of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with HER2+ cancer by fluorescence in situ hybridization of a biopsy or surgical specimen of the cancer.

[0690] In some embodiments, according to Guidelines, e.g., 2018 The patient is diagnosed with or has been diagnosed with HER2+ cancer according to 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), which is incorporated herein by reference in its entirety.

[0691] In some embodiments, according to Guidelines, e.g., 2018 Guidelines, for example, as described in Wolff et al., Human epidermal growth factor receptor 2 detection in breast cancer, Arch Pathol Lab Med 142:1364–82 (2018), wherein the patient is diagnosed with or has been diagnosed with HER2+ cancer with an IHC score of 2+, which is incorporated herein by reference in its entirety. In some embodiments, the patient is diagnosed with or has been diagnosed with HER2+ cancer with an IHC score of 2+ within 12 months of treatment (e.g., within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month).

[0692] In some embodiments, the patient has a cancer that is not HER2-positive but shows some degree of HER2-expression. In some embodiments, the patient is diagnosed with or has been diagnosed with a HER2-low expressing cancer. In some embodiments, according to Guidelines, e.g., 2018 Guidelines, for example, such as Wolff et al., Human epidermal growth factor receptor 2 test in breast cancer, Arch Pathol Lab Med 142: described in 1364-82 (2018), which is incorporated herein by reference in its entirety, low HER2 cancer has a HER2 expression classification scored as 1+ or 2+, but lacks HER2 amplification. In some embodiments, the cancer of low HER2 carries HER2 gain. In some embodiments, the cancer of low HER2 does not carry HER2 gain.

[0693] In some embodiments, the patient is or has been diagnosed with a HER2+ cancer by genetic analysis, eg, by identifying a HER2-mutated cancer, eg, a somatic mutation in the HER2 (ERBB2) gene.

[0694] In some embodiments, the patient has a cancer comprising one or more mutations listed in Table 7: an insertion or deletion polymorphism of the HER2 gene, a copy number variation of the HER2 gene, a methylation mutation of the HER2 gene, or a combination thereof. In some embodiments, the mutation is a HER2 activating mutation.

[0695] In some embodiments, the patient has a chromosomal translocation associated with cancer, such as HER2+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, such as HER+ cancer.

[0696] Table 7. HER2 (ERBB2) mutations (relative to the human genome assembly reference build GRCh38.p13 (ncbi.nlm.nih.gov / Assembly / 88331))

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706] In some embodiments, the HER2+ cancer is HER2+ breast cancer, gastric cancer, gastroesophageal (GEJ) cancer, or lung cancer.

[0707] In some embodiments, the breast cancer is in situ (non-invasive). In some embodiments, the breast cancer is invasive. In some embodiments, the breast cancer is metastatic.

[0708] In some embodiments, the breast cancer is a carcinoma (e.g., adenocarcinoma). In some embodiments, the breast cancer is a ductal carcinoma. In some embodiments, the breast cancer is an invasive lobular carcinoma. In some embodiments, the breast cancer is an inflammatory breast cancer. In some embodiments, the breast cancer is Paget's disease of the breast. In some embodiments, the breast cancer is a phyllodes tumor.

[0709] In some embodiments, the gastric cancer is an adenocarcinoma. In some embodiments, the gastric cancer is an intestinal adenocarcinoma. In some embodiments, the gastric cancer is a diffuse adenocarcinoma. In some embodiments, the gastric cancer is a lymphoma. In certain cases, the gastric cancer is a mucosa-associated lymphoid tissue lymphoma. In some embodiments, the gastric cancer is a gastrointestinal stromal tumor. In some embodiments, the gastric cancer is a gastric carcinoid. In some embodiments, the gastric cancer is a type I, type II, or type III ECL-cell carcinoid. In some embodiments, the gastric cancer is hereditary diffuse gastric cancer.

[0710] In some embodiments, the GEJ cancer is GEJ adenocarcinoma.

[0711] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as metastatic NSCLC. In some embodiments, the patient has NSCLC with a HER2 activating mutation.

[0712] In some embodiments, the patient is refractory to or has relapsed after treatment (eg, trastuzumab or a biosimilar thereof).

[0713] In some embodiments, the patient is refractory to or has relapsed after 1 or more prior systemic therapies. In some embodiments, the patient is refractory to or has relapsed after 2 or more prior systemic therapies.

[0714] In some embodiments, according to Guidelines, e.g., 2018 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), which is incorporated herein by reference in its entirety, are when a patient is diagnosed with IHC 3+ or IHC 2+ / ISH+ cancer and the patient has failed or relapsed after prior treatment with HER2-targeted therapy.

[0715] In some embodiments, according to Guidelines, e.g., 2018 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), which is incorporated herein by reference in its entirety, are provided when a patient is diagnosed with an IHC 1+ or IHC 2+ / ISH- cancer (e.g., breast cancer) that expresses HER2, the patient's cancer is unresectable, metastatic, or the patient has failed or relapsed after prior treatment.

[0716] In some embodiments, the patient is ineffective or relapsed after treatment with pertuzumab (or its FDA-approved biosimilar), trastuzumab (or its FDA-approved biosimilar) and docetaxel (or its pharmaceutically acceptable salt). In some embodiments, pertuzumab (or its FDA-approved biosimilar) is administered as an intravenous injection of 840 mg on day 1, followed by an intravenous injection of 420 mg. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered as an intravenous injection of 7 mg / kg on day 1, followed by an intravenous injection of 6 mg / kg every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously via trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection. In some embodiments, docetaxel (or its pharmaceutically acceptable salt) is administered as an intravenous injection of 75-100 mg / m2 on day 1, with a cycle of 21 days.

[0717] In some embodiments, the patient is ineffective or relapses after treatment with pertuzumab (or its FDA-approved biosimilar), trastuzumab (or its FDA-approved biosimilar) and paclitaxel (or its pharmaceutically acceptable salt). In some embodiments, pertuzumab (or its FDA-approved biosimilar) is administered as 840 mg intravenously on day 1, followed by 420 mg intravenously, with a cycle every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered as 4 mg / kg intravenously on day 1, followed by 2 mg / kg intravenously every week, or as 8 mg / kg intravenously on day 1, followed by 6 mg / kg intravenously every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously with trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection. In some embodiments, paclitaxel (or a pharmaceutically acceptable salt thereof) is administered weekly at 80 mg / m2 intravenously on day 1, or at 175 mg / m2 intravenously on day 1, in cycles of 21 days.

[0718] In some embodiments, the patient is ineffective or relapses after treatment with tucatinib (or a pharmaceutically acceptable salt thereof), trastuzumab (or its FDA-approved biosimilar) and capecitabine (or its medically acceptable salt). In some embodiments, 300 mg of tucatinib (or its FDA-approved biosimilar) is taken orally twice a day on days 1-21. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously at 8 mg / kg on day 1, and then at 6 mg / kg every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously via trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection. In some embodiments, capecitabine (or its FDA-approved biosimilar) is administered orally at a dose of 1000 mg / m2 twice a day on days 1-14. In some embodiments, tucatinib (or an FDA-approved biosimilar thereof), trastuzumab (or an FDA-approved biosimilar thereof), and capecitabine (or a pharmaceutically acceptable salt thereof) are administered in a cycle every 21 days.

[0719] In some embodiments, the patient is refractory or relapsed after treatment with trastuzumab emtansine (T-DM1) (or its FDA-approved biosimilar). In some embodiments, trastuzumab emtansine (T-DM1) (or its FDA-approved biosimilar) is administered as an intravenous injection of 3.6 mg / kg on day 1, with a cycle of 21 days.

[0720] In some embodiments, the patient is refractory to or has relapsed from treatment with trastuzumab (or its FDA-approved biosimilar). In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered as an intravenous injection of 5.4 mg / kg on day 1, with a cycle of 21 days.

[0721] In some embodiments, the patient is ineffective or relapsed after treatment with paclitaxel / carboplatin (or a pharmaceutically acceptable salt thereof) and trastuzumab (or its FDA-approved biosimilar). In some embodiments, carboplatin / paclitaxel (or its pharmaceutically acceptable salt) is administered intravenously on day 1 at carboplatin AUC 6 and intravenously on day 1 at paclitaxel 175 mg / m2, with a cycle of 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously on day 1 at 4 mg / kg, followed by 2 mg / kg intravenously weekly, or 8 mg / kg intravenously on day 1, followed by 6 mg / kg intravenously every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously with trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oy sk injection.

[0722] In some embodiments, the patient is ineffective or relapsed after treatment with paclitaxel / carboplatin (or a pharmaceutically acceptable salt thereof) and trastuzumab (or its FDA-approved biosimilar). In some embodiments, carboplatin / paclitaxel (or its pharmaceutically acceptable salt) is administered intravenously at carboplatin AUC 2 and paclitaxel 80 mg / m2 on days 1, 8, and 15, with a cycle every 28 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously at 4 mg / kg on day 1, followed by 2 mg / kg intravenously weekly, or at 8 mg / kg intravenously on day 1, followed by 6 mg / kg intravenously every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously with trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection.

[0723] In some embodiments, the patient is ineffective or relapses after treatment with trastuzumab (or its FDA-approved biosimilar) and paclitaxel (or its pharmaceutically acceptable salt). In some embodiments, paclitaxel (or its pharmaceutically acceptable salt) is administered intravenously at 175 mg / m2 on day 1, cycled every 21 days, or administered weekly at 80-90 mg / m2 on day 1. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously at 4 mg / kg on day 1, then intravenously at 2 mg / kg weekly, or intravenously at 8 mg / kg on day 1, then intravenously at 6 mg / kg every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously with trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection.

[0724] In some embodiments, the patient is ineffective or relapsed after treatment with trastuzumab (or its FDA-approved biosimilar) and docetaxel (or its pharmaceutically acceptable salt). In some embodiments, docetaxel (or its pharmaceutically acceptable salt) is administered intravenously at 80-100 mg / m2 on the first day, once every 21 days, or 35 mg / m2 on days 1, 8, and 15, administered weekly. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously at 4 mg / kg on day 1, then 2 mg / kg weekly, or 8 mg / kg on day 1, then 6 mg / kg every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously with trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection.

[0725] In some embodiments, the patient is ineffective or relapses after treatment with trastuzumab (or its FDA-approved biosimilar) and vinorelbine (or its pharmaceutically acceptable salt). In some embodiments, vinorelbine (or its pharmaceutically acceptable salt) is administered at 25 mg / m2 intravenously on the first day of each week, or 20-35 mg / m2 intravenously on the 1st and 8th days, and is administered once every 21 days, or 25-30 mg / m2 intravenously on the 1st, 8th and 15th days, and is administered once every 28 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered at 4 mg / kg intravenously on the first day, then at 2 mg / kg intravenously weekly, or at 8 mg / kg intravenously on the first day, then at 6 mg / kg intravenously every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously via trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection.

[0726] In some embodiments, the patient is ineffective or relapses after treatment with trastuzumab (or its FDA-approved biosimilar) and capecitabine (or its pharmaceutically acceptable salt). In some embodiments, capecitabine (or its pharmaceutically acceptable salt) is administered orally on days 1-14 at 1000-1250 mg / m2 twice a day, with a cycle every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously at 4 mg / kg on day 1, followed by 2 mg / kg intravenously weekly, or 8 mg / kg intravenously on day 1, followed by 6 mg / kg intravenously every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously with trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection.

[0727] In some embodiments, the patient is ineffective or relapses after treatment with lapatinib (or a pharmaceutically acceptable salt thereof) and capecitabine (or a medically acceptable salt thereof). In some embodiments, lapatinib (or a pharmaceutically acceptable salt thereof) is orally administered at 1250 mg / m2 per day for 1-21 days. In some embodiments, capecitabine (or a pharmaceutically acceptable salt thereof) is orally administered at 1000 mg / m2 twice a day for 1-14 days, with a cycle of administration every 21 days.

[0728] In some embodiments, the patient is invalid or relapses after treatment with trastuzumab (or its FDA-approved biosimilar) and lapatinib (or its pharmaceutically acceptable salt). In some embodiments, the lapatinib (or its pharmaceutically acceptable salt) administered is orally administered at 1000 mg / m2 every day. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered intravenously at 4 mg / kg on the first day, followed by intravenous administration of 2 mg / kg per week, or intravenous administration of 8 mg / kg on the first day, followed by intravenous administration of 6 mg / kg every 21 days. In some embodiments, trastuzumab (or its FDA-approved biosimilar) is administered subcutaneously by trastuzumab (or its FDA-approved biosimilar) and hyaluronidase-oysk injection.

[0729] In some embodiments, the patient is refractory or relapsed after treatment with neratinib (or a pharmaceutically acceptable salt thereof) and capecitabine (or a pharmaceutically acceptable salt thereof). In some embodiments, neratinib is taken orally at a dose of 240 mg / m2 daily on days 1-21. 2 In some embodiments, capecitabine is administered at 750 mg / m2 on days 1-14. 2 The drug is administered orally twice daily for a 21-day cycle.

[0730] In some embodiments, the patient has an oxygen saturation level of at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% as measured by pulse oximetry within 1, 5, 10, 15, 20, 30, 45, 60, or 90 minutes, or within 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.

[0731] In some embodiments, the patient has a left ventricular ejection fraction (LVEF) of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65% measured within 1 day, 1 week or 1 month after treatment.

[0732] In some embodiments, the patient has an absolute neutrophil count (ANC) of at least 1000 / mm3 measured within 1 day, 1 week, or 1 month after treatment. 3 (1.0x109 / L), 1500 / mm 3 , 2000 / mm 3 , 2500 / mm 3 、3000 / mm 3 、3500 / mm 3 , 4000 / mm 3 , 4500 / mm3 , 5000 / mm 3 , 5500 / mm 3 or 6000 / mm 3 In some embodiments, the patient's ANC measured within 1 day, 1 week, or 1 month after treatment is 1000 / mm 3 Up to 6000 / mm 3 , 1000 / mm 3 Up to 5500 / mm 3 , 1000 / mm 3 Up to 5000 / mm 3 , 1000 / mm 3 Up to 4500 / mm 3 , 1000 / mm 3 Up to 4000 / mm 3 , 1000 / mm 3 Up to 3500 / mm 3 , 1000 / mm 3 Up to 3000 / mm 3 , 1000 / mm 3 Up to 2500 / mm 3 , 1000 / mm 3 Up to 2000 / mm 3 , 1000 / mm 3 Up to 1500 / mm 3 、1500 / mm 3 Up to 6000 / mm 3 、1500 / mm 3 Up to 5500 / mm 3 、1500 / mm 3 Up to 5000 / mm 3 、1500 / mm 3 Up to 4500 / mm 3 、1500 / mm 3 Up to 4000 / mm 3 、1500 / mm 3 Up to 3500 / mm 3 、1500 / mm 3 Up to 3000 / mm 3 、1500 / mm 3 Up to 2500 / mm 3 、1500 / mm 3 Up to 2000 / mm 3 , 2000 / mm 3 Up to 6000 / mm 3 , 2000 / mm 3 Up to 5500 / mm 3 , 2000 / mm3 to 5000 / mm 3 、2000 / mm 3 to 4500 / mm 3 、2000 / mm 3 to 4000 / mm 3 、2000 / mm 3 to 3500 / mm 3 、2000 / mm 3 to 3000 / mm 3 、2000 / mm 3 to 2500 / mm 3 、2500 / mm 3 to 6000 / mm 3 、2500 / mm 3 to 5500 / mm 3 、2500 / mm 3 to 5000 / mm 3 、2500 / mm 3 to 4500 / mm 3 、2500 / mm 3 to 4000 / mm 3 、2500 / mm 3 to 3500 / mm 3 、2500 / mm 3 to 3000 / mm 3 、3000 / mm 3 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​, 4000 / mm 3 Up to 6000 / mm 3 , 4000 / mm 3 Up to 5500 / mm 3 , 4000 / mm 3 Up to 5000 / mm 3 , 4000 / mm 3 Up to 4500 / mm 3 , 4500 / mm 3 Up to 6000 / mm 3 , 4500 / mm 3 Up to 5500 / mm 3 , 4500 / mm 3 Up to 5000 / mm 3 , 5000 / mm 3 Up to 6000 / mm 3 , 5000 / mm 3 Up to 5500 / mm 3 , or 5500 / mm 3 Up to 6000 / mm 3 .

[0733] In some embodiments, the patient's platelet count is at least 75,000 / mm3 measured within 1 day, 1 week, or 1 month after treatment. 3 (75x10 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 some embodiments, the patient has a platelet count of 75,000 / mm3 measured within 1 day, 1 week, or 1 month after treatment. 3 Up to 400,000 / mm 3 , 75000 / mm 3 Up to 375,000 / mm 3 , 75000 / mm 3 Up to 350,000 / mm 3, 75000 / mm 3 to 300000 / mm 3 , 75000 / mm 3 to 275000 / mm 3 , 75000 / mm 3 to 250000 / mm 3 , 75000 / mm 3 to 225000 / mm 3 , 75000 / mm 3 to 200000 / mm 3 , 75000 / mm 3 to 175000 / mm 3 , 75000 / mm 3 to 150000 / mm 3 , 75000 / mm 3 to 125000 / mm 3 , 75000 / mm 3 to 100000 / mm 3 , 100000 / mm 3 to 400000 / mm 3 , 100000 / mm 3 to 375000 / mm 3 , 100000 / mm 3 to 350000 / mm 3 , 100000 / mm 3 to 325000 / mm 3 , 100000 / mm 3 to 300000 / mm 3 , 100000 / mm 3 to 275000 / mm 3 , 100000 / mm 3 to 2500时0 / mm 3 , 100000 / mm 3 to 225000 / mm 3 , 100000 / mm 3 to 200000 / mm 3 , 100000 / mm 3 to 175000 / mm 3 , 100000 / mm 3 to 150000 / mm 3 [[ID=SO]], 100000 / mm 3 to 125000 / mm 3 , 125000 / mm 3 to 400000 / mm It should be noted that there seems to be a small error in the original text where "2500时0 / mm" in line might be a typo. I've translated it as is based on the rules, but it might need to be corrected in the original source.3 、125,000 / mm 3 to 375,000 / mm 3 、125,000 / mm 3 to 350,000 / mm 3 、125,000 / mm 3 to 325,000 / mm 3 、125,000 / mm 3 to 300,000 / mm 3 、125,000 / mm 3 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​To 375000 / mm 3 、175000 / mm 3 To 350000 / mm 3 、175000 / mm 3 To 325000 / mm 3 、175000 / mm 3 To 300000 / mm 3 、175000 / mm 3 To 275000 / mm 3 、175000 / mm 3 To 250000 / mm 3 、175000 / mm 3 To 225000 / mm 3 、175000 / mm 3 To 200000 / mm 3 、200000 / mm 3 To 400000 / mm 3 、200000 / mm 3 To 375000 / mm 3 、200000 / mm 3 To 350000 / mm 3 、200000 / mm 3 To 325000 / mm 3 、200000 / mm 3 To 300000 / mm 3 、200000 / mm 3 To 275000 / mm 3 、200000 / mm 3 To 250000 / mm 3 、200000 / mm 3 To 225000 / mm 3 、225000 / mm 3 To 400000 / mm 3 、225000 / mm 3 To 375000 / mm 3 、225000 / mm 3 To 350000 / mm 3 、225000 / mm 3 To 325000 / mm 3 、225000 / mm 3 To 3​​​​​​3 to 250,000 / mm 3 、250,000 / mm 3 to 400,000 / mm 3 、250,000 / mm 3 to 375,000 / mm 3 、250,000 / mm 3 to 350,000 / mm 3 、275,000 / mm 3 to 325,000 / mm 3 、275,000 / mm 3 to 300,000 / mm 3 、300,000 / mm 3 to 400,000 / mm 3 、300,000 / mm 3 to 375,000 / mm 3 、300,000 / mm 3 to 350,000 / mm 3 、300,000 / mm 3 to 325,000 / mm 3 、325,000 / mm 3 to 400,000 / mm 3 、the 350,000 / mm 3 to 400,000 / mm 3 、the 350,000 / mm 3 to 375,000 / mm 3 、or 375,000 / mm 3 to 400,000 / mm 3 。

[0734] In some embodiments, the patient's hemoglobin level measured within 1 day, 1 week, or 1 month after treatment, with or without prior transfusion, 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. In some embodiments, the patient's hemoglobin level measured within 1 day, 1 week, or 1 month after treatment, with or without prior blood transfusion, is 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, 9.0 to 16.0, 9.0 to 15.0, 8.0 to 14.0, 8.0 to 13.0, 8.0 to 12.0, to 15.0, 9.0 to 14.0, 9.0 to 13.0, 9.0 to 12.0, 9.0 to 11.0, 9.0 to 10.0, 10.0 to 20.0, 10.0 to 19.0, 10.0 to 18.0, 10.0 to 17.0, 10.0 to 16.0, 10.0 to 15.0, 10.0 to 14.0, 10.0 to 13.0, 10.0 to 12.0, 10.0 to 11.0, 11.0 to 20.0, 11.0 to 19.0, 11.0 to 18.0, 11.0 to 17.0, 11.0 to 16.0, 11.0 to 15.0, 11.0 to 14.0, 11.0 to 13.0, 11.0 to 12.0, 12.0 to 20.0, 12.0 to 19.0, 12.0 to 18.0, 12.0 to 17.0, 12.0 to 16.0, 12.0 to 15.0, 12.0 to 14.0, 12.0 to 13.0, 13.0 to 20.0, 13.0 to 19.0, 13.0 to 18.0, 13.0 to 17.0, 13.0 to 16.0, 13.0 to 15.0, 13.0 to 14.0, 14.0 to 20.0, or 19.0 to 20.0, 14.0 to 19.0, 14.0 to 18.0, 14.0 to 17.0, 14.0 to 16.0, 14.0 to 15.0, 15.0 to 20.0, 15.0 to 19.0, 15.0 to 18.0, 15.0 to 17.0, 15.0 to 16.0, 16.0 to 20.0, 16.0 to 19.0, 16.0 to 18.0, 16.0 to 17.0, 17.0 to 20.0, 17.0 to 19.0, 17.0 to 18.0, 18.0 to 20.0, 18.0 to 19.0, or 19.0 to 20.0.

[0735] In some embodiments, the patient has a creatinine clearance of 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 using the Cockcraft-Gault equation, or an estimated glomerular filtration rate (eGFR) of mL / min / 1.73 m2 according to the Modification of Diet in Renal Disease (MDRD) equation, measured within 1 day, 1 week, or 1 month after treatment. 2 In some embodiments, the patient has a creatinine clearance of 45 to 150, 45 to 125, 45 to 100, 45 to 75, 75 to 150, 75 to 100, 100 to 150, 100 to 125, or 125 to 150 mL / min using the Cockcraft-Gault equation, or an estimated glomerular filtration rate (eGFR) of mL / min / 1.73 m2 measured within 1 day, 1 week, or 1 month after treatment according to the Modification of Diet in Renal Disease (MDRD) equation. 2 .

[0736] In some embodiments, 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 measured within 1 day, 1 week, or 1 month of treatment. In some embodiments, the patient's total serum bilirubin measured within 1 day, 1 week, or 1 month of 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 to 3.5, 0.2 to 3.0, 0.2 to 2.5, 0.2 to 2.0, 0.2 to 1.5, 0.2 to 1.0, 0.2 to 0.5, 0.2 to 0.4, 0.2 to 0.3, 0.3 to 5.0, 0.3 to 4.5, 0.3 to 4.0, 0.3 to 3.5, 0.3 to 3.0, 0.3 to 2.5, 0.3 to 2.0, 0.3 to 1.5, 0.3 to 1.0, 0.3 to 0.5, 0.3 to 0.4, 0.4 to 5.0, 0.4 to 4.5, 0.4 to 4.0, 0.4 to 3.5, 0.4 to 3.0, 0.4 to 2.5, 0.4 to 2.0, 0.4 to 1.5, 0. 4 to 1.0, 0.4 to 0.5, 0.5 to 5.0, 0.5 to 4.5, 0.5 to 4.0, 0.5 to 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, 1.0 to 2.5, 1.0 to 2.0, 1.0 to 1.5, 1.5 to 5.0, 1.5 to 4.5, 1.5 to 4.0, 1.5 to 4.0, 1.5 to 3.5 , 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.0, 2.0 to 5.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2.5, 2.5 to 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, 3.5 to 5.0, 3.5 to 4.5, 3.5 to 4.0, 4.0 to 5.0, 4.0 to 4.5, 4.5 to 5.0 mg / dL.

[0737] In some embodiments, when the patient has known Gilbert's syndrome, the patient's serum total 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 measured within 1 day, 1 week, or 1 month of treatment. In some embodiments, when the patient has known Gilbert's syndrome, the patient's serum total bilirubin measured within 1 day, 1 week, or 1 month of 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 to 3.5, 0.2 to 3.0, 0.2 to 2.5 , 0.2 to 2.0, 0.2 to 1.5, 0.2 to 1.0, 0.2 to 0.5, 0.2 to 0.4, 0.2 to 0.3, 0.3 to 5.0, 0.3 to 4.5, 0.3 to 4.0, 0.3 to 3.5, 0.3 to 3.0, 0.3 to 2.5, 0.3 to 2.0, 0.3 to 1.5, 0.3 to 1.0, 0.3 to 0.5, 0.3 to 0.4, 0.4 to 5.0, 0.4 to 4.5, 0.4 to 4.0, 0.4 to 3.5, 0.4 to 3.0, 0.4 to 2.5, 0.4 to 2.0, 0 .4 to 1.5, 0.4 to 1.0, 0.4 to 0.5, 0.5 to 5.0, 0.5 to 4.5, 0.5 to 4.0, 0.5 to 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, 1.0 to 2.5, 1.0 to 2.0, 1.0 to 1.5, 1.5 to 5.0, 1.5 to 4.5, 1.5 to 4.0, 1.5 to 4.0 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.0, 2.0 to 5.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2.5, 2.5 to 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, 3.5 to 5.0, 3.5 to 4.5, 3.5 to 4.0, 4.0 to 5.0, 4.0 to 4.5, 4.5 to 5.0 mg / dL.

[0738] In some embodiments, when the patient does not have known Gilbert's syndrome, the patient's serum total bilirubin measured within 1 day, 1 week, or 1 month of treatment is less than 2.5, 2.0, 1.5, 1.0, 0.5, 0.4, 0.3, 0.2, or 0.1 mg / dL. In some embodiments, when the patient does not have known Gilbert's syndrome, the patient's serum total bilirubin measured within 1 day, 1 week, or 1 month of treatment is 0.1 to 2.5, 0.1 to 2.0, 0.1 to 1.5, 0.1 to 1.0, 0.1 to 0.5, 0.1 to 0.4, 0.1 to 0.3, 0.1 to 0.2, 0.2 to 2.5, 0.2 to 2.0, 0.2 to 1.5, 0.2 to 1.0, 0.2 to 0.5, 0.2 to 0.4, 0.2 to 0.3, 0.1 to 0.2, 0.3 to 2.5, 0.3 to 2.0, 0.3 to 1.5, 0.3 to 1.0, 0.3 to 0.5, 0.3 to 0.4, 0.4 to 2.5, 0.4 to 2.0, 0.4 to 1.5, 0.4 to 1.0, 0.4 to 0.5, 0.5 to 2.5, 0.5 to 2.0, 0.5 to 1.5, 0.5 to 1.0, 1.0 to 2.5, 1.0 to 2.0, 1.0 to 1.5, 1.5 to 2.5, 1.5 to 2.0, or 2.0 to 2.5 mg / dL.

[0739] In some embodiments, the patient's liver transaminases (aspartate transaminase / alanine transaminase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month after treatment 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) of the test. In some embodiments, the patient's liver transaminases (aspartate transaminase / alanine transaminase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month of treatment are less than the upper limit of normal to 5.0 times the upper limit of normal. In some embodiments, the patient has liver transaminases (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month after treatment that are 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 above the upper limit of normal (ULN) of the test. 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, 1.0 to 2.5, 1.0 to 2.0, 1.0 to 1.5, 1.5 to 5.0, 1.5 to 4.5, 1.5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.0, 2.0 or 4.5 to 5.0, 4.0 to 5.0, 4.0 to 5.0, 5.0 to 6.5, 2.0 to 4.5, 2.5 to 5.0, 6.0 to 7.5, 2.5 to 5.0, 7.0 to 8.5, 2.5 to 5.0, 8.0 to 9.5, 3.0 to 10.0, 9.0 to 11.0, 9.0 to 12.0, 9.0 to 13.0, 9.0 to 14.0, 9.0 to 15.0, 9.0 to 16.0, 9.0 to 17.0, 9.0 to 18.0, 9.0 to 19.0, 10.0 to 19.0, 11.0 to 18.0, 11.0 to 19.0

[0740] In some embodiments, when the patient has liver metastases, the patient's liver transaminase (aspartate transaminase / alanine transaminase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month after treatment is 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) of the test. In some embodiments, when the patient has liver metastases, the patient's liver transaminase (aspartate transaminase / alanine transaminase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month of treatment is less than the upper limit of normal to 5.0 times the upper limit of normal. In some embodiments, the patient has liver transaminases (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month after treatment that are 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, 0.5o3.5, 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 , 1.0 to 2.5, 1.0 to 2.0, 1.0 to 1.5, 1.5 to 5.0, 1.5 to 4.5, 1.5 to 4.0, 1.5 to 3.5, 1.5 to 3.0, 1.5 to 2.5, 1.5 to 2.0, 2.0 to 5.0, 2.0 to 4.5, 2.0 to 4.0, 2.0 to 3.5, 2.0 to 3.0, 2.0 to 2 .5, 2.5 to 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 4.0, 4.0 to 5.0, 4.0 to 4.5, or 4.5 to 5.0 times.

[0741] In some embodiments, when the patient does not have liver metastases, the patient's liver transaminases (aspartate aminotransferase / alanine aminotransferase-alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month of treatment 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) of the test. In some embodiments, when the patient does not have liver metastases, the patient's liver transaminases (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month of treatment are less than the upper limit of normal to 5.0 times the upper limit of normal. In some embodiments, when the patient does not have liver metastases, the patient has liver transaminases (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (AST / ALT / ALP)) measured within 1 day, 1 week, or 1 month of treatment that are 0 to 2.5, 0 to 2.0, 0 to 1.5, 0 to 1.0, 0 to 0.5, 0.5 to 2.5, 0.5 to 2.0, 0.5 to 1.5, 0.5 to 1.0, 1.0 to 2.5, 1.0 to 2.0, 1.0 to 1.5, 1.5 to 2.5, 1.5 to 2.0, or 2.0 to 2.5 times the upper limit of normal (ULN).

[0742] In some embodiments, the patient does not have active nervous system metastases. In some embodiments, the patient does have active nervous system metastases.

[0743] In some embodiments, the patient has no clinically relevant CNS disease or history, such as seizures, cerebral ischemia / hemorrhage, dementia, cerebellar disease, cerebral edema, posterior reversible encephalopathy syndrome (PRES), or any autoimmune disease involving the CNS.

[0744] In some embodiments, the patient does not have active human immunodeficiency virus (HIV) infection, eg, as evidenced by a positive HIV polymerase chain reaction (PCR) test, within 1 day, 1 week, or 1 month after treatment.

[0745] In some embodiments, the patient has no active HBV or hepatitis C virus (HCV) infection within 1 day, 1 week, or 1 month after treatment, e.g., based on laboratory testing. In some embodiments, the patient's viral HBV load is below the institutional limit of quantification (LOQ), and the subject is receiving stable viral suppression therapy. In some embodiments, the patient's HCV RNA viral load is below the institutional LOQ, and therapeutic antiviral therapy has been completed.

[0746] Lymphocyte depletion

[0747] In some embodiments, the patient is lymphodepleted prior to treatment.

[0748] Schematic lymphodepleting chemotherapy regimens and associated beneficial biomarkers are described in WO 2016 / 191756 and WO 2019 / 079564, which are incorporated herein by reference in their entirety. In certain embodiments, the lymphodepleting chemotherapy regimen comprises administering cyclophosphamide (at a dose of 200 mg / m 2 / day to 2000 mg / m 2 / day) and fludarabine (dose 20 mg / m 2 / day to 900 mg / m 2 / sky).

[0749] In some embodiments, lymphocyte depletion comprises administering or administering about 250 to about 500 mg / m 2 of cyclophosphamide, for example, about 250 to about 500, 250, 400, 500, about 250, about 400, or about 500 mg / m 2 Cyclophosphamide.

[0750] In some embodiments, lymphocyte depletion comprises administering or administering about 20 mg / m 2 / day to approximately 40 mg / m 2 Fludarabine / day, for example, 30 or about 30 mg / m 2 / sky.

[0751] In some embodiments, lymphocyte depletion comprises administration of cyclophosphamide and fludarabine.

[0752] In some embodiments, cyclophosphamide (250 mg / m 2 / day) and fludarabine (30 mg / m 2 / day) causes lymphatic depletion in patients.

[0753] In some embodiments, cyclophosphamide (500 mg / m 2 / day) and fludarabine (30 mg / m 2 / day) to deplete the patient's lymphocytes.

[0754] In some embodiments, lymphocyte depletion occurs no more than 5 days before the first dose of NK cells. In some embodiments, lymphocyte depletion occurs no more than 7 days before the first dose of NK cells.

[0755] In some embodiments, lymphocyte depletion occurs daily for 3 consecutive days, starting 5 days before the first dose of NK cells (ie, from day -5 to day -3).

[0756] In some embodiments, lymphodepletion occurs on day -5, day -4, and day -3.

[0757] In some embodiments involving repeated or multiple administrations, the patient undergoes lymphocyte depletion as described above prior to each or all administrations. In some embodiments, the patient undergoes lymphocyte depletion two, three, four, five, or six times.

[0758] Drug administration

[0759] NK cells

[0760] In some embodiments, NK cells (e.g., NK cells described herein, e.g., 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, and in some cases, no further manipulation is required when the cryoprotectant is compatible with immediate administration. For a given individual, the treatment regimen typically includes administering multiple aliquots or doses of NK cells over time, including doses taken from the same batch or donor.

[0761] The NK cells can be administered in any suitable manner, such as by intravenous bolus, injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subinjection, subtenon's fascia capsule injection, retrobulbar injection, peribulbar injection or posterior scleral injection. In certain embodiments, they are administered parenterally, intrapulmonary and intranasally, and if local treatment is required, they can also be administered intralesionally. Parenteral infusion includes intramuscular injection, intravenous injection, arterial injection, intraperitoneal injection or subcutaneous injection. In certain embodiments, a given dose is given by a single bolus of cells. In some embodiments, a given dose is given by multiple boluses of cells, for example, within a period of no more than 3 days, or by continuous infusion of cells. In some embodiments, the administration of cell dose or any additional therapy, such as lymphocyte depletion therapy, interventional therapy and / or combination therapy, is carried out by outpatient delivery.

[0762] In the context of adoptive cell therapy, the administration of a given "dose" can include administering a given amount or number of cells as a single composition and / or a single uninterrupted administration, such as a single injection or continuous infusion. A given "dose" can also include administering a given amount or number of cells as divided doses or as multiple compositions over a specified time period, such as no more than 3 days, which are provided in multiple separate compositions or infusions. Thus, in some cases, a dose is a single or continuous administration of a specified number of cells given or initiated at a single time point. However, in some cases, the dose is administered as multiple injections or infusions over a period of no more than three days, such as once a day for three or two days, or as multiple infusions in one day.

[0763] In some embodiments, NK cells are administered to a patient in a range of 1 million or about 1 million to 100 billion or about 100 billion cells. In some embodiments, NK cells (e.g., NK cells described herein, e.g., CAR-NK cells described herein) are administered in a dose of 5x10 6 or about 5x10 6 to 1x10 9 or about 1x10 9 In some embodiments, NK cells are administered at a dose of 5x10 6 or about 5x10 6 , 1x10 7 or about 1x10 7 , 3x10 7 or about 3x10 7 , 1x10 8 or about 1x10 8 , 3x10 8 or about 3x10 8 , 1x10 9 or about 1x10 9In some embodiments, the NK cells are administered at a dose of 1 million or about 1 million to 20 billion or about 20 billion cells per dose (e.g., 5 million or about 5 million cells, 25 million or about 25 million cells, 50 million or about 50 million cells, 75 million or about 75 million cells, 100 million or about 100 million cells, 200 million or about 200 million cells, 300 million or about 300 million cells, 400 million or about 400 million cells, 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 2 billion cells, 3 billion or about 3 billion cells, 4 billion or about 4 billion cells, 5 billion cells or about 5 billion cells, 6 billion cells or about 6 billion cells, 7 billion cells or about 7 billion cells, 8 billion cells or about 8 billion cells, 9 billion cells or about 9 billion cells, 10 billion cells or about 10 billion cells, or a range defined by any two of the foregoing values), 10 million cells or about 10 million cells to 20 billion cells or about 20 billion cells (e.g., 25 million cells or about 25 million cells, 50 million cells or about 50 million cells, 75 million cells or about 75 million cells, 100 million cells or about 100 million cells, 200 million cells or about 200 million cells, 300 million cells or about 300 million cells, 400 million cells or about 400 million cells , 500 million or about 500 million cells, 1 billion or about 1 billion cells, 2 billion or about 2 billion cells, 3 billion or about 3 billion cells, 4 billion or about 4 billion cells, 5 billion or about 5 billion cells, 6 billion or about 6 billion cells, 7 billion or about 7 billion cells, 8 billion or about 8 billion cells, 9 billion or about 9 billion cells, 10 billion or about 10 billion cells, 20 billion or about 20 billion cells, or a range defined by any two of the foregoing values), in some cases between 100 million or about 100 million cells and 50 billion or about 50 billion cells (e.g., 150 million or about 150 million cells). or about 40 billion cells), or any value in between these ranges.

[0764] Therefore, in some embodiments, the NK cells are administered at a dose of or about 1 x 10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x106 , or 9x10 6 cells / dose, or approximately 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , or 9x10 7 cells / dose, or approximately 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , or 9x10 8 cells / dose, or approximately 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 cells / dose, or approximately 1x10 10 or 2x10 10 cells / dose.

[0765] Thus, in some embodiments, the NK cells are administered at a dose of at least or at least about 1 x 10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , or 9x10 6 cells / dose, at least or at least about 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , or 9x10 7cells / dose, at least or at least about 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , or 9x10 8 cells / dose, at least or at least about 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , or 9x10 9 cells / dose, or at least or at least about 1x10 10 , or 2x10 10 cells / dose.

[0766] In some embodiments, the NK cells are administered at a dose comprising between 1 million or about 1 million and 20 billion or about 20 billion CAR-expressing NK cells per dose (e.g., 5 ...

Claims

1. A method comprising administering 1 million to 100 billion natural killer (NK) cells to a patient in need thereof, characterized in that The NK cell comprises a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain, the domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) Nucleic acid encoding IL-15.

2. The method according to claim 1, characterized in that: The NK cell administration dose includes from 1 billion to 100 billion NK cells, optionally, the dose includes from or about 1 million to or about 20 billion cells (such as or about 5 million cells, or about 25 million cells, or about 50 million cells, or about 75 million cells, or about 100 million cells, or about 200 million cells, or about 300 million cells, or about 400 million cells, or about 500 million cells, or about 1 billion cells, or about 2 billion cells, or about 3 billion cells, or about 400 million cells, or about 500 million cells, or about 1 billion cells, or about 2 billion cells, or about 3 billion cells, or about 5 billion cells, or about 100 million cells). For example, 4 billion cells or about 5 billion cells, 6 billion cells or about 7 billion cells, 8 billion cells or about 9 billion cells, 10 billion cells or about 10 billion cells, or a range defined by any two of the foregoing values), 10 million to 20 billion cells or about 25 million cells, 50 million cells or about 75 million cells, 100 million cells or about 200 million cells, 300 million cells or about 300 million cells, or about 100 million cells. In some embodiments, the present invention relates to a cell population of at least 400 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 2 billion cells, at or about 3 billion cells, at or about 4 billion cells, at or about 5 billion cells, at or about 6 billion cells, at or about 7 billion cells, at or about 8 billion cells, at or about 9 billion cells, at or about 10 billion cells, at or about 20 billion cells, or a range defined by any two of the foregoing values), and in some embodiments, from or about 100 million to or about 50 billion cells (at or about 150 million cells). cells, at or about 200 million cells, at or about 300 million cells, at or about 400 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 2 billion cells, at or about 3 billion cells, at or about 4 billion cells, at or about 5 billion cells, at or about 6 billion cells, at or about 7 billion cells, at or about 8 billion cells, at or about 9 billion cells, at or about 10 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells).

3. The method according to claim 1, characterized in that: The NK cell administration dose includes at least 1×10 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, with each dose being at least 1×10 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, with each dose being at least 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 ,7×10 8 , 8×10 8 、or9×10 8 Each dose should be at least 1×10 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 cells, or at least 1×10 per dose 10 or 2×10 10 cells.

4. The method according to claim 1, characterized in that: The NK cell administration dose includes about 1×10 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 Each dose is about 1×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 ,7×10 7 , 8×10 7 or ×10 7 Each dose is about 1×10 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 Each dose is about 1×10 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 cells, or approximately 1 × 10 per dose 10 or 2×10 10 cells.

5. The method according to claim 1, characterized in that: The dosage of the NK cells includes a dosage of from or about 1 million to or about 20 billion CAR-expressing NK cells (such as from or about 5 million cells, from or about 25 million CAR-expressing NK cells, from or about 50 million CAR-expressing NK cells, from or about 75 million CAR-expressing NK cells, from or about 100 million CAR-expressing NK cells, from or about 200 million CAR-expressing NK cells, from or about 300 million CAR-expressing NK cells, from or about 400 million CAR-expressing NK cells, from or about 500 million CAR-expressing NK cells, from or about 1 billion CAR-expressing NK cells, from or about 2 billion CAR-expressing NK cells, The present invention relates to NK cells of at least 1 billion CAR-expressing cells, NK cells of at least 1 billion CAR-expressing cells, NK cells of at least 2 billion CAR-expressing cells, NK cells of at least 3 billion CAR-expressing cells, NK cells of at least 4 billion CAR-expressing cells, NK cells of at least 5 billion CAR-expressing cells, NK cells of at least 6 billion CAR-expressing cells, NK cells of at least 7 billion CAR-expressing cells, NK cells of at least 8 billion CAR-expressing cells, NK cells of at least 9 billion CAR-expressing cells, NK cells of at least 10 billion CAR-expressing cells, NK cells of at least 20 billion CAR-expressing cells, or a range defined by any two of the foregoing values).

6. The method according to claim 1, characterized in that: The NK cell administration dose includes 1×10 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, with doses including or approximately 1×10 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 CAR-expressing cells, with doses including or approximately 1×10 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 CAR-expressing cells, with doses including or approximately 1×10 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 a dose comprising or about 1×10 10 or about 2×10 10 CAR-expressing cells.

7. The method according to claim 1, characterized in that: The NK cells are administered in a dosage of at least or about 1×10 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, with each dose being at least or about 1×10 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 CAR-expressing cells, with each dose being at least or about 1×10 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 CAR-expressing cells, with each dose being at least or about 1×10 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, with each dose being at least or about 1×10 10 CAR-expressing cells.

8. A method comprising administering NK cells to a patient in need thereof, characterized in that The NK cell comprises a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain, the domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, The dosage of NK cells is 1×10 5 Up to 10×10 8 cells / kg.

9. The method according to claim 8, characterized in that The cell dose includes or is about 1×10 5 cells / kg and about 1×10 8 cells / kg, such as or about 1.5×10 5 cells / kg and about 1.5×10 7 cells / kg, or about 4×10 5 cells / kg and about 4×10 6 cells / kg.

10. The method according to claim 8, characterized in that: The NK cell administration dose includes or is about 1×10 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 9.5×10 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 9.5×10 6 cells / kg, or about 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 1×10 8 , 1.5×10 8 or 2×10 8 cells / kg.

11. The method according to claim 8, characterized in that The NK cell administration dose includes at least or at least about 1×10 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 9.5×10 5 cells / kg, at least or about 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, at least or about 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 at least or about 1×10 8 or 1.5 × 10 8 cells / kg.

12. A method comprising administering multiple doses of NK cells to a patient in need thereof, characterized in that The NK cell comprises a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain, the domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, and The administration dosage is once every 1 to 16 weeks.

13. The method according to claim 12, characterized in that: The NK cells are administered once every 2 weeks, 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.

14. A method comprising administering NK cells to a patient in need thereof, characterized in that The NK cell comprises a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain, the domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, The patients met one or more of the following criteria: (i) the patient's Eastern Cooperative Oncology Group (ECOG) performance status was 0 to 4 at the time of treatment; (ii) the patient is refractory or has relapsed to one or more previous systemic therapies; (iii) the patient's blood oxygen saturation is at least 92% as measured by pulse oximetry; (iv) the patient has a left ventricular ejection fraction (LVEF) of at least 50%; (v) The patient has an absolute neutrophil count (ANC) of at least 1000 / mm 3 (1.0×10 9 / L); (vi) the patient has a platelet count of at least 75,000 / mm 3 (75×10 9 / L); (vii) with or without prior metastasis, the patient's hemoglobin level is at least 8.0 g / dL; (viii) the patient's creatinine clearance is at least 45 mL / min according to the Cockcraft-Gault formula, or the patient's estimated glomerular filtration rate (eGFR) is at least mL / min / 1.73 m according to the Modification of Diet in Renal Disease (MDRD) formula 2 ; (ix) the patient's total serum bilirubin is at least 5 mg / dL; or (x) The patient's liver transferase (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (AST / ALT / ALP)) is less than or equal to 5 times the upper limit of normal (ULN) of the test.

15. The method according to claim 14, characterized in that: The patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, 2, 3, or 4 while receiving treatment.

16. The method according to claim 14, characterized in that: The patients were refractory or had relapsed after 2 or more prior systemic therapies.

17. The method according to claim 14, characterized in that: The patient's blood oxygen saturation is at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% as measured by pulse oximetry.

18. The method according to claim 14, characterized in that: The patient has a left ventricular ejection fraction (LVEF) of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64% or 65%.

19. The method of claim 14, wherein: The patient has an absolute neutrophil count (ANC) of at least 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 .

20. The method of claim 14, wherein: The patient has a platelet count of at least 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 .

21. The method of claim 14, wherein: With or without prior metastasis, 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.

22. The method of claim 14, wherein: The patient has a creatinine clearance of 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 using the Cockcraft-Gault formula or an estimated glomerular filtration rate (eGFR) of at least mL / min / 1.73 m according to the Modification of Diet in Renal Disease (MDRD) formula 2 .

23. The method of claim 14, wherein: The patient has a total serum bilirubin of at least 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.

24. The method of claim 14, wherein: The patient's liver transferase (aspartate aminotransferase / alanine aminotransferase / alkaline phosphatase (AST / ALT / ALP)) is 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) of the test.

25. A method comprising administering NK cells to a patient in need thereof, characterized in that The NK cell comprises a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain, the domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, The patients met one or more of the following criteria: (i) the patient does not have active nervous system metastases; (ii) the patient has no history or condition of clinically relevant CNS disease, such as seizures (e.g., epilepsy), cerebrovascular ischemia / hemorrhage, dementia, cerebellar disease, cerebral edema, post-reversible encephalopathy syndrome (PRES), or any autoimmune disease involving the CNS; (iii) the patient does not have 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), as determined by laboratory testing; (v) the patient's HBV viral load is below the institutional limit of quantification (LOQ) and the subject is receiving stable viral suppression therapy; or (vi) The patient's HCV RNA viral load is below the institutional LOQ and has completed curative antiviral treatment.

26. A method comprising administering NK cells to a patient in need thereof, characterized in that The NK cell comprises a polynucleotide comprising: a) a nucleic acid encoding an anti-human epidermal growth factor receptor 2 (HER2) chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an extracellular antigen binding domain, the domain comprising an anti-HER2 antibody or an antigen-binding fragment thereof; and b) a nucleic acid encoding IL-15, The patients met one or more of the following criteria: (i) the patient is diagnosed or has been diagnosed with HER2+ cancer with an IHC value of 2+; (ii) the patient's cancer is not HER2-dependent but has some degree of HER2 expression; (iii) the patient is diagnosed or has been diagnosed with low HER2 cancer; (iv) the patient is diagnosed or has been diagnosed with low HER2 cancer, with a HER2 expression grade score of 1+ or 2+; (v) the patient is or has been diagnosed with HER2-low cancer without HER2 amplification; (vi) the patient suffers from a cancer comprising a HER2 activating mutation; or (vii) The patient has been diagnosed with a HER2-expressing cancer that is IHC 1+ or IHC 2+ / ISH- and is refractory or has relapsed after prior treatment.

27. The method of claim 26, wherein: The patient is or has been diagnosed with a HER2-low cancer with a HER2 expression grade score of 1+ or 2+ without HER2 amplification.

28. The method of claim 27, wherein: The HER2-low cancer has HER2 gain.

29. The method of claim 27, wherein: The HER2-low cancer lacks HER2 gain.

30. The method of claim 26, wherein: The patients have been diagnosed with IHC 3+ or IHC 2+ / ISH+ cancer that is refractory or has relapsed after prior treatment with HER2-targeted therapy.

31. The method of claim 26, wherein: The patient has been diagnosed with HER2-expressing cancer that is IHC 3+ or IHC 2+ / ISH+, and the patient's cancer is unresectable or has metastasized, or the patient is refractory to previous treatment or has relapsed.

32. The method according to any one of claims 1 to 31, characterized in that: The anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain complementary determining region 1 (CDRL1) comprising SEQ ID NO: 34, a light chain complementary determining region 2 (CDRL2) comprising SEQ ID NO: 36, a light chain complementary determining region 3 (CDRL3) comprising SEQ ID NO: 38, a heavy chain complementary determining region 1 (CDRH1) comprising SEQ ID NO: 44, a heavy chain complementary determining region 2 (CDRH2) comprising SEQ ID NO: 46, and a heavy chain complementary determining region 3 (CDRH3) comprising SEQ ID NO:

48.

33. The method according to any one of claims 1 to 32, characterized in that: The nucleic acid encoding the anti-HER2 antibody or antigen-binding fragment thereof encodes CDRL1 encoded by SEQ ID NO:35, CDRL 2 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.

34. The method according to any one of claims 1 to 33, characterized in that: The anti-HER2 antibody or antigen-binding fragment thereof comprises a light chain variable (V L ) region, and a heavy chain variable (V H )district.

35. The method according to any one of claims 1 to 34, characterized in that The anti-HER2 antibody or antigen-binding fragment thereof comprises a V encoding SEQ ID NO: 33 L A nucleic acid encoding a V region containing SEQ ID NO: 37 H nucleic acid in the region.

36. The method according to any one of claims 1 to 35, characterized in that The anti-HER2 antibody or antigen-binding fragment thereof comprises V L Area and V H Area, the V L The V region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:32, wherein the V H The region includes an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

42.

37. The method according to any one of claims 1 to 36, characterized in that The anti-HER2 antibody or antigen-binding fragment thereof is an antigen-binding fragment.

38. The method according to any one of claims 1 to 37, characterized in that The antigen-binding fragment includes a single-chain Fv (scFv).

39. The method according to any one of claims 1 to 38, characterized in that The V L Area V H The amino terminus of the region.

40. The method according to any one of claims 1 to 38, characterized in that The V L Area V H The carboxyl terminus of the region.

41. The method according to any one of claims 1 to 40, characterized in that The V L The zone is connected to the V H The areas are connected.

42. The method of claim 41, wherein: The flexible linker comprises the amino acid sequence shown in SEQ ID NO:

40.

43. The method of claim 42, wherein: The flexible linker is encoded by a nucleic acid sequence comprising SEQ ID NO:

41.

44. The method according to any one of claims 38 to 43, characterized in that The scFv comprises the amino acid sequence shown in SEQ ID NO:

30.

45. The method of claim 44, wherein: The scFv is encoded by a nucleic acid sequence comprising SEQ ID NO:

31.

46. ​​The method of claim 44, wherein: The scFv comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

30.

47. The method according to any one of claims 1 to 46, characterized in that The anti-HER2 CAR specifically binds to the human epidermal growth factor receptor 2 (HER2) protein.

48. The method of claim 47, wherein: The HER2 protein includes the amino acid sequence of SEQ ID NO:

62.

49. The method according to any one of claims 1 to 48, characterized in that The CAR comprises a transmembrane domain, optionally a CD28 transmembrane domain.

50. The method of claim 49, wherein: The CD28 transmembrane domain includes the amino acid sequence shown in SEQ ID NO:

53.

51. The method of claim 50, wherein: The CD28 transmembrane domain is encoded by a nucleic acid sequence comprising SEQ ID NO:54 or SEQ ID NO:

55.

52. The method according to any one of claims 1 to 51, characterized in that The CAR also includes a hinge domain between the extracellular antigen binding domain and the transmembrane domain.

53. The method of claim 52, wherein: The hinge domain comprises at least a portion of the CD8α hinge domain.

54. The method of claim 53, wherein: The CD8α hinge domain includes the amino acid sequence shown in SEQ ID NO:

50.

55. The method of claim 54, wherein: The CD8α hinge domain is encoded by a nucleic acid sequence comprising SEQ ID NO:51 or SEQ ID NO:

52.

56. The method of claim 54, wherein: The CD8α hinge domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

50.

57. The method according to any one of claims 1 to 56, characterized in that The CAR includes an intracellular signaling region, and optionally, the intracellular signaling region includes a CD28 intracellular signaling domain, an OX40L intracellular signaling domain, and a CD 3-zeta (CD3ζ) signaling domain.

58. The method of claim 57, wherein: The intracellular signaling region includes a CD28 intracellular signaling domain and a CD3-zeta signaling domain.

59. The method according to any one of claims 57 to 58, characterized in that The intracellular signaling region includes the OX40L intracellular signaling domain.

60. The method of claim 57 or 59, wherein: The OX40L intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:

10.

61. The method of claim 57 or 59, wherein: The OX40L intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:

10.

62. The method of claim 57 or 59, wherein: The OX40L intracellular signaling domain is encoded by a nucleic acid sequence comprising SEQ ID NO:11 or SEQ ID NO:

12.

63. The method according to any one of claims 57 to 62, characterized in that The intracellular signaling region includes the CD28 intracellular signaling domain.

64. The method of claim 63, wherein: The CD28 intracellular signaling domain includes the amino acid sequence shown in SE QID NO:

5.

65. The method of claim 63, wherein: The CD28 intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SE QID NO:

5.

66. The method of claim 63, wherein: The CD28 intracellular signaling domain is encoded by a nucleic acid sequence comprising SEQ ID NO:6 or SEQ ID NO:

7.

67. The method according to any one of claims 57 to 66, characterized in that The intracellular signaling region includes a CD3-zeta intracellular signaling domain.

68. The method of claim 67, wherein: The CD3-zeta intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:

13.

69. The method of claim 67, wherein: The CD3-zeta intracellular signaling domain comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

13.

70. The method of claim 67, wherein: The CD3-zeta intracellular signaling domain is encoded by a nucleic acid sequence comprising SEQ ID NO:14 or SEQ ID NO:

15.

71. The method according to any one of claims 57 to 70, characterized in that The intracellular signal region includes the amino acid sequence shown in SEQ ID NO:

25.

72. The method according to any one of claims 57 to 70, characterized in that The intracellular signaling region comprises an amino acid sequence that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

25.

73. The method according to any one of claims 1 to 72, characterized in that The CAR comprises the amino acid sequence shown in SEQ ID NO:

56.

74. The method of claim 73, wherein: The CAR is encoded by a nucleic acid sequence comprising SEQ ID NO:

57.

75. The method according to any one of claims 1 to 74, characterized in that The IL-15 comprises the amino acid sequence shown in SEQ ID NO:

22.

76. The method of claim 75, wherein: The IL-15 is encoded by a nucleic acid sequence comprising SEQ ID NO: 23 or SEQ ID NO:

24.

77. The method according to any one of claims 1 to 76, characterized in that The polynucleotide encodes a polyprotein including CAR and IL-15.

78. The method according to any one of claims 1 to 77, characterized in that The polynucleotide also includes a nucleic acid encoding a self-cleaving peptide, optionally, a T2A self-cleaving peptide.

79. The method of claim 78, wherein: The CAR is linked to IL-15 via the self-cleaving peptide.

80. The method of claim 79, wherein: The self-cleaving peptide is able to induce ribosome jumping between CAR and IL-15.

81. The method according to any one of claims 1 to 80, characterized in that The polynucleotide also includes a nucleic acid encoding a signal sequence.

82. The method of claim 81, wherein: The signal sequence includes the amino acid sequence shown in SEQ ID NO:

27.

83. The method of claim 81, wherein: The nucleic acid encoding the signal sequence includes the nucleic acid sequence shown in SEQ I DNO:

28.

84. The method according to any one of claims 1 to 83, characterized in that The polynucleotide encodes a polyprotein comprising the amino acid sequence shown in SEQ I DNO:

59.

85. The method according to any one of claims 1 to 83, characterized in that The polynucleotide comprises the nucleic acid sequence shown in SEQ ID NO:60 or SEQ ID NO:

61.

86. The method according to any one of claims 1 to 85, characterized in that The NK cells are umbilical cord blood NK cells.

87. The method according to any one of claims 1 to 86, characterized in that The NK cells include the KIR-B haplotype.

88. The method according to any one of claims 1 to 87, characterized in that The NK cells express CD16 with a V / V polymorphism at F158.

89. The method according to any one of claims 1 to 88, characterized in that The NK cells are administered as part of a pharmaceutical composition.

90. The method of claim 89, wherein: The pharmaceutical composition includes a pharmaceutically acceptable excipient.

91. The method of claim 89, wherein: The pharmaceutical composition comprises: (a) human albumin; (b) dextran; (c) glucose; (d) DMSO; and (e) Buffer.

92. The method of claim 91, wherein: The pharmaceutical composition includes 30 to 50 mg / mL of human albumin.

93. The method of claim 91, wherein: The pharmaceutical composition includes 40 mg / mL of human albumin.

94. The method according to any one of claims 91 to 93, characterized in that The pharmaceutical composition includes 20 to 30 mg / mL of dextran.

95. The method according to any one of claims 91 to 94, characterized in that The pharmaceutical composition includes 25 mg / mL of dextran.

96. The method according to any one of claims 91 to 95, characterized in that The dextran is dextran 40.

97. The method according to any one of claims 91 to 96, characterized in that The pharmaceutical composition includes 12 to 15 mg / mL of glucose.

98. The method according to any one of claims 91 to 97, characterized in that The pharmaceutical composition includes 12.5 mg / mL of glucose.

99. The method according to any one of claims 91 to 98, characterized in that The pharmaceutical composition includes at least 27.5 g / L of glucose.

100. The method according to any one of claims 91 to 99, characterized in that The pharmaceutical composition includes 50 to 60 ml / mL of DMSO.

101. The method according to any one of claims 91 to 100, characterized in that The pharmaceutical composition included 55 mg / mL of DMSO.

102. The method according to any one of claims 91 to 101, characterized in that The pharmaceutical composition comprises 40 to 60% v / v of buffer.

103. The method according to any one of claims 91 to 102, characterized in that The buffer is phosphate buffered saline.

104. The method of claim 91, wherein: The pharmaceutical composition comprises: (a) about 40 mg / mL human albumin; (b) about 25 mg / mL of dextran 40; (c) about 12.5 mg / mL of glucose; (d) about 55 mg / mL of DMSO; and (e) Approximately 0.5 mL / mL of phosphate buffered saline.

105. The method according to any one of claims 89 to 104, characterized in that The pharmaceutical composition also includes 0.5 mL / mL of water.

106. The method according to any one of claims 1 to 85, characterized in that The subject has a disease or disorder associated with HER2.

107. A method as claimed in any one of the preceding claims, characterized in that The HER2-related disease or disorder is cancer.

108. The method of claim 107, wherein: The cancer expresses HER2.

109. The method of claim 87, wherein: The cancer is a HER2+ cancer.

110. The method of claim 108 or 109, wherein: The cancer is or includes a solid tumor.

111. The method according to any one of claims 107 to 109, characterized in that The cancer is or includes bladder cancer, breast adenocarcinoma, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, cervical squamous cell carcinoma, gastric adenocarcinoma, bile duct cancer, ovarian cancer, renal papillary cell carcinoma and combinations thereof.

112. The method according to any one of claims 107 to 109, characterized in that The cancer is or includes breast cancer.

113. The method according to any one of claims 107 to 109, characterized in that The breast cancer is non-invasive.

114. The method according to any one of claims 107 to 109, characterized in that The breast cancer is aggressive.

115. The method according to any one of claims 107 to 109, characterized in that The breast cancer is metastatic.

116. The method according to any one of claims 107 to 109, characterized in that The cancer is or includes gastric cancer.

117. The method according to any one of claims 107 to 109, characterized in that The cancer is or includes ovarian cancer.

118. The method according to any one of claims 107 to 109, characterized in that The cancer is or includes gastroesophageal cancer.

119. The method of any one of claims 107 to 109, wherein the cancer is or comprises lung cancer.

120. The method of claim 119, wherein: The lung cancer is non-small cell lung cancer (NSCL C).

121. The method of claim 120, wherein: The patient has a HER2 activating mutation.

122. A method as claimed in any one of the preceding claims, characterized in that It also includes administering lympholysis chemotherapy to the subject prior to treatment.

123. The method of claim 122, wherein: The lymphoablative chemotherapy is non-myeloablative chemotherapy.

124. The method of claim 122 or 123, wherein: The myeloablative chemotherapy comprises treatment with at least one of cyclophosphamide and fludarabine.

125. The method of claim 124, wherein: The myeloablative chemotherapy includes treatment with cyclophosphamide and fludarabine.

126. The method according to any one of claims 122 to 123, characterized in that 100 and 500 mg / m2 / day 2 Cyclophosphamide between.

127. The method of claim 126, wherein: 250 mg / m2 / day 2 of cyclophosphamide.

128. The method of claim 126, wherein: 500 mg / m2 / day 2 of cyclophosphamide.

129. The method according to any one of claims 124 to 128, characterized in that 10 and 50 mg / m daily 2 Fludarabine between.

130. The method of claim 129, wherein: 30 mg / m2 / day 2 of fludarabine.

131. The method according to any one of claims 106 to 130, characterized in that Also included is administering IL-2 to the subject.

132. The method of claim 131, wherein: Patients were given 1×10 6 IU / m 2 of IL-2.

133. The method of claim 131, wherein: Patients were given 1×10 7 IU of IL-2.

134. The method of claim 131, wherein: Patients were given 6 × 10 7 IU of IL-2.

135. The method according to any one of claims 132 to 134, characterized in that IL-2 was administered within 1-4 hours of cell administration.

136. The method according to any one of claims 132 to 134, characterized in that IL-2 was administered at least 1-4 hours after administration of the cells.

137. The method according to any one of claims 106 to 136, characterized in that Multiple administrations of cells are included.

138. The method according to any one of claims 106 to 137, characterized in that Administration of cells 3, 4 or 8 times was included.

139. The method according to any one of claims 106 to 137, characterized in that Administration of cells weekly, every 2 weeks, every 3 weeks, or every 4 weeks is included.

140. The method according to any one of claims 106 to 139, characterized in that Also included is administering Pertuzumab to the subject.

141. The method according to any one of claims 106 to 140, characterized in that Also included is administering trastuzumab to the subject.

142. The method according to any one of claims 106 to 141, characterized in that Also included is administering necituzumab to the subject.

143. The method according to any one of claims 106 to 142, characterized in that Also included is administering magituximab to the subject.

144. The method according to any one of claims 106 to 143, characterized in that Also included is administering a taxane to a subject.

145. The method of claim 144, wherein: The taxane is at least one of paclitaxel, docetaxel and cabazitaxel.

146. The method according to any one of claims 106 to 145, characterized in that Also included is administering endocrine therapy to the subject.

147. The method of claim 146, wherein: The endocrine comprises at least one of an aromatase inhibitor, fulvestrant and tamoxifen.

148. The method according to any one of claims 106 to 147, characterized in that Also included is administering a checkpoint inhibitor to the subject.

149. The method of claim 148, wherein: The checkpoint inhibitors inhibit CTLA-4, PD-1 or PD-L1.

150. The method of claim 148 or 149, wherein: The checkpoint inhibitor is or includes ipilimumab.

151. The method according to any one of claims 148 to 150, characterized in that The checkpoint inhibitor is or includes nivolumab.

152. The method according to any one of claims 148 to 151, characterized in that The checkpoint inhibitor is or includes pembrolizumab.

153. The method according to any one of claims 148 to 152, characterized in that The checkpoint inhibitor is or includes cemiplimab.

154. The method according to any one of claims 148 to 153, characterized in that The checkpoint inhibitor is or includes atezolizumab.

155. The method according to any one of claims 148 to 150, characterized in that The checkpoint inhibitor is or includes avelumab.

156. The method according to any one of claims 148 to 150, characterized in that The checkpoint inhibitor is or includes durvalumab.

157. The method according to any one of claims 1 to 156, characterized in that Also includes: A second treatment moiety is administered to the subject.

158. The method of claim 157, wherein: The second therapeutic moiety includes a lymph-clearing chemotherapeutic agent.

159. The method according to any one of claims 157 to 158, characterized in that The second therapeutic portion comprises IL-2.

160. The method according to any one of claims 157 to 159, characterized in that The second therapeutic portion includes at least one of pertuzumab, trastuzumab, necituzumab, and makituximab.

161. The method according to any one of claims 157 to 160, characterized in that The second therapeutic portion comprises a taxane.

162. The method according to any one of claims 157 to 161, characterized in that The second therapeutic portion comprises a checkpoint inhibitor.

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