Docket No.49755-0045WO1 TREATMENT OF CANCER WITH NK CELLS AND BENDAMUSTINE [0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/467,860, filed on May 19, 2023. The entire contents of the foregoing are incorporated herein by reference. BACKGROUND [0002] Targeted therapies, including antibody therapy, have revolutionized cancer treatment. One mechanism of action by which antibody therapy induces cytotoxicity is through antibody dependent cell-mediated cytotoxicity (ADCC). Many cancer patients are unable to mount a robust ADCC response. A reduced ADCC response may render any of the indicated monoclonal antibody therapeutics significantly less effective for these patients, which could prevent these patients from responding or lead to relapse. Thus, a reduced ADCC response could negatively impact their clinical outcomes. [0003] Despite recent discoveries and developments of several anti-cancer agents, there is still a need for improved methods and therapeutic agents due to poor prognosis for many types of cancers, including Non-Hodgkin Lymphomas. [0004] Although allogeneic NK cells have been used clinically since 2005, their utility has been limited by challenges with product sourcing, scalability, and dose-to-dose variability. [0005] Lymphodepletion has been shown to increase CAR-T cell proliferation, cytotoxicity, engraftment, and persistence. As a result, most adoptive cell therapy protocols, including NK cell therapy protocols, include some form of pre-treatment or conditioning with a lymphodepletion or chemotherapy regimen. Cyclophosphamide and fludarabine (cy/flu) lymphodepletion is the most common protocol. [0006] The cy/flu lymphodepletion causes many of the adverse events that patients experience independent of the CAR-T or NK cell therapy itself, including hematological toxicities, severe lymphocytopenia, low neutrophil and platelet counts, low hemoglobin levels, high infection rates, and others. These adverse events limit the frequency of cy/flu treatment. Importantly, the effect of cy/flu may begin to wear off after 2-3 weeks resulting in lymphorecovery in the middle of a multi-dose cell therapeutic cycle before the patient is able to tolerate another round of cy/flu. This isn’t an issue with CAR-T therapies because they are delivered as a single infusion and repeat dosing isn’t an option.
Docket No.49755-0045WO1 [0007] Doctors are often hesitant to subject patients to cy/flu lymphodepletion at all, which makes adoptive cell therapy in earlier lines of therapy less attractive than existing second- and third-line alternatives. Further, cy/flu use is generally limited to hematological indications. Thus, finding an alternative to cy/flu could greatly reduce the number of adverse events that patients experience and make NK cell therapies more attractive in earlier lines of therapy. [0008] The present invention addresses these and other deficiencies in the art. SUMMARY [0009] NK cells are immune cells that can engage tumor cells through a complex array of receptors on their cell surface, as well as through antibody-dependent cellular cytotoxicity (ADCC). To initiate ADCC, NK cells engage with antibodies via the CD16 receptor on their surface. NK cells may have an advantage over other immune cells, such as the T cells used in CAR-T cell therapy and other cell therapies. In an exemplary advantage, NK cells can be used as allogeneic therapies, meaning that NK cells from one donor can be safely used in one or many patients without the requirement for HLA matching, gene editing, or other genetic manipulations. Allogeneic NK cells with anti-tumor activity can be administered safely to patients without many of the risks associated with T cell therapies, such as severe cytokine release syndrome (CRS), and neurological toxicities or graft versus host disease (GvHD). [0010] Allogeneic NK cells may provide an important treatment option for cancer patients. In one exemplary advantage, NK cells have been well tolerated without evidence of graft-versus-host disease, neurotoxicity or cytokine release syndrome associated with other cell-based therapies. In another exemplary advantage, NK cells do not require prior antigen exposure or expression of a specific antigen to identify and lyse tumor cells. In another exemplary advantage, NK cells have the inherent ability to bridge between innate immunity and engender a multi-clonal adaptive immune response resulting in long-term anticancer immune memory. All of these features contribute to the potential for NK cell efficacy as cancer treatment options. [0011] 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) that signal and recruit T cells to tumors. Through direct killing of tumor cells, NK cells also expose tumor antigens for recognition by the adaptive immune system.
Docket No.49755-0045WO1 [0012] Additionally, cords with preferred characteristics for enhanced clinical activity (e.g., high-affinity CD16 and Killer cell Immunoglobulin-like Receptor (KIR) B-haplotype) can be selected by utilizing a diverse umbilical cord blood bank as a source for NK cells. [0013] The administration of the allogenic NK cells, as described herein, can enhance patients’ ADCC responses, e.g., when undergoing monoclonal antibody therapy. [0014] The administration of the allogenic NK cells, as described herein, in combination with Bendamustine [0015] The administration of the allogenic NK cells, as described herein, in combination with bendamustine and an antibody, e.g., a an antibody targeting human CD20 (e.g., bendamustine plus rituximab (BR)) can confer meaningful clinical benefit, through ADCC enhancement and repeated dosing of chemotherapy, particularly in patients who have failed a prior line, e.g., containing a CD20-directed therapy. Moreover, the combination can enable further development of the therapy as an earlier line of treatment for patients with, e.g., lymphoma or CLL. [0016] Thus, described herein, are methods for treating a patient suffering from a neoplastic disorder, comprising: administering bendamustine or a pharmaceutically acceptable salt thereof to the patient; administering an antibody to the patient; and administering natural killer (NK) cells to the patient, optionally wherein the NK cells are a population of expanded natural killer cells comprising a KIR-B haplotype and homozygous for a CD16158V polymorphism. [0017] Also provided herein are methods for treating a patient suffering from a CD20+ cancer, comprising: administering bendamustine or a pharmaceutically acceptable salt thereof to the patient; administering an antibody targeted to human CD20 to the patient; and administering natural killer (NK) cells to the patient, wherein the NK cells are a population of expanded natural killer cells comprising a KIR-B haplotype and homozygous for a CD16 158V polymorphism. [0018] In some embodiments, the cancer is non-Hodgkins lymphoma (NHL). In some embodiments, the NHL is indolent NHL. In some embodiments, the NHL is aggressive NHL. In some embodiments, the indolent NHL is selected from the group consisting of Follicular lymphoma, Lymphoplasmacytic lymphoma/Waldenström macroglobulinemia, Gastric MALT, Non-gastric MALT, Nodal marginal zone lymphoma, Splenic marginal zone lymphoma, Small-cell lymphocytic lymphoma (SLL), and Chronic lymphocytic lymphoma (CLL). In some embodiments, the aggressive NHL is selected from the group consisting of Diffuse large B-cell lymphoma, Mantle cell lymphoma, Transformed follicular lymphoma,
Docket No.49755-0045WO1 Follicular lymphoma (Grade IIIB), Transformed mucosa-associated lymphoid tissue (MALT) lymphoma, Primary mediastinal B-cell lymphoma, Richter’s Syndrome or Richter’s Transformation, Lymphoblastic lymphoma, and High-grade B-cell lymphomas with translocations of MYC and BCL2, optionally further comprising a translocation of BCL6. In some embodiments, the Small-cell lymphocytic lymphoma (SLL) or Chronic lymphocytic lymphoma (CLL) comprises nodal or splenic involvement. In some embodiments, the patient has relapsed after treatment with an anti-CD20 antibody. In some embodiments, the patient has experienced disease progression after treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T). [0019] In some embodiments, the patient is administered 1 x 10^8 to 1 x 10^10 NK cells. In some embodiments, the patient is administered 1 x 10^9 to 8 x 10^9 NK cells. In some embodiments, the patient is administered 4 x 10^8, 1 x 10^9, 4 x 10^9, or 8 x 10^9 NK cells. [0020] In some embodiments, the patient is administered 100 to 500 mg/m2 of the antibody. In some embodiments, the patient is administered 375 or 500 mg/m2 of the antibody. In some embodiments, the antibody is rituximab. In some embodiments, the patient is not subjected to lymphodepleting chemotherapy prior to treatment. In some embodiments, the patient is administered 90 mg/m2 of the bendamustine or pharmaceutically acceptable salt thereof. In some embodiments, the method further comprises administering IL-2. In some embodiments, the patient is administered 1 × 106 IU/m2 of IL-2. In some embodiments, the patient is administered 6 million IU of IL-2. In some embodiments, administration of IL-2 occurs within 1-4 hrs of administration of the NK cells. [0021] In some embodiments, administration comprises a first treatment cycle, the first treatment cycle comprising: a first treatment day comprising administration of the antibody and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells; a fourth treatment day comprising administration of the NK cells; and optionally, a fifth treatment day comprising administration of the NK cells. In some embodiments, the first treatment day is day 1 of the treatment cycle, the second treatment day is day 2 of the treatment cycle, the third treatment day is day 5 of the treatment cycle, the fourth treatment day is day 8 of the treatment cycle, and the fifth treatment day is day 15 of the treatment cycle. In some embodiments, the treatment cycle is a 28-day treatment cycle. In some embodiments, the antibody is administered on the first treatment day and, optionally, on the fourth treatment day. In some embodiments, IL-2 is administered on one or more days that the NK cells are administered.
Docket No.49755-0045WO1 In some embodiments, IL-2 is administered on each day that the NK cells are administered. In some embodiments, IL-2 is not administered on one or more days that the NK cells are administered. In some embodiments, IL-2 is not administered on any days. [0022] Also provided herein are methods for treating a patient suffering from a neoplastic disorder, optionally a CD20+ cancer, the method comprising: a first treatment cycle comprising or consisting of: a first treatment day comprising administration of an antibody, optionally targeted to human CD20, and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells and IL-2; a fourth treatment day comprising administration of the NK cells, IL- 2, and the antibody; a fifth treatment day comprising administration of the NK cells and IL-2. In some embodiments, the method further comprises: a second treatment cycle comprising or consisting of: a first treatment day comprising administration of the antibody and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells; a fourth treatment day comprising administration of the NK cells; and a fifth treatment day comprising administration of the NK cells. In some embodiments, the method further comprises: a third treatment cycle comprising or consisting of: a first treatment day comprising administration of the antibody and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells; a fourth treatment day comprising administration of the NK cells; and a fifth treatment day comprising administration of the NK cells. In some embodiments, the method further comprises: a fourth treatment cycle comprising or consisting of: a first treatment day comprising administration of the antibody and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells; and a fourth treatment day comprising administration of the NK cells. In some embodiments, the method further comprises: a fifth treatment cycle comprising or consisting of: a first treatment day comprising administration of the antibody and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells; and a fourth treatment day comprising
Docket No.49755-0045WO1 administration of the NK cells. In some embodiments, the method further comprises: a sixth treatment cycle comprising or consisting of: a first treatment day comprising administration of the antibody and bendamustine or a pharmaceutically acceptable salt thereof; a second treatment day comprising administration of bendamustine or a pharmaceutically acceptable salt thereof; a third treatment day comprising administration of the NK cells; and a fourth treatment day comprising administration of the NK cells. In some embodiments, the treatment cycle is 28 days long and the first treatment day is day 1/28, the second treatment day is day 2/28, the third treatment day is day 5/28, the fourth treatment day is day 8/28, and the fifth treatment day is day 15/28. [0023] In some embodiments, the bendamustine or pharmaceutically acceptable salt thereof is administered at 90 mg/m2 per dose. In some embodiments, the NK cells are administered at from 1 to 10 billion cells per dose, optionally 1 to 4 billion cells per dose. In some embodiments, the IL-2 is administered at 6 million IU per dose. In some embodiments, the antibody is rituximab. In some embodiments, the antibody is administered at 375 mg/m2 during the first treatment cycle and 500 mg/m2 during the second, third, fourth, fifth, and sixth treatment cycles. In some embodiments, the method consists of six treatment cycles. [0024] In some embodiments, the NK cells are not genetically modified. In some embodiments, at least 70% of the NK cells are CD56+ and CD16+. In some embodiments, at least 85% of the NK cells are CD56+ and CD3-. In some embodiments, 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+ and 1% or less of the NK cells are CD14+. In some embodiments, the allogenic NK cells are expanded natural killer cells. In some embodiments, the expanded natural killer cells are expanded umbilical cord blood natural killer cells. In some embodiments, the expanded natural killer cells comprise at least 60%, e.g., at least 70%, at least 80%, at least 90% at least 95%, at least 99%, or 100% CD16+ cells. In some embodiments, the expanded natural killer cells 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. In some embodiments, the expanded natural killer cells 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. In some embodiments, the expanded natural killer cells 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. In some embodiments, the expanded natural killer cells 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. In some embodiments, the expanded natural killer cells 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. In some
Docket No.49755-0045WO1 embodiments, the expanded natural killer cells comprise less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD3+ cells. In some embodiments, the expanded natural killer cells comprise less than 20% or less, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD14+ cells. In some embodiments, the expanded natural killer cells comprise less than 20% or less, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD19+ cells. In some embodiments, the expanded natural killer cells comprise less than 20% or less, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD38+ cells. In some embodiments, the expanded natural killer cells do not comprise a CD16 transgene. In some embodiments, the expanded natural killer cells do not express an exogenous CD16 protein. In some embodiments, the expanded natural killer cells are not genetically engineered. [0025] In some embodiments, the expanded natural killer cells are derived from the same umbilical cord blood donor. In some embodiments, the population is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express a membrane bound IL-21, a mutated TNFα, and a 4-1BBL gene to produce expanded natural killer cells, thereby producing the population of expanded natural killer cells. In some embodiments, the population is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express a membrane bound IL-21, a mutated TNFα, and a 4-1BBL gene to produce a master cell bank population of expanded natural killer cells; and (d) expanding the master cell bank population of expanded natural killer cells by culturing with a second plurality of Hut78 cells engineered to express a membrane bound IL-21, a mutated TNFα, and a 4-1BBL gene to produce expanded natural killer cells; thereby producing the population of expanded natural killer cells. In some embodiments, the method further comprises, after step (c), (i) freezing the master cell bank population of expanded natural killer cells in a plurality of containers; and (ii) thawing a container comprising an aliquot of the master cell bank population of expanded natural killer cells, wherein expanding the master cell bank population of expanded natural killer cells in step (d) comprises expanding the aliquot of the master cell bank population of expanded natural killer cells. In some embodiments, the umbilical cord blood is from a donor with the KIR-B haplotype and homozygous for the CD16158V polymorphism. In some embodiments, the method
Docket No.49755-0045WO1 comprises expanding the natural killer cells from umbilical cord blood at least 10,000 fold, e.g., 15,000 fold, 20,000 fold, 25,000 fold, 30,000 fold, 35,000 fold, 40,000 fold, 45,000 fold, 50,000 fold, 55,000 fold, 60,000 fold, 65,000 fold, or 70,000 fold. In some embodiments, the population of expanded natural killer cells is not enriched or sorted after expansion. [0026] In some embodiments, the percentage of NK cells expressing CD16 in the population of expanded natural killer cells is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKG2D in the population of expanded natural killer cells is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp30 in the population of expanded natural killer cells is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp44 in the population of expanded natural killer cells is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp46 in the population of expanded natural killer cells is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. In some embodiments, the percentage of NK cells expressing DNAM-1 in the population of expanded natural killer cells is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0027] In some embodiments, the patient has previously been treated with a combination of bendamustine and rituximab (BR). In some embodiments, the patient has not previously been treated with a combination of bendamustine and rituximab (BR). [0028] In some embodiments, the patient has previously been treated with a combination of fludarabine and cyclophosphamide (FC). In some embodiments, the patient has not previously been treated with a combination of fludarabine and cyclophosphamide (FC). In some embodiments, the patient has previously been treated with a combination of fludarabine, cyclophosphamide, and rituximab (FCR). In some embodiments, the patient has not previously been treated with a combination of fludarabine, cyclophosphamide, and rituximab (FCR). In some embodiments, the patient has previously been treated with a combination of rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, and presdnisone or prednisolone (R-CHOP). In some embodiments, the patient has not previously been treated with a combination of rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, and presdnisone or prednisolone (R-CHOP).
Docket No.49755-0045WO1 [0029] 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 are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. [0030] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. INCORPORATION BY REFERENCE [0031] 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. BRIEF DESCRIPTION OF THE DRAWINGS [0032] The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0033] FIG.1 shows an example of a treatment regimine for NK cells (e.g., as described herein, e.g., AB-101) in combination with rituximab and bendamustine. Abbreviations: BR = Bendamustine plus rituximab; IL-2 = interleukin 2; NHL = Non-Hodgkin lymphoma; R/R = relapsed/refractory; DLT = dose limiting toxicity; EOT = end of treatment. In this example, a Rituximab dose on Day 8 is given in Cycle 1 only. In this example, IL-2 doses given are Cycle 1 only. [0034] FIG.2 shows lympho-recovery kinetics post cyclophosphamide/fludarabine. Absolute count of total T (a) and NK (b) cells in peripheral blood of NHL patients pre (C1
Docket No.49755-0045WO1 pre-LD) and post lymphodepletion assessed by flow cytometry. All blood samples were collected prior to any drug administration. Patients were given either cyclophosphamide 250 mg/m2/day (black circle) or 500 mg/m2/day (blue square) with fludarabine 30 mg/m2/day for three consecutive days. DETAILED DESCRIPTION [0035] Provided herein are, amongst other things, Natural Killer (NK) cells, e.g., expanded and stimulated NK cells, methods for producing the NK cells, pharmaceutical compositions comprising the NK cells, and methods of treating patients suffering, e.g., from cancer, with the NK cells. In some cases, the method comprises administering NK cells, bendamustine, and an antibody (e.g., an antibody described herein) to a patient in need thereof, e.g, a patient suffering from a disorder described herein, e.g., a cancer. I. EXPANSION AND STIMULATION OF NATURAL KILLER CELLS [0036] In some cases, the NK cells are expanded and stimulated, e.g., as described in WO2022216813, which is hereby incorporated by reference in its entirety. [0037] In some cases, e.g., after having been ex vivo expanded and stimulated, e.g., as described herein, the expanded and stimulated NK cell populations not only have a number/density (e.g., as described above) that could not occur naturally in the human body, but they also differ in their phenotypic characteristics, (e.g., gene expression and/or surface protein expression) with the starting source material or other naturally occurring populations of NK cells. [0038] In some cases, the starting NK cell source is a sample derived from a single individual, e.g., a single cord blood unit that has not been ex vivo expanded. Therefore, in some cases, the expanded and stimulated NK cells share a common lineage, i.e., they all result from expansion of the starting NK cell source, and, therefore, share a genotype via clonal expansion of a population of cells that are, themselves, from a single organism. Yet, they could not occur naturally at the density achieved with ex vivo expansion and also differ in phenotypic characteristics from the starting NK cell source. [0039] In some cases, the population of expanded and stimulated NK cells comprises at least 100 million expanded natural killer cells, e.g., 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, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 9- billion, 100
Docket No.49755-0045WO1 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, or 10 trillion expanded natural killer cells. [0040] In some 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. [0041] In some embodiments, the expanded and stimulated NK cells do not comprise a CD16 transgene. [0042] In some embodiments, the expanded and stimulated NK cells do not express an exogenous CD16 protein. [0043] The expanded and stimulated NK cells can be characterized, for example, by surface expression, e.g., of one or more of CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44. [0044] The surface protein expression levels stated herein, in some cases are achieved without positive selection on the particular surface protein referenced. For example, in some cases, the NK cell source, e.g., a single cord unit, comprises both the KIR B allele of the KIR receptor family and the 158 V/V variant of CD16 and is + enriched and CD3(+) depleted, e.g., by gating on CD56+CD3- expression, but no other surface protein expression selection is carried out during expansion and stimulation. [0045] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single 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. [0046] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single 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. [0047] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single 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. [0048] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single 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.
Docket No.49755-0045WO1 [0049] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single 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. [0050] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single 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. [0051] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises 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. [0052] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises 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. [0053] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises 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. [0054] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises 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. [0055] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises 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. [0056] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% CD3-CD14- CD19-CD16+CD56- cells. [0057] As described herein, the inventors have demonstrated that, surprisingly, the NK cells expanded and stimulated by the methods described herein express CD16 at high levels
Docket No.49755-0045WO1 throughout the expansion and stimulation process, resulting in a cell population with high CD16 expression. The high expression of CD16 obviates the need for engineering the expanded cells to express CD16, which is important for initiating ADCC, and, therefore, a surprising and unexpected benefit of the expansion and stimulation methods described herein. Thus, in some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells. [0058] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises both the KIR B allele of the KIR receptor family and the 158 V/V variant of CD16 and comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD16+ NK cells. [0059] In some embodiments, the percentage of expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, expressing CD16 is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0060] In some embodiments, the percentage of expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, expressing NKG2D is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0061] In some embodiments, the percentage of expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, expressing NKp30 is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0062] In some embodiments, the percentage of expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, expressing DNAM-1 is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0063] In some embodiments, the percentage of expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, expressing NKp44 is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0064] In some embodiments, the percentage of expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above,
Docket No.49755-0045WO1 expressing NKp46 is the same or higher than the percentage of natural killer cells in the seed cells from umbilical cord blood. [0065] As described herein, the inventors have also demonstrated that, surprisingly, the NK cells expanded and stimulated by the methods described herein express CD38 at low levels. CD38 is an effective target for certain cancer therapies (e.g., multiple myeloma and acute myeloid leukemia). See, e.g., Jiao et al., “CD38: Targeted Therapy in Multiple Myeloma and Therapeutic Potential for Solid Cancers,” Expert Opinion on Investigational Drugs 29(11):1295–1308 (2020). [0066] Thus, in some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of 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. [0067] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises both the KIR B allele of the KIR receptor family and the 158 V/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. [0068] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises both the KIR B allele of the KIR receptor family and the 158 V/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. [0069] In some embodiments, the expanded and stimulated NK cells, e.g., from expansion and stimulation of a single cord blood unit, e.g., as described above, comprises both the KIR B allele of the KIR receptor family and the 158 V/V variant of CD16 and comprise: 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 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+
Docket No.49755-0045WO1 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% 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. [0070] In some embodiments, the NK cell is engineered to alter, e.g., reduce, expression of one or more inhibitor receptor genes. [0071] In some embodiments, the inhibitory receptor gene is a HLA-specific inhibitory receptor. In some embodiments, the inhibitory receptor gene is a non-HLA-specific inhibitory receptor. [0072] In some 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. [0073] Also provided herein are pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein. [0074] In some cases, the dosage unit comprises between 100 million and 1.5 billion cells, e.g., 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, or 1.5 billion. [0075] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. [0076] In some embodiments, the pharmaceutical composition comprises: a) natural killer cell(s) described herein; and b) a cryopreservation composition. Suitable cryopreservation compositions are described herein.
Docket No.49755-0045WO1 [0077] In some embodiments, the composition is frozen. In some 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. [0078] In some 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 being thawed. [0079] In some embodiments, the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cell(s), e.g., the engineered NK cells described herein. [0080] In some embodiments, the pharmaceutical composition further comprises: c) a buffer solution. Suitable buffer solutions are described herein, e.g., as for cryopreservation compositions. [0081] In some embodiments, the pharmaceutical composition comprises from or from about 1x107 to or to about 1x109 cells/mL. In some embodiments, the pharmaceutical composition comprises 1x108 cells/mL. In some embodiments, the pharmaceutical composition comprises about 1x108 cells/mL. [0082] In some embodiments, the pharmaceutical composition comprises from or from about 1x108 to or to about 1x1010 cells/mL. [0083] In some embodiments, the pharmaceutical composition further comprises an antibody or antigen binding fragment thereof, e.g., an antibody described herein. [0084] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. [0085] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with
Docket No.49755-0045WO1 acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [0086] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. [0087] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated 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 yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0088] Examples of suitable pharmaceutical compositions are described, for example, in WO2017/135631 and WO2022/0133061, each of which is hereby incorporated by reference in its entirety.
Docket No.49755-0045WO1 II. ANTIBODIES [0089] The methods described herein comprise administering an antibody or antigen binding fragment thereof, e.g., an antibody or antigen binding fragment thereof capable of activating NK cells to elicit ADCC activity. [0090] In some embodiments, the antibody is a full length antibody comprising an Fc domain including at least one heavy chain. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody. [0091] In some embodiments, the antibody or antigen binding fragment thereof is selected from a CD20 targeting antibody, a CD38 targeting antibody, a CD19 targeting antibody, a HER-2 targeting antibody, and combinations thereof. [0092] In some embodiments, the antibody is a CD20 targeted antibody. In some embodiments, the CD20 targeted antibody is rituximab or a biosimilar thereof. Rituximab (e.g., Rituxan®) is one example of a CD20 targeted antibody useful in the presently described methods. [0093] Rituximab is a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of immune system B cells. It is used to treat diseases characterized by excessive numbers of B cells, overactive B cells, or dysfunctional B cells. This includes, e.g., disorders described herein, such as, for example, many lymphomas, leukemias, transplant rejection, and autoimmune disorders. In some embodiments, the CD20 targeting antibody is a CD20 targeting antibody selected from Table 1 or a combination thereof.
Docket No.49755-0045WO1 Table 3. Exemplary HER2 Targeting Antibodies Name Internal Name Antigen Company Reference Nemeth et al Br J 6 t b , in 3 n g
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference X
MT-1522 HT-19 HER2/n Adimab, Mer Trial ID: T k d NCT02952729
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference N
M-02 HER2/n Suzhou Trial ID: N b NCT04674722 et t 5
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference Hong et al., Cancer H
ER2/ Bi Th C L d - t n
consisting of trastuzumab (or a biosimilar thereof), margetuximab (or a biosimilar thereof), pertuzumab (or a biosimilar thereof), trastuzumab emtansine (or a biosimilar thereof), PF- 05280014 (or a biosimilar thereof), trastuzumab-anns (or a biosimilar thereof), HLX02 (or a biosimilar thereof), trastuzumab-dkst (or a biosimilar thereof), Hervycta (or a biosimilar thereof), and combinations thereof. [0101] In some embodiments, the HER2 targeting antibody is trastuzumab or a biosimilar thereof. In some embodiments, the HER2 targeting antibody is trastuzumab. [0102] In some embodiments, the EGFR targeting antibody is an EGFR targeting antibody selected from Table 4, or a combination thereof. Table 4. Exemplary EGFR Targeting Antibodies N
ame Internal Name Antigen(s) Company Reference l., 0 1- l., ol -
Docket No.49755-0045WO1 N
ame Internal Name Antigen(s) Company Reference Diaz-Serrano et Prtrzz IMC- l Ft r Onol. - . 1- ., 9 - , . 4 r. ): ol 4- 8 -
Docket No.49755-0045WO1 N
ame Internal Name Antigen(s) Company Reference U.Penn. patent WO2014130657 nti-EGFRIII
EGFR UP nn A1 - - 3 7 -
Docket No.49755-0045WO1 N
ame Internal Name Antigen(s) Company Reference duligotumab, Hill et al., Clin RG7597 Anti- Cn r R 2018 - 9 7- 3 ., 5 4 - t 7 -
, g g y g p consisting of Cetuximab (or a biosimilar thereof), Panitumumab (or a biosimilar thereof), Necitumumab (or a biosimilar thereof), and combinations thereof.
Docket No.49755-0045WO1 [0104] In some embodiments, the EGFR targeting antibody is selected from Cetuximab or a biosimilar thereof. In some embodiments, the EGFR targeting antibody is Cetuximab. In some embodiments, the biosimilar of Cetuximab is selected from the group consisting of CDP1 (Dragonboat Biopharmcaeutical Co., Ltd, Shanghai, China; clinicaltrials.gov: NCT04739111 (2021), NCT04151810 (2019), NCT03881787 (2019), and NCT03491709 (2018), CMAB009 (Shanghai BioMab Pharmaceuticals Ltd; Shanghai Zhangjiang Biotechnology Co. Ltd; clinicaltrials.gov: NCT03206151 (2017), NCT01550055 (2009); WO2016110226; US20180030139; US10118966; Shi et al., “CMAB009 plus irinotecan versus irinotecan-only as second-line treatment after fluoropyrimidine and oxaliplatin failure in KRAS wild-type metastatic colorectal cancer patients: promising findings from a prospective, open-label, randomized, phase III trial,” Cancer Comm (Lond) 39:29 (2019)), KL-140 (Sichuan Kelum Pharmaceutical Co., Ltd; clinicaltrials.gov NCT03426371), ABP 494 (Allergan, Inc.; Amgen), HLX05 (Shanghai Henlius Biotech Co. Ltd.; WO2015184403; US20170218073), and STI-001 (Mabtech; Sorrento Therapeutics; WO2017120613; US20170247458). III. PHARMACEUTICAL COMPOSITIONS [0105] Provided herein are pharmaceutical compositions comprising the natural killer cells described herein and dosage units of the pharmaceutical compositions described herein. [0106] In some cases, the dosage unit comprises between 100 million and 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, 1.5 billion, 2 billion, 2.5 billion, 3 billion, 3.5 billion, 4 billion, 4.5 billion, 5 billion, or thereabouts. [0107] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. [0108] In some embodiments, the pharmaceutical composition comprises: a) natural killer cell(s) described herein; and b) a cryopreservation composition. [0109] Suitable cryopreservation compositions are described herein. Examples of suitable cryopreservation compositions are as follows:
Docket No.49755-0045WO1 Table 5. Exemplary Cryopreservation Compositions Exemplary Range v/v% Excipient Concentration Range Exemplary Solution in Cryopreservation
Exemplary v/v% in Final Concentration in Excipient l i ii i i 0;
Docket No.49755-0045WO1 Table 7. Exemplary Cryopreservation Composition #2 Exemplary v/v% Final Concentration in Excipient in 0;
Exemplary v/v% Final Concentration in 0;
[0110] In some embodiments, the pharmaceutical composition comprises: a) a cryopreservation composition described herein; and b) therapeutic cell(s). [0111] In some embodiments, the therapeutic cell(s) are animal cell(s). In some embodiments, the therapeutic cell(s) are human cell(s).
Docket No.49755-0045WO1 [0112] In some embodiments, the therapeutic cell(s) are immune cell(s). In some embodiments, the immune cell(s) are selected from basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages, neutrophils, dendritic cells, natural killer cells, B cells, T cells, and combinations thereof. [0113] In some embodiments, the immune cell(s) are natural killer (NK) cells. In some embodiments, the natural killer cell(s) are expanded and stimulated by a method described herein. [0114] In some embodiments, the pharmaceutical composition further comprises: c) a buffer solution. Suitable buffer solutions are described herein, e.g., as for cryopreservation compositions. [0115] In some embodiments, the pharmaceutical composition comprises from or from about 1x10
7 to or to about 1x10
9 cells/mL. In some embodiments, the pharmaceutical composition comprises 1x10
8 cells/mL. In some embodiments, the pharmaceutical composition comprises about 1x10
8 cells/mL. [0116] In some embodiments, the pharmaceutical composition further comprises an antibody or antigen binding fragment thereof, e.g., an antibody described herein. [0117] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. [0118] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Docket No.49755-0045WO1 [0119] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. [0120] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated 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 yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. IV. METHODS OF TREATMENT [0121] The NK cells described herein find use for treating cancer or other proliferative disorders. [0122] Thus, also provided herein are methods of treating a patient suffering from a disorder, e.g., a disorder associated with a cancer, e.g., a NHL, comprising administering the
Docket No.49755-0045WO1 NK cells, e.g., the NK cells described herein, and a CD20 targeting antibody, e.g., an antibody described herein, e.g., rituximab. [0123] Also provided herein are methods of preventing, reducing and/or inhibiting the recurrence, growth, proliferation, migration and/or metastasis of a cancer cell or population of cancer cells in a subject in need thereof, comprising administering the NK cells, e.g., the NK cells described herein, and a CD20 targeting antibody, e.g., an antibody described herein, e.g., rituximab. [0124] Also provided herein are methods of enhancing, improving, and/or increasing the response to an anticancer therapy in a subject in need thereof, comprising administering the NK cells, e.g., the NK cells described herein, and a CD20 targeting antibody, e.g., an antibody described herein, e.g., rituximab. [0125] Also provided herein are methods for inducing the immune system in a subject in need thereof comprising administering the NK cells, e.g., the NK cells described herein, and a CD20 targeting antibody, e.g., an antibody described herein, e.g., rituximab. [0126] The methods described herein include methods for the treatment of disorders associated with abnormal apoptotic or differentiative processes, e.g., cellular proliferative disorders or cellular differentiative disorders, e.g., cancer, including both solid tumors and hematopoietic cancers. Generally, the methods include administering a therapeutically effective amount of a treatment as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. In some embodiments, the methods include administering a therapeutically effective amount of a treatment comprising an NK cells, e.g., NK cells described herein, and a CD20 targeting antibody, e.g., an antibody described herein, e.g., rituximab. [0127] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disorder associated with abnormal apoptotic or differentiative processes. For example, a treatment can result in a reduction in tumor size or growth rate. Administration of a therapeutically effective amount of a compound described herein for the treatment of a condition associated with abnormal apoptotic or differentiative processes will result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in metastasis, increased survival, and/or decreased morbidity and mortality, among other things. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible
Docket No.49755-0045WO1 individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. [0128] As used herein, the terms "inhibition”, as it relates to cancer and/or cancer cell proliferation, refer to the inhibition of the growth, division, maturation or viability of cancer cells, and/or causing the death of cancer cells, individually or in aggregate with other cancer cells, by cytotoxicity, nutrient depletion, or the induction of apoptosis. [0129] As used herein, “delaying” development of a disease or disorder, or one or more symptoms thereof, means to defer, hinder, slow, retard, stabilize and/or postpone development of the disease, disorder, or symptom thereof. This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease, disorder, or symptom thereof. For example, a method that “delays” development of cancer is a method that reduces the probability of disease development in a given time frame and/or reduces extent of the disease in a given time frame, when compared to not using the method. Such comparisons may be based on clinical studies, using a statistically significant number of subjects. [0130] As used herein, “prevention” or “preventing” refers to a regimen that protects against the onset of the disease or disorder such that the clinical symptoms of the disease do not develop. Thus, “prevention” relates to administration of a therapy (e.g., administration of a therapeutic substance) to a subject before signs of the disease are detectable in the subject and/or before a certain stage of the disease (e.g., administration of a therapeutic substance to a subject with a cancer that has not yet metastasized). The subject may be an individual at risk of developing the disease or disorder, or at risk of disease progression, e.g., cancer metastasis. Such as an individual who has one or more risk factors known to be associated with development or onset of the disease or disorder. For example, an individual may have mutations associated with the development or progression of a cancer. Further, it is understood that prevention may not result in complete protection against onset of the disease or disorder. In some instances, prevention includes reducing the risk of developing the disease or disorder. The reduction of the risk may not result in complete elimination of the risk of developing the disease or disorder. [0131] An “increased” or “enhanced” amount (e.g., with respect to antitumor response, cancer cell metastasis) refers to an increase that is 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
Docket No.49755-0045WO1 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or level described herein. 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 an amount or level described herein. [0132] A “decreased” or “reduced” or “lesser” amount (e.g., with respect to tumor size, cancer cell proliferation or growth) refers to a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) an amount or level described herein. It may also include a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, at least 100%, at least 150%, at least 200%, at least 500%, or at least 1000% of an amount or level described herein. A. Disorders [0133] Methods and manufactured compositions disclosed herein find use in targeting a number of disorders, such as cellular proliferative disorders. A benefit of the approaches herein is that allogenic cells are used in combination with exogenous antibody administration to target specific proliferating cells targeted by the exogenous antibody. Unlike previous therapies, such as chemo or radiotherapy, using the approaches and pharmaceutical compositions herein, one is able to specifically target cells exhibiting detrimental proliferative activity, potentially without administering a systemic drug or toxin that impacts proliferating cells indiscriminately. [0134] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin. [0135] As used herein, the terms “cancer”, “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective
Docket No.49755-0045WO1 of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. [0136] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. [0137] The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. [0138] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. [0139] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. In some cases, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol.11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell
Docket No.49755-0045WO1 lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease. [0140] In some embodiments, the cancer is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid, cardiac tumors, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer (e.g., intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumor, hepatocellular cancer, histiocytosis, Hodgkin lymphomas, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, pleuropulmonary blastoma, and tracheobronchial tumor), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic cancer, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasms, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myeloproliferative neoplasms, nasal cavity and paranasal 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, pheochromocytomas, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland
Docket No.49755-0045WO1 cancer, sarcoma (e.g., childhood rhabdomyosarcoma, childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach cancer, T-cell lymphomas, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thryomoma and thymic carcinomas, thyroid cancer, tracheobronchial tumors, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, and Wilms tumor. [0141] In some embodiments, the cancer is a solid tumor. [0142] In some embodiments, the cancer is metastatic. [0143] In some embodiments, the disorder is selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, follicular lymphoma, granulomastosis with polyangiitis, microscopic polyangiitis, multiple sclerosis, non-Hodkin’s Lymphoma, Pemphigus Vulgaris, Rheumatoid Arthritis, and combinations thereof. [0144] In some embodiments, the cancer is a CD20+ cancer, a CD19+ cancer, an EGFR+ cancer, a HER2+ cancer, or a combination thereof. [0145] In some embodiments, the cancer is a CD20+ cancer. [0146] In some embodiments, the CD20+ cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). [0147] In some embodiments, the CD20+ cancer is selected from the group consisting of indolent or aggressive non-Hodgkin’s lymphoma (NHL). In some embodiments, the CD20+ cancer is relapsed or refractory indolent or aggressive NHL of B-cell origin. Among the aggressive and indolent subtypes are those in Table 9. Table 9. Exemplary Aggressive and Indolent NHL Aggressive Subtype Indolent Subtype Diff l B ll l h F lli l l h G d I II d IIIA) ,
Docket No.49755-0045WO1 Aggressive Subtype Indolent Subtype Small-cell lymphocytic lymphoma L h bl i l h SLL /Ch i l h i l k i CLL) e
CD19+ cancer is a B-cell cancer. In some embodiments, the CD19+ cancer is selected from the group consisting of B-cell lymphoma, leukemia, and combinations thereof. [0149] In some embodiments, the cancer is a CD38+ cancer. [0150] In some embodiments, the CD38+ cancer is selected from the group consisting of glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, liver cancer, pancreatic cancer, renal cancer, urothelial cancer, prostate cancer, testis cancer, breast cancer, cervical cancer, ovarian cancer, melanoma, lymphoma, and combinations thereof. [0151] In some embodiments, the CD38+ cancer is prostate cancer. [0152] In some embodiments, the CD38+ cancer is lymphoma. [0153] In some embodiments, the CD38+ cancer is multiple myeloma. [0154] In some embodiments, the cancer is an EGFR+ cancer. In some embodiments, the EGFR+ cancer is selected from the group consisting of glioblastoma, lung adenocarcinoma, non-small cell lung cancer, lower grade glioma, colorectal adenocarcinoma, high-grade glioma, multiple myeloma, breast adenocarcinoma, pilocityc astrocytoma, and combinations thereof. [0155] In some embodiments, the cancer is a HER2+ cancer. [0156] In some embodiments, the HER2+ cancer is selected from the group consisting of bladder cancer, breast adenocarcinoma, colorectal adenocarcinoma, non-small cell lung cancer, esophageal cancer, cervix squamous cancer, stomach adenocarcinoma, cholangiocarcinoma, ovary cancer, renal papillary cell carcinoma, and combinations thereof. [0157] In some embodiments, the HER2+ cancer is selected from the group consisting of breast cancer, gastric cancer, and ovarian cancer. [0158] 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.
Docket No.49755-0045WO1 B. Patients [0159] Suitable patients for the compositions and methods herein include those who are suffering from, who have been diagnosed with, or who are suspected of having a cellular proliferative and/or differentiative disorder, e.g., a cancer. Patients subjected to technology of the disclosure herein generally respond better to the methods and compositions herein, in part because the pharmaceutical compositions are allogeneic and target cells identified by the antibodies, rather than targeting proliferating cells generally. As a result, there is less off- target impact and the patients are more likely to complete treatment regimens without substantial detrimental off-target effects. [0160] In some embodiments, the methods of treatment provided herein may be used to treat a subject (e.g., human, monkey, dog, cat, mouse) who has been diagnosed with or is suspected of having a cellular proliferative and/or differentiative disorder, e.g., a cancer. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. [0161] As used herein, a subject refers to a mammal, including, for example, a human. [0162] In some embodiments, the mammal is selected from the group consisting of an armadillo, an ass, 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, an ox, a pig, a polar bear, a porcupine, a puma, a rabbit, a raccoon, a rat, a rhinoceros, a sheep, a squirrel, a tiger, a walrus, a weasel, a wolf, a zebra, a goat, a horse, and combinations thereof. [0163] In some embodiments, the mammal is a human. [0164] The subject, e.g., the human subject, can be a child, e.g., from or from about 0 to or to about 14 years in age. The subject can be a youth, e.g., from or from about 15 to or to about 24 years in age. The subject can be an adult, e.g., from or from about 25 to or to about 64 years in age. The subject can be a senior, e.g, 65+ years in age. [0165] In some embodiments, the subject may be a human who exhibits one or more symptoms associated with a cellular proliferative and/or differentiative disorder, e.g., a cancer, e.g., a tumor. Any of the methods of treatment provided herein may be used to treat cancer at various stages. By way of example, the cancer stage includes but is not limited to early, advanced, locally advanced, remission, refractory, reoccurred after remission and progressive. In some embodiments, the subject is at an early stage of a cancer. In other embodiments, the subject is at an advanced stage of cancer. In various embodiments, the
Docket No.49755-0045WO1 subject has a stage I, stage II, stage III or stage IV cancer. The methods of treatment described herein can promote reduction or retraction of a tumor, decrease or inhibit tumor growth or cancer cell proliferation, and/or induce, increase or promote tumor cell killing. In some embodiments, the subject is in cancer remission. The methods of treatment described herein can prevent or delay metastasis or recurrence of cancer. [0166] In some embodiments, the subject suffers from low numbers of NK cells. Some subjects with low numbers of NK cells are unable to mount robust ADCC responses when treated with antibodies, including rituximab. Resistance to rituximab can result even without CD20 antigen loss. In some cases, low NK cell numbers can result in or contribute to resistance to rituximab. In some cases, low NK cell numbers are associated with relapsed or refractory NHL. Thus, these patients may benefit from the use of the compositions and methods described herein. [0167] In some embodiments, the subject is at risk, or genetically or otherwise predisposed (e.g., risk factor), to developing a cellular proliferative and/or differentiative disorder, e.g., a cancer, that has or has not been diagnosed. [0168] As used herein, an “at risk” individual is an individual who is at risk of developing a condition to be treated, e.g., a cellular proliferative and/or differentiative disorder, e.g., a cancer. Generally, an “at risk” subject may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a disease or condition and are known in the art. For example, an at risk subject may have one or more risk factors, which are measurable parameters that correlate with development of cancer. A subject having one or more of these risk factors has a higher probability of developing cancer than an individual without these risk factor(s). In general, risk factors may include, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposure. In some embodiments, the subjects at risk for cancer include, for example, those having relatives who have experienced the disease, and those whose risk is determined by analysis of genetic or biochemical markers. [0169] In addition, the subject may be undergoing one or more standard therapies, such as chemotherapy, radiotherapy, immunotherapy, surgery, or combination thereof. Accordingly, one or more kinase inhibitors may be administered before, during, or after administration of chemotherapy, radiotherapy, immunotherapy, surgery or combination thereof.
Docket No.49755-0045WO1 [0170] In certain embodiments, the subject may be a human who is (i) substantially refractory to at least one chemotherapy treatment, or (ii) is in relapse after treatment with chemotherapy, or both (i) and (ii). In some of embodiments, the subject is refractory to at least two, at least three, or at least four chemotherapy treatments (including standard or experimental chemotherapies). In some embodiments, at least one of such therapies is or includes an anti-CD20 monoclonal antibody therapy. In some embodiments, the subject has previously undergone an autologous hematopoietic stem cell transplant. In some embodiments, the subject has previously been treated with a CAR-T therapy. In some embodiments, the subject has previously been administered an investigational drug or agent. [0171] In some embodiments, the patient is or has been diagnosed with a disorder selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B- cell lymphoma, follicular lymphoma, granulomastosis with polyangiitis, microscopic polyangiitis, multiple sclerosis, non-Hodkin's Lymphoma, Pemphigus Vulgaris, Rheumatoid Arthritis, and combinations thereof. [0172] In some embodiments, the patient is or has been diagnosed with a CD20+ cancer. [0173] In some embodiments, the patient is or has been diagnosed with a CD20+ cancer by immunohistochemical staining of a biopsy or surgical sample of the cancer. In some embodiments, the patient is or has been diagnosed with a CD20+ cancer by chromogenic in situ hybridization. In some embodiments, the patient is or has been diagnosed with a CD20+ cancer by fluorescent in situ hybridization of a biopsy or surgical sample of the cancer. [0174] In some embodiments, the patient is or has been diagnosed with a CD20+ cancer by genetic analysis, e.g., by identifying a CD20 mutated cancer, e.g., a somatic mutation, e.g., a somatic mutation in the CD20 (MS4A1) gene. [0175] In some embodiments, the patient has a cancer comprising one or more mutation, insertion or deletion polymorphisms in the CD20 gene, a copy number variation of the CD20 gene, a methylation mutation of the CD20 gene, or combinations thereof. [0176] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., a CD20+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., a CD20+ cancer. [0001] In some embodiments, the patient is or has been diagnosed with a CD19+ cancer. [0002] In some embodiments, the patient is or has been diagnosed with a CD19+ cancer by immunohistochemical staining of a biopsy or surgical sample of the cancer. In some embodiments, the patient is or has been diagnosed with a CD19+ cancer by chromogenic in
Docket No.49755-0045WO1 situ hybridization. In some embodiments, the patient is or has been diagnosed with a CD19+ cancer by fluorescent in situ hybridization of a biopsy or surgical sample of the cancer. [0003] In some embodiments, the patient is or has been diagnosed with a CD19+ cancer by genetic analysis, e.g., by identifying a CD19mutated cancer, e.g., a somatic mutation, e.g., a somatic mutation in the CD19 gene. [0004] In some embodiments, the patient has a cancer comprising one or more mutations, insertion, or deletion polymorphism in the CD19 gene, a copy number variation of the CD19 gene, a methylation mutation of the CD19 gene, or combinations thereof. [0005] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., a CD19+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., a CD19+ cancer. [0006] In some embodiments, the patient is diagnosed with or has been diagnosed with a CD38+ cancer by immunohistochemical staining of a biopsy or surgical sample of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with a CD38+ cancer by chromogenic in situ hybridization. In some embodiments, the patient is diagnosed with or has been diagnosed with a CD38+ cancer by fluorescent in situ hybridization of a biopsy or surgical sample of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with a CD38+ cancer by genetic analysis, e.g., by identifying a CD38 mutated cancer, e.g., a somatic mutation in the CD38 gene. [0007] In some embodiments, the patient is a multiple myeloma patient is not a candidate for autologous hematopoietic stem cell transplant (ASCT). In some embodiments, the patient is relapsed or refractory after having received ASCT. [0008] In some cases, the patient is a multiple myeloma patient whose cancer is relapsed or refractory after having received a treatment selected from the group consisting of one or more steroids, alkylating agents, and/or anthracyclines, proteasome inhibitors, or immunomodulatory drugs. [0009] In some embodiments, the patient has a cancer comprising one or more mutation, insertion, or deletion polymorphisms in the CD38 gene, a copy number variation of the CD38 gene, a methylation mutation of the CD38 gene, or combinations thereof. [0010] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., a CD38+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., a CD38+ cancer.
Docket No.49755-0045WO1 [0011] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., a CD38+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., a CD38+ cancer. [0012] In some embodiments, the patient is diagnosed with or has been diagnosed with EGFR+ cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with an EGFR+ cancer by immunohistochemical staining of a biopsy or surgical sample of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with an EGFR+ cancer by chromogenic in situ hybridization. In some embodiments, the patient is diagnosed with or has been diagnosed with an EGFR+ cancer by fluorescent in situ hybridization of a biopsy or surgical sample of the cancer. In some embodiments, the patient is diagnosed with or has been diagnosed with an EGFR+ cancer by genetic analysis, e.g., by identifying an EGFR mutated cancer, e.g., a somatic mutation in the EGFR gene. [0013] In some embodiments, the patient has a cancer comprising one or more mutation, insertion, or deletion polymorphisms in the EGFR gene, a copy number variation of the EGFR gene, a methylation mutation of the EGFR gene, or combinations thereof. [0014] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., an EGFR+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., a EGFR + cancer. [0015] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., an EGFR+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., an EGFR+ cancer. [0016] In some embodiments, the patient is refractory to or has a recurrence after treatment with an EGFR inhibitor. In some embodiments, the patient is refractory to or has a recurrence after treatment with a chemotherapy drug. [0017] In some embodiments, the chemotherapy drug is selected from the group consisting of cisplatin, docetaxel, carboplatin, gemcitabine, cisplatin, pemetrexed, or combinations thereof. [0018] In some embodiments, the patient is refractory to or has a recurrence after treatment with a tyrosine kinase inhibitor. In some embodiments, the tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlontinib, afatinib, osimertinib, and combinations thereof. [0019] In some embodiments, the patient has a cancer that is resistant to an EGFR specific antibody (e.g., as described herein). In some cases, the EGFR specific antibody is an
Docket No.49755-0045WO1 EGFR antagonist, such as cetuximab. In some cases, the EGFR specific antibody is an EGFR antagonist that also has an ADCC mechanism, such as cetuximab. [0020] Cetuximab resistance, for example, is associated with Ras pathway mutations (e.g., KRAS/NRAS/HRAS/BRAFI/PIK3CA. See, e.g., Song et al., “Qualitative Ras Pathway Signature for Cetixumab Therapy Reveals Resistance Mechanism in Colorectal Cancer,” The FEBS Journal 287:5236–48 (2020); and Rampias et al., “RAS/PI3K Crosstalk and Cetuximab Resistance in Head and Neck Squamos Cell Carcinoma,” Clin Cancer Res 20(11):2933-46 (2014); , which are hereby incorporated by reference in their entirety. Thus, in some embodiments, the patient has a Ras-activated cancer. For example, in some embodiments, the patient is not a Ras Pathway wild-type patient (e.g., the patient has mutations in or affecting expression of one or more of KRAS, NRAS, BRAF, HRAS, or PIK3CA). [0021] In some embodiments, the patient is diagnosed with or has been diagnosed with a HER2+ cancer by genetic analysis, e.g., by identifying a HER2 mutated cancer, e.g., a somatic mutation in the HER2 (ERBB2) gene. [0022] In some embodiments, the patient has a cancer comprising one or more mutation, insertion or deletion polymorphisms in the HER2 gene, a copy number variation of the HER2 gene, a methylation mutation of the HER2 gene, or combinations thereof. [0023] In some embodiments, the patient has a chromosomal translocation associated with cancer, e.g., a HER2+ cancer. In some embodiments, the patient has a fusion gene associated with cancer, e.g., a HER+ cancer. [0024] In some embodiments, the patient is refractory to or has a recurrence of HER2+ cancer after treatment, e.g., with trastuzumab or a biosimilar thereof. [0025] In some embodiments, the patient is refractory to or has a recurrence after treatment with pertuxumab (or FDA-approved biosimilar thereof), trastuzumab (or FDA- approved biosimilar thereof) and docetaxel (or pharmaceutically acceptable salt thereof). In some embodiments, the pertuzumab (or FDA-approved biosimilar thereof) is administered at 840 mg IV day 1 followed by 420 mg IV. In some embodiments, the trastuzumab (or FDA- approved biosimilar thereof) is administered at 7 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. In some embodiments, the docetaxel (or pharmaceutically acceptable salt thereof) is administered at 75-100 mg/m2 IV day 1 cycled every 21 days.
Docket No.49755-0045WO1 [0026] In some embodiments, the patient is refractory to or has a recurrence after treatment with pertuxumab (or FDA-approved biosimilar thereof), trastuzumab (or FDA- approved biosimilar thereof), and paclitaxel (or pharmaceutically acceptable salt thereof). In some embodiments, the pertuxumab (or FDA-approved biosimilar thereof) is administered at 840 mg IV day 1 followed by 420 mg IV, cycled every 21 days. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered at 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. In some embodiments, the paclitaxel (or pharmaceutically acceptable salt thereof) is administered at 80 mg/m2 IV day 1 weekly or 175 mg/m2 day 1 cycled every 21 days. [0027] In some embodiments, the patient is refractory to or has a recurrence after treatment with tucatinib (or pharmaceutically acceptable salt thereof), trastuzumab (or FDA- approved biosimilar thereof), and capecitabine (or pharmaceutically acceptable salt thereof). In some embodiments, the tucatinib (or FDA-approved biosimilar thereof) is administered at 300 mg orally twice daily on days 1-21. In some embodiments, the trastuzumab (or FDA- approved biosimilar thereof) is administered at 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. In some embodiments, the capecitabine (or FDA-approved biosimilar thereof) is administered at 1000 mg/m2 orally twice daily on days 1-14. In some embodiments, the administration of tucatinib (or FDA- approved biosimilar thereof), trastuzumab (or FDA-approved biosimilar thereof), and capecitabine (or pharmaceutically acceptable salt thereof) is cycled every 21 days. [0028] In some embodiments, the patient is refractory to or has a recurrence after treatment with ado-trastuzumab emtansine (T-DM1) (or FDA-approved biosimilar thereof). In some embodiments, the ado-trastuzumab emtansine (T-DM1) (or FDA-approved biosimilar thereof) is administered at 3.6 mg/kg IV day 1, cycled every 21 days. [0029] In some embodiments, the patient is refractory to or has a recurrence after treatment with fam-trastuzumab deruxtecan-nxki (or FDA-approved biosimilar thereof). In some embodiments, the fam-trastuzumab deruxtecan-nxki (or FDA-approved biosimilar thereof) is administered at 5.4 mg/kg IV day 1, cycled every 21 days.
Docket No.49755-0045WO1 [0030] In some embodiments, the patient is refractory to or has a recurrence after treatment with paclitaxel/carboplatin (or pharmaceutically acceptable salts thereof) and trastuxumab (or FDA-approved biosimilar thereof). In some embodiments, the carboplatin/paclitaxel (or pharmaceutically acceptable salts thereof) is administered at AUC 6 IV day 1 carboplatin and 175 mg/m2 IV day 1 paclitaxel), cycled every 21 days. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase- oysk injection for subcutaneous administration. [0031] In some embodiments, the patient is refractory to or has a recurrence after treatment with paclitaxel/carboplatin (or pharmaceutically acceptable salts thereof) and trastuxumab (or FDA-approved biosimilar thereof). In some embodiments, the carboplatin/paclitaxel (or pharmaceutically acceptable salts thereof) is administered at AUC 2 IV carboplatin and 80 mg/m2 IV day 1 paclitaxel), days 1, 8, and 15, cycled every 28 days. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. [0032] In some embodiments, the patient is refractory to or has a recurrence after treatment with trastuzumab (or FDA-approved biosimilar thereof) and paclitaxel (or pharmaceutically acceptable salt thereof). In some embodiments, the paclitaxel (or pharmaceutically acceptable salt thereof) is administered at 175 mg/m2 IV day 1 cycled every 21 days or 80-90 mg/m2 IV day 1 weekly. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. [0033] In some embodiments, the patient is refractory to or has a recurrence after treatment with trastuzumab (or FDA-approved biosimilar thereof) and docetaxel (or pharmaceutically acceptable salt thereof). In some embodiments, the docetaxel (or pharmaceutically acceptable salt thereof) is administered at 80-100 mg/m2 IV day 1 cycled
Docket No.49755-0045WO1 every 21 days or 35 mg/m2 IV days 1, 8, and 15 weekly. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. [0034] In some embodiments, the patient is refractory to or has a recurrence after treatment with trastuzumab (or FDA-approved biosimilar thereof) and vinorelbine (or pharmaceutically acceptable salt thereof). In some embodiments, the vinorelbine (or pharmaceutically acceptable salt thereof) is administered at 25 mg/m2 IV day 1 weekly or 20- 35 mg/m2 IV days 1 and 8, cycled every 21 days, or 25-30 mg/m2 IV days 1, 8, and 15, cycled every 28 days. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. [0035] In some embodiments, the patient is refractory to or has a recurrence after treatment with trastuzumab (or FDA-approved biosimilar thereof) and capecitabine (or pharmaceutically acceptable salt thereof). In some embodiments, the capecitabine (or pharmaceutically acceptable salt thereof) is administered at 1000-1250 mg/m2 PO twice daily days 1-14 cycled every 21 days. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase-oysk injection for subcutaneous administration. [0036] In some embodiments, the patient is refractory to or has a recurrence after treatment with lapatinib (or pharmaceutically acceptable salt thereof) and capecitabine (or pharmaceutically acceptable salt thereof). In some embodiments, the lapatinib (or pharmaceutically acceptable salt thereof) is administered at 1250 mg/m2 PO daily days 1-21. In some embodiments, the capecitabine (or pharmaceutically acceptable salt thereof) is administered at 1000 mg/m2 PO twice daily days 1-14, cycled every 21 days. [0037] In some embodiments, the patient is refractory to or has a recurrence after treatment with trastuzumab (or FDA-approved biosimilar thereof) and lapatinib (or
Docket No.49755-0045WO1 pharmaceutically acceptable salt thereof). In some embodiments, the administered (or pharmaceutically acceptable salt thereof) is administered at 1000 mg/m2 PO daily. In some embodiments, the trastuzumab (or FDA-approved biosimilar thereof) is administered 4 mg/kg IV day 1 followed by 2 mg/kg IV weekly or 8 mg/kg IV day 1 followed by 6 mg/kg IV day 1 every 21 days. In some embodiments, the trastuxumab (or FDA-approved biosimilar thereof) is administered as a trastuzumab (or FDA-approved biosimilar thereof) and hyaluronidase- oysk injection for subcutaneous administration. In some embodiments, the patient is refractory to or has a recurrence after treatment with neratinib (or pharmaceutically acceptable salt thereof) and capecitabine (or pharmaceutically acceptable salt thereof). In some embodiments, the neratinib is administered at 240 mg/m2 PO daily on days 1-21. In some embodiments, the capecitabine is administered at 750 mg/m2 PO twice daily on days 1-14, cycled every 21 days. [0038] In some embodiments, the patient is refractory to or has recurrent after treatment with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone), e.g., at least 4 cycles of R-CHOP, a second line of chemotherapy, e.g., ICE (ifosfamide, carboplatin, and etoposide) or DHAP (dexamethasone, high-dose Ara-C cytarabine, and platinol) with or without an approved therapeutic mAb (e.g., rituximab). C. Administration [0039] The methods described herein include the administration of one or more of: NK cells, antibody (e.g., as described herein), bendamustine (e.g., as described herein), and cytokines (e.g., as described herein). In some embodiments, one or more of the NK cells, antibody, bendamustine, and/or cytokines are each administered within a single treatment cycle. In some embodiments, administration of one or more of the NK cells, antibody, bendamustine, and/or cytokines is on the same day within a treatment cycle. In some embodiments, administration the NK cells, antiody, bendamustine, and/or cytokines is on different days within a treatment cycle. [0040] In some cases, a patient is administered one or more of: NK cells, antibodies, bendamustine, and cytokines over multiple treatment cycles, which can have the same or different administration schedules. 1. Treatment Cycles [0041] In some embodiments, administration (e.g., a therapeutic cycle for a patient) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 treatment cycles. In some embodiments, each
Docket No.49755-0045WO1 treatment cycle independently comprises from 1 to 30 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, each treatment cycle comprises 28 days. [0042] Each cycle can be preceded by a debulking or pretreatment dose of antibody. [0043] In some cases, the cycles comprising more administrations of the NK cells, antibodies, bendamustine, and/or cytokines, followed by additional cycles comprising less frequent administrations, including, e.g., administration every month, every other month, or every third month. Such doses can help the patient maintain a response to the therapy. [0044] In some embodiments, the first NK cells administration is after (e.g., the day after) a day that bendamustine is administered, e.g., after the last day that bendmustine is administered. For example, in a 28 day treatment cycle where bendamustine is administered only on days 1 and 2, NK cells are administered on any one or more of days 3–28. In some embodiments, both bendamustine and the antibody are administered before the first administration of NK cells. For example, in a 28 day treatment cycle, bendamustine and the antibody are each administered on days 1 and/or 2 of the treatment cycle, and NK cells are administered on any one or more of days 3–28. In some embodiments, bendamustine and rituximab are administered on day 1, bendamustine is administered on day 2, and NK cells are administered on any one or more of days 3–28 (e.g., on any one or more of days 5, 8, and 15). [0045] In some embodiments, bendamustine and rituximab are administered on day 1, bendamustine is administered on day 2, NK cells are administered on any one or more of days 5, 8, and 15, the antibody is optionally administered with the second dose of NK cells (e.g., on day 8). In some embodiments (e.g., of a 28 day treatment cycle), bendamustine and rituximab are administered on day 1, bendamustine is administered on day 2, NK cells and optionally a cytokine (e.g., IL-2) are administered on day 5, NK cells, optionally the antibody, and optionally a cytokine (e.g., IL-2) are administered on 8, NK cells, and, optionally, NK cells and optionally a cytokine (e.g., IL-2) are administered on day 15. In some embodiments, the 28 day treatment cycle is repeated 1, 2, 3, 4, 5, or 6 times in a therapeutic cycle. [0046] In some embodiments, e.g., of a therapeutic cycle comprising 6 treatment cycles, e.g., 28 day treatment cycles, the NK cells and optionally a cytokine (e.g., IL-2) are administered on day 15 during one or more treatment cycles (e.g., treatment cycles 1, 2, and 3, e.g., of a therapeutic cycle comprising 6 treatment cycles), but not during the subsequent treatment cycles (e.g., treatment cycles 4, 5, and 6, e.g., of a therapeutic cycle comprising 6
Docket No.49755-0045WO1 treatment cycles). In some embodiments, e.g., of a therapeutic cycle comprising 6 treatment cycles, cytokines are administered during treatment cycles 1, 2, 3, 4, 5 and/or 6. In some embodiments, e.g., of a therapeutic cycle comprising 6 treatment cycles, cytokines are administered only during the first treatment cycle. [0047] In some embodiments, e.g., of a therapeutic cycle comprising 6 treatment cycles, e.g., 28 day treatment cycles, the antibody is administered after the last bendamustine treatment during one or more treatment cycles (e.g., treatment cycles 1, 2, and 3, e.g., of a therapeutic cycle comprising 6 treatment cycles), but not during the subsequent treatment cycles (e.g., treatment cycles 4, 5, and 6, e.g., of a therapeutic cycle comprising 6 treatment cycles). For example, for a therapeutic cycle comprising 628 day treatment cycles in which bendamustine is administered on days 1 and 2, and NK cells are administered on days 5, 8, and optionally 15, the antibody is administered on day 8 during cycles 1, 2, and 3, but not during cycles 4, 5, and 6. 2. Bendamustine [0048] In some embodiments, Bendamustine (4-[5-[bis(2-chloroethyl)amino]-1- methylbenzimidazol-2-yl]butanoic acid) or a pharmaceutically acceptable salt thereof (e.g., a pharmaceutical composition comprising bendamustine or a pharmaceutically acceptable salt thereof) is administered, e.g., to a patient described herein, e.g., in combination with NK cells and/or an antibody described herein. [0049] In some embodiments, the bendamustine is administered at or at about 10 to 500 mg/m
2, e.g., at or at about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg/m
2 per dose, preferably 90 mg/m
2 per dose. [0050] In some embodiments, the bendamustine is administered intravenously. In some embodiments, the bendamustine is administered intravenously via syringe or via gravity IV infusion. In some embodiments, the infusion is administered over 1 to 60 minutes, e.g., over or over about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 minutes. [0051] In some embodiments, the bendamustine is administered from 10 minutes to 24 hours, e.g., 10, 20, 30, 40, 50 or 60 minutes or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
Docket No.49755-0045WO1 17, 18, 19, 20, 21, 22, 23, or 24 hours after completion of antibody administration if antibody administration is scheduled for the same day. 3. NK Cells [0052] In some embodiments, the NK cells are administered as part of a pharmaceutical composition, e.g., a pharmaceutical composition described herein. Cells are administered after thawing, in some cases without any further manipulation in cases where their cryoprotectant is compatible for immediate administration. For a given individual, a treatment regimen often comprises administration over time of multiple aliquots or doses of NK cells drawn from a common batch or donor. [0053] In some embodiments, the NK cells, e.g., the NK cells described herein are administered at or at about 1 x 10
8 to or to about 8 x 10
9 NK cells per dose, including 1 x 10
8, 2 x 10
8, 3 x 10
8, 4 x 10
8, 5 x 10
8, 6 x 10
8, 7 x 10
8, 8 x 10
8, 9 x 10
8, 1 x 10
9, 2 x 10
9, 3 x 10
9, 4 x 10
9, 5 x 10
9, 6 x 10
9, 7 x 10
9, or 8 x 10
9 cells per dose. In some embodiments, the NK cells are administered at or at about 1 x 10
8, at or at about 1 x 10
9, at or at about 4 x 10
9, or at or at about 8 x 10
9 NK cells per dose. [0054] In some embodiments, the NK cells are administered weekly. In some embodiments, the NK cells are administered weekly for or for about four weeks. In some embodiments, the NK cells are administered weekly for or for about 8 weeks. [0055] In some embodiments, the NK cells are cryopreserved in an infusion-ready media, e.g., a cryopreservation composition suitable for intravenous administration, e.g., as described herein. [0056] In some embodiments, the NK cells are cryopreserved in vials containing from or from about 1 x 10
8 to or to about 8 x 10
9 cells per vial, including 1 x 10
8, 2 x 10
8, 3 x 10
8, 4 x 10
8, 5 x 10
8, 6 x 10
8, 7 x 10
8, 8 x 10
8, 9 x 10
8, 1 x 10
9, 2 x 10
9, 3 x 10
9, 4 x 10
9, 5 x 10
9, 6 x 10
9, 7 x 10
9, or 8 x 10
9 cells per vial. In some embodiments, the NK cells are cryopreserved in vials containing a single dose. In some embodiments, the NK cells are cryopreserved in vials containing less than a single dose. In some of such cases, multiple vials can be thawed simultaneously or separately, and can be combined prior to administration or administered separately. [0057] In some embodiments, the cells are thawed, e.g., in a 37°C water bath, prior to administration. [0058] In some embodiments, the thawed vial(s) of NK cells are aseptically transferred to a single administration vessel, e.g., administration bag using, e.g., a vial adapter and a sterile
Docket No.49755-0045WO1 syringe. The NK cells can be administered to the patient from the vessel through a Y-type blood/solution set filter as an IV infusion, by gravity. [0059] In some embodiments, the NK cells are administered as soon as practical, preferably less than 90 minutes, e.g., less than 80, 70, 60, 50, 40, 30, 20, or 10 minutes after thawing. In some embodiments, the NK cells are administered within 30 minutes of thawing. [0060] In some embodiments, the pharmaceutical composition is administered intravenously via syringe or via gravity IV infusion. In some embodiments, the infusion is administered in or in about 1 minute, 2 minutes, 3, minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. In some embodiments, the infusion is administered over or over about 1 minute, 2 minutes, 3, minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. [0061] In some embodiments, 1 mL, 4 mL, or 10 mL of drug product is administered to the patient intravenously via syringe. 4. Antibodies [0062] In some embodiments, the NK cell(s) described herein, e.g., the pharmaceutical compositions comprising NK cell(s) described herein, are administered in combination with an antibody, e.g., an antibody described herein, e.g., a CD20 targeting antibody, e.g., rituximab. In some embodiments, an antibody is administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, an antibody is administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition. Antibodies can be administered prior to, subsequent to, or simultaneously with administration of the NK cells. [0063] In some embodiments, the antibody is administered before the NK cells. In some embodiments, the antibody is administered after the NK cells. [0064] In some embodiments, the NK cells are administered or are administered at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes after completing administration of the antibody. In some embodiments, the NK cells are administered within 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes after completing administration of the antibody. [0065] In some embodiments, the antibody is administered preferably at least 1 hour prior to administration of NK cells. [0066] In some embodiments, the antibody is administered at from 100 to 1,000 mg/m
2, e.g., from 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300,
Docket No.49755-0045WO1 100 to 200, 200 to 1,000, 200 to 900, 200 to 800, 200 to 700, 200 to 600, 200 to 500, 200 to 400, 200 to 300, 300 to 1,000, 300 to 900, 300 to 800, 300 to 700, 300 to 600, 300 to 500, 300 to 400, 400 to 1,000, 400 to 900, 400 to 800, 400 to 700, 400 to 600, 400 to 500, 500 to 1,000, 500 to 900, 500 to 800, 500 to 900, 500 to 800, 500 to 700, 500 to 600, 600 to 1,000, 600 to 900, 600 to 800, 600 to 700, 700 to 1,000, 700 to 900, 700 to 800, 800 to 1,000, 800 to 900, or 900 to 1,000 mg/m
2. [0067] Rituximab is preferably administered at 375 mg/m
2 or 500 mg/m
2, preferably at least 1 hour prior to each administration of NK cells. [0068] In some embodiments, the antibody is administered intravenously. In some embodiments, the antibody is administered intravenously via syringe or via gravity IV infusion. 5. Cytokines [0069] In some embodiments, a cytokine is administered to the patient. [0070] In some embodiments, the cytokine is administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the cytokine is administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition. [0071] In some embodiments, the cytokine is IL-2. Some tumor microenvironments can become deprived of certain cytokines, including IL-2. In such cases, it can be advantageous to administer a cytokine, such as IL-2, to the patient as part of a treatment regimen involving NK cells. In some cases, the presence of the cytokine, such as IL-2, at the tumor site can increase, enhance, or support the cytotoxicity of the NK cells. In some cases, the cytokine, such as IL-2, can enhance the survival, persistence, or expansion of the NK cells in the patient’s body. [0072] In some embodiments, the IL-2 is administered subcutaneously. [0073] In some embodiments, the IL-2 is administered from between 1 to 4 or about 1 to about 4 hours following the conclusion of NK cell administration. In some embodiments, the IL-2 is administered at least 1 hour following the conclusion of NK cell administration. In some embodiments, the IL-2 is administered no more than 4 hours following the conclusion of NK cell administration. In some embodiments, the IL-2 is administered at least 1 hour after and no more than 4 hours following the conclusion of NK cell administration. [0074] In some embodiments, IL-2 is administered at each or a subset of NK cell administrations within a treatment cycle. In some embodiments, IL-2 is administered at each
Docket No.49755-0045WO1 or a subset of NK cell administrations for one or more treatment cycles, followed by one or more treatment cycles without IL-2 administration. [0075] In some embodiments, the IL-2 is administered at up to 10 million IU/M
2, e.g., up to 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, or 10 million IU/m
2. [0076] In some embodiments, the IL-2 is administered at or at about 0.5 million, 1 million, at or at about 2 million, at or at about 3 million, at or at about 4 million, at or at about 5 million, at or at about 6 million, at or at about 7 million, at or at about 8 million, at or at about 9 million, at or at about 10 million IU/m
2 [0077] In some embodiments, the IL-2 is administered at or at about 1 x 10
6 IU/M
2. In some embodiments, the IL-2 is administered at or at about 2 x 10
6 IU/m
2. In some embodiments, the IL-2 is administered at or at about 6 x 10
6 IU/m
2. [0078] In some embodiments, less than 1 x 10
6 IU/m
2 IL-2 is administered to the patient. [0079] In some embodiments, a flat dose of IL-2 is administered to the patient. In some embodiments, a flat dose of 1 million IU or about 1 million IU is administered to the patient. In some embodiments, a flat dose of 2 million IU or about 2 million IU is administered to the patient. In some embodiments, a flat dose of 3 million IU or about 3 million IU is administered to the patient. In some embodiments, a flat dose of 4 million IU or about 4 million IU is administered to the patient. In some embodiments, a flat dose of 5 million IU or about 5 million IU is administered to the patient. In some embodiments, a flat dose of 6 million IU or about 6 million IU is administered to the patient. In some embodiments, a flat dose of 7 million IU or about 7 million IU is administered to the patient. In some embodiments, a flat dose of 8 million IU or about 8 million IU is administered to the patient. In some embodiments, a flat dose of 9 million IU or about 9 million IU is administered to the patient. [0080] In some embodiments, IL-2 is not administered to the patient. D. Dosing [0081] An “effective amount” is an amount sufficient to effect beneficial or desired results. For example, a therapeutic amount is one that achieves the desired therapeutic effect. This amount can be the same or different from a prophylactically effective amount, which is an amount necessary to prevent onset of disease or disease symptoms. An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage)
Docket No.49755-0045WO1 depends on the therapeutic compounds selected. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. [0082] Dosage, toxicity and therapeutic efficacy of the therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. [0083] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography, flow cytometry, or a molecular assay. E. Combination Therapies [0084] In some embodiments, the method comprises administering the NK cells described herein and a CD20 targeted antibody in combination with another therapy, e.g., an
Docket No.49755-0045WO1 additional antibody, an NK cell engager, an antibody drug conjugate (ADC), a chemotherapy drug, e.g., a small molecule drug, an immune checkpoint inhibitor, and combinations thereof. 1. Small Molecule / Chemotherapy Drugs [0085] In some embodiments, the additional therapy is a small molecule drug. In some embodiments, the additional therapy is a chemotherapy drug. In some embodiments, the additional therapy is a small molecule chemotherapy drug. Such small molecule drugs can include existing standard-of-care treatment regimens to which adoptive NK cell therapy is added. In some cases, the use of the NK cells described herein can enhance the effects of small molecule drugs, including by enhancing the efficacy, reducing the amount of small molecule drug necessary to achieve a desired effect, or reducing the toxicity of the small molecule drug. [0086] In some embodiments, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-acetyloxy- 1,9,12-trihydroxy-15-[(2R,3S)-2-hydroxy-3-[(2-methylpropan-2-yl)oxycarbonylamino]-3- phenylpropanoyl]oxy-10,14,17,17-tetramethyl-11-oxo-6- oxatetracyclo[11.3.1.0
3,10.0
4,7]heptadec-13-en-2-yl] benzoate (docetaxel) or a pharmaceutically acceptable salt thereof. [0087] In some embodiments, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4,12- diacetyloxy-15-[(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoyl]oxy-1,9-dihydroxy- 10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0
3,10.0
4,7]heptadec-13-en-2-yl] benzoate (paclitaxel) or a pharmaceutically acceptable salt thereof. [0088] In some embodiments, the drug is 6-N-(4,4-dimethyl-5H-1,3-oxazol-2-yl)-4-N-[3- methyl-4-([1,2,4]triazolo[1,5-a]pyridin-7-yloxy)phenyl]quinazoline-4,6-diamine (tucatinib) or a pharmaceutically acceptable salt thereof. [0089] In some embodiments, the drug is pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5- methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) or a pharmaceutically acceptable salt thereof. [0090] In some embodiments, the drug is azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) (carboplatin) or a pharmaceutically acceptable salt thereof. [0091] In some embodiments, the drug is methyl (1R,9R,10S,11R,12R,19R)-11- acetyloxy-12-ethyl-4-[(12S,14R)-16-ethyl-12-methoxycarbonyl-1,10- diazatetracyclo[12.3.1.0
3,11.0
4,9]octadeca-3(11),4,6,8,15-pentaen-12-yl]-10-hydroxy-5- methoxy-8-methyl-8,16-diazapentacyclo[10.6.1.0
1,9.0
2,7.0
16,19]nonadeca-2,4,6,13-tetraene-10- carboxylate (vinorelbine) or a pharmaceutically acceptable salt thereof.
Docket No.49755-0045WO1 [0092] In some embodiments, the drug is N-[3-chloro-4-[(3- fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]furan-2- yl]quinazolin-4-amine (lapatinib) or a pharmaceutically acceptable salt thereof. [0093] In some embodiments, the drug is (E)-N-[4-[3-chloro-4-(pyridin-2- ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide (neratinib) or a pharmaceutically acceptable salt thereof. [0094] In some embodiments, the drug is 6-acetyl-8-cyclopentyl-5-methyl-2-[(5- piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one (palbociclib) or a pharmaceutically acceptable salt thereof. [0095] In some embodiments, the drug is 7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1- ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide (ribociclib) or a pharmaceutically acceptable salt thereof. [0096] In some embodiments, the drug is N-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2- yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine (abemaciclib) or a pharmaceutically acceptable salt thereof. [0097] In some embodiments, the drug is (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)- 1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy- 15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0
4,9]hexatriaconta- 16,24,26,28-tetraene-2,3,10,14,20-pentone (everolimus) or a pharmaceutically acceptable salt thereof. [0098] In some embodiments, the drug is (2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2- methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide (alpelisib) or a pharmaceutically acceptable salt thereof. [0099] In some embodiments, the drug is 4-[[3-[4-(cyclopropanecarbonyl)piperazine-1- carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one (olaparib) or a pharmaceutically acceptable salt thereof. [0100] In some embodiments, the drug is (11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2- methyl-1,2,4-triazol-3-yl)-2,3,10-triazatricyclo[7.3.1.0
5,13]trideca-1,5(13),6,8-tetraen-4-one (talazoparib) or a pharmaceutically acceptable salt thereof. [0101] In some embodiments, the drug is N-[2-[2-(dimethylamino)ethyl-methylamino]-4- methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamid (osimertinib) or a pharmaceutically acceptable salt thereof.
Docket No.49755-0045WO1 [0102] In some embodiments, the drug is N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3- morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib) or a pharmaceutically acceptable salt thereof. [0103] In some embodiments, the drug is N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)quinazolin-4-amine (erlotinib) or a pharmaceutically acceptable salt thereof. [0104] In some embodiments, the drug is (E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)- oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide (afatinib) or a pharmaceutically acceptable salt thereof. [0105] In some embodiments, the drug is azane;dichloroplatinum (cisplatin, platinol) or a pharmaceutically acceptable salt thereof. [0106] In some embodiments, the drug is azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) (carboplatin) or a pharmaceutically acceptable salt thereof [0107] In some embodiments, the drug is 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy- 5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one (gemcitabine) or a pharmaceutically acceptable salt thereof. [0108] In some embodiments, the drug is (2S)-2-[[4-[2-(2-amino-4-oxo-3,7- dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid (pemetrexed) or a pharmaceutically acceptable salt thereof. [0109] In some embodiments, the drug is N,N-bis(2-chloroethyl)-2-oxo-1,3,2λ
5- oxazaphosphinan-2-amine (cyclophosphamide) or a pharmaceutically acceptable salt thereof. [0110] In some embodiments, the drug is (2R,3S,4S,5R)-2-(6-amino-2-fluoropurin-9-yl)- 5-(hydroxymethyl)oxolane-3,4-diol (fludarabine) or a pharmaceutically acceptable salt thereof. [0111] In some embodiments, the drug is (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6- methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H- tetracene-5,12-dione (doxorubicin) or a pharmaceutically acceptable salt thereof. [0112] In some embodiments, the drug is methyl (1R,9R,10S,11R,12R,19R)-11- acetyloxy-12-ethyl-4-[(13S,15S,17S)-17-ethyl-17-hydroxy-13-methoxycarbonyl-1,11- diazatetracyclo[13.3.1.0
4,12.0
5,10]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5- methoxy-8,16-diazapentacyclo[10.6.1.0
1,9.0
2,7.0
16,19]nonadeca-2,4,6,13-tetraene-10- carboxylate (vincristine) or a pharmaceutically acceptable salt thereof. [0113] In some embodiments, the drug is (8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2- hydroxyacetyl)-10,13-dimethyl-6,7,8,9,12,14,15,16-octahydrocyclopenta[a]phenanthrene- 3,11-dione (prednisone) or a pharmaceutically acceptable salt thereof.
Docket No.49755-0045WO1 [0114] In some embodiments, the drug is N,3-bis(2-chloroethyl)-2-oxo-1,3,2λ
5- oxazaphosphinan-2-amine (ifosfamide) or a pharmaceutically acceptable salt thereof. [0115] In some embodiments, the drug is (5S,5aR,8aR,9R)-5-[[(2R,4aR,6R,7R,8R,8aS)- 7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4- hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[6,5- f][1,3]benzodioxol-8-one (etopside) or a pharmaceutically acceptable salt thereof. [0116] In some embodiments, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro- 11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16- octahydrocyclopenta[a]phenanthren-3-one (dexamethasone) or a pharmaceutically acceptable salt thereof. [0117] In some embodiments, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro- 11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16- octahydrocyclopenta[a]phenanthren-3-one (cytarabine) or a pharmaceutically acceptable salt thereof. 2. NK Cell Engagers [0118] In some embodiments, the additional therapy is an NK cell engager, e.g., a bispecific or trispecific antibody. [0119] In some embodiments, the NK cell engager is a bispecific antibody against CD16 and a disease-associated antigen, e.g., cancer-associated antigen, e.g., an antigen of cancers described herein. In some embodiments, the NK cell engager is a trispecific antibody against CD16 and two disease-associated antigens, e.g., cancer-associated antigens, e.g., antigens of cancers described herein. [0120] In some embodiments, the NK cells, e.g., the NK cells described herein, e.g., AB- 101 cells, are administered in combination with a CD20 targeting antibody as well as a therapy selected from the group consisting of cyclophosphamide, doxorubicin, vincristine, prednisone, dexamethasone, cytarabine, e.g., high-dose Ara-C cytarabine, platinol, and combinations thereof (e.g., R-CHOP, ICE, or DHAP). 3. Checkpoint Inhibitors [0121] In some embodiments, the additional therapy is an immune checkpoint inhibitor. [0122] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and combinations thereof.
Docket No.49755-0045WO1 [0123] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a VISTA inhibitor, a BTLA inhibitor, a TIM-3 inhibitor, a KIR inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD-96 inhibitor, a SIRPα inhibitor, and combinations thereof. [0124] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a LAG-3 (CD223) inhibitor, a TIM-3 inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, an A2aR inhibitor, a CD73 inhibitor, a NKG2A inhibitor, a PVRIG/PVRL2 inhibitor, a CEACAM1 inhibitor, a CEACAM 5 inhibitor, a CEACAM 6 inhibitor, a FAK inhibitor, a CCL2 inhibitor, a CCR2 inhibitor, a LIF inhibitor, a CD47 inhibitor, a SIRPα inhibitor, a CSF-1 inhibitor, an M-CSF inhibitor, a CSF-1R inhibitor, an IL-1 inhibitor, an IL-1R3 inhibitor, an IL-RAP inhibitor, an IL-8 inhibitor, a SEMA4D inhibitor, an Ang-2 inhibitor, a CELVER-1 inhibitor, an Axl inhibitor, a phsphatidylserine inhibitor, and combinations thereof. [0125] In some embodiments, the immune checkpoint inhibitor is selected from those shown in Table 10, or combinations thereof. Table 10. Exemplary Immune Checkpoint Inhibitors Target Inhibitor L
AG525 (IMP701), REGN3767 (R3767), BI 754,091, tebotelimab
Docket No.49755-0045WO1 [0126] In some embodiments, the immune checkpoint inhibitor is an antibody. [0127] In some embodiments, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, toripalimab, cemiplimab-rwlc, sintilimab, and combinations thereof. [0128] In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, durvalumab, avelumab, and combinations thereof. [0129] In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, the PD-1 inhibitor is selected from the group of inhibitors shown in Table 11. Table 11. Exemplary PD-1 Inhibitor Antibodies Name Internal Name Antigen Company nivolumab Opdivo, ONO-4538, PD-1 BMS, Medarex, Ono n
Docket No.49755-0045WO1 Name Internal Name Antigen Company F520 PD-1 Shandong New Time e
Docket No.49755-0045WO1 Name Internal Name Antigen Company camrelizumab SHR-1210 PD-1 Incyte, Jiangsu u
, ors shown in Table 12. Table 12. Exemplary PD-L1 Inhibitor Antibodies Name Internal Name Antigen Company I
mfinzi, MEDI-4736, AstraZeneca, Celgene, Me i
Docket No.49755-0045WO1 Name Internal Name Antigen Company IBI322 IBI-322 CD47, PD-L1 Innovent a
i
[0131] In some embodiments, the CTLA-4 inhibitor is selected from the group of inhibitors shown in Table 13. Exemplary CTLA4 Inhibitor Antibodies Name Internal Name Antigen Company
Docket No.49755-0045WO1 Name Internal Name Antigen Company vudalimab XmAb20717 CTLA4, PD-1 Xencor n i
[0132] In some embodiments, the immune checkpoint inhibitor is a small molecule drug. Small molecule checkpoint inhibitors are described, e.g., in WO2015/034820A1, WO2015/160641A2, WO2018/009505 A1, WO2017/066227 A1, WO2018/044963 A1, WO2018/026971 A1, WO2018/045142 A1, WO2018/005374 A1, WO2017/202275 A1, WO2017/202273 A1, WO2017/202276 A1, WO2018/006795 A1, WO2016/142852 A1, WO2016/142894 A1, WO2015/033301 A1, WO2015/033299 A1, WO2016/142886 A2, WO2016/142833 A1, WO2018/051255 A1, WO2018/051254 A1, WO2017/205464 A1, US2017/0107216 A1, WO2017/070089A1, WO2017/106634A1, US2017/0174679 A1, US2018/0057486 A1, WO2018/013789 A1, US2017/0362253 A1, WO2017/192961 A1, WO2017/118762 A1, US2014/199334 A1, WO2015/036927 A1, US2014/0294898 A1, US2016/0340391 A1, WO2016/039749 A1, WO2017/176608 A1, WO2016/077518 A1, WO2016/100608 A1, US2017/0252432 A1, WO2016/126646 A1, WO2015/044900 A1,
Docket No.49755-0045WO1 US2015/0125491 A1, WO2015/033303 A1, WO2016/142835 A1, WO2019/008154 A1, WO2019/008152 A1, and WO2019023575A1. [0133] In some embodiments, the PD-1 inhibitor is 2-[[4-amino-1-[5-(1-amino-2- hydroxypropyl)-1,3,4-oxadiazol-2-yl]-4-oxobutyl]carbamoylamino]-3-hydroxypropanoic acid (CA-170). [0134] In some embodiments, the immune checkpoint inhibitor is (S)-1-(3-Bromo-4-((2- bromo-[1,1′-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic Acid. [0135] In some embodiments, the immune checkpoint inhibitor is a peptide. See, e.g., Sasikumar et al., “Peptide and Peptide-Inspired Checkpoint Inhibitors: Protein Fragments to Cancer Immunotherapy,” Medicine in Drug Discovery 8:100073 (2020). V. VARIANTS [0136] In some embodiments, the nucleotide or amino acid sequences described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the nucleotide or amino acid sequence of an exemplary sequence (e.g., as provided herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent. [0137] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid "identity" is equivalent to nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. [0138] Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various
Docket No.49755-0045WO1 ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); "BestFit" (Smith and Waterman, Advances in Applied Mathematics, 482- 489 (1981)) as incorporated into GeneMatcher PlusTM, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403- 10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for target proteins or nucleic acids, the length of comparison can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence. [0139] For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. [0140] Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. VI. DEFINITIONS [0141] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [0142] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically
Docket No.49755-0045WO1 disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [0143] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof. [0144] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context. [0145] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. [0146] The term “in vivo” is used to describe an event that takes place in a subject’s body. [0147] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay. [0148] The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed. [0149] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value. [0150] As used herein, the term "buffer solution" refers to an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa.
Docket No.49755-0045WO1 [0151] As used herein, the term "cell culture medium" refers to a mixture for growth and proliferation of cells in vitro, which contains essential elements for growth and proliferation of cells such as sugars, amino acids, various nutrients, inorganic substances, etc. [0152] A buffer solution, as used herein, is not a cell culture medium. [0153] As used herein, the term “bioreactor” refers to a culture apparatus capable of continuously controlling a series of conditions that affect cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH, and temperature. [0154] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Some vectors are suitable for delivering the nucleic acid molecule(s) or polynucleotide(s) of the present application. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as expression vectors. [0155] The term “operably linked” refers to two or more nucleic acid sequence or polypeptide elements that are usually physically linked and are in a functional relationship with each other. For instance, a promoter is operably linked to a coding sequence if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case, the coding sequence should be understood as being “under the control of” the promoter. [0156] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “engineered cells,” “transformants,” and “transformed cells,” which include the primary engineered (e.g., transformed) cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. [0157] As appropriate, the host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein, as described herein. [0158] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Docket No.49755-0045WO1 VII. EXAMPLES [0159] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1: AB-101 [0160] AB-101 is a universal, off-the-shelf, cryopreserved allogeneic cord blood derived NK cell therapy product comprising ex vivo expanded and activated effector cells designed to enhance ADCC anti-tumor responses in patients, e.g., patients treated with monoclonal antibodies or NK cell engagers. AB-101 was prepared as described, for example, in WO2022/133056. Example 2: Lympho-Recovery with Cy/Flu [0161] FIG.1 shows lympho-recovery kinetics post cyclophosphamide/fludarabine. Absolute count of total T (a) and NK (b) cells in peripheral blood of NHL patients pre (C1 pre-LD) and post lymphodepletion assessed by flow cytometry. All blood samples were collected prior to any drug administration. Patients were given either cyclophosphamide 250 mg/m2/day (black circle) or 500 mg/m2/day (blue square) with fludarabine 30 mg/m2/day for three consecutive days. Lymphodepletion chemotherapy (cyclophosphamide/fludarabine) decreased the total number of immune cells in the peripheral blood of NHL patients up to day 3, after which host immune cells gradually started to reconstitute. There was no significant difference in the depth or reconstitution kinetics between cyclophosphamide 250 vs.500 cohort. Example 3: AB-101 in combination with Bendamustine and Rituximab (BR) [0162] An example of a patient dosing schedule of NK cells (here, AB-101), in combination with bendamustine and rituximab, e.g., for patients with NHL, e.g., R/R NHL, is shown in FIG.2. OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference CHO-H01 CD20 Cho Pharma Trial ID: NCT03221348 1 7 9 1 9- 7 st 9 5 5 k 5 6 8 2 7
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference PF-05280586 Rituximab-Pfizer CD20 Pfizer Sharman et al., 3 8 6 5 . 9 3 9 1
consisting of rituximab (or a biosimilar thereof), obinutuzumab (or a biosimilar thereof), ofatumumab (or a biosimilar thereof), ocrelizumab (or a biosimilar thereof), ibritumomab (or a biosimilar thereof), veltuzumab (or a biosimilar thereof), tositumomab (or a biosimilar thereof), ublituximab (or a biosimilar thereof), and combinations thereof. [0095] In some embodiments, the CD20 targeting antibody is selected rituximab or a biosimilar thereof. In some embodiments, the CD20 targeting antibody is rituximab. [0096] In some embodiments, the antibody is a CD19 targeting antibody. In some embodiments, the CD19 targeting antibody is selected from the group consisting of blinatumomab (or a biosimilar thereof), tafasitamab (or a biosimilar thereof), inebilizumab (or a biosimilar thereof), loncastuximab tesirine (or a biosimilar thereof), and combinations thereof. [0097] CD38 targeting antibodies suitable for use in the methods described herein include, but are not limited to, those in Table 2, and combinations thereof.
Docket No.49755-0045WO1 Table 2. CD38 targeting antibodies Name Internal Name Antig Company Reference en daratumumab Darzalex CD38 Genmab Jansse Usmani et al Blood 2019 J. l.
consisting of daratumumab (or a biosimilar thereof), isatuximab (or a biosimilar thereof), and combinations thereof. [0099] In some embodiments, the HER2 targeting antibody is an EGFR targeting antibody selected from Table 3, or a combination thereof.
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference XMT-1522 HT-19 HER2/n Adimab, Mer Trial ID: T k d NCT02952729
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference NM-02 HER2/n Suzhou Trial ID: N b NCT04674722 et t 5
Docket No.49755-0045WO1 Name Internal Name Antigen Company Reference Hong et al., Cancer HER2/ Bi Th C L d - t n
consisting of trastuzumab (or a biosimilar thereof), margetuximab (or a biosimilar thereof), pertuzumab (or a biosimilar thereof), trastuzumab emtansine (or a biosimilar thereof), PF- 05280014 (or a biosimilar thereof), trastuzumab-anns (or a biosimilar thereof), HLX02 (or a biosimilar thereof), trastuzumab-dkst (or a biosimilar thereof), Hervycta (or a biosimilar thereof), and combinations thereof. [0101] In some embodiments, the HER2 targeting antibody is trastuzumab or a biosimilar thereof. In some embodiments, the HER2 targeting antibody is trastuzumab. [0102] In some embodiments, the EGFR targeting antibody is an EGFR targeting antibody selected from Table 4, or a combination thereof. Table 4. Exemplary EGFR Targeting Antibodies Name Internal Name Antigen(s) Company Reference l., 0 1- l., ol -
Docket No.49755-0045WO1 Name Internal Name Antigen(s) Company Reference Diaz-Serrano et Prtrzz IMC- l Ft r Onol. - . 1- ., 9 - , . 4 r. ): ol 4- 8 -
Docket No.49755-0045WO1 Name Internal Name Antigen(s) Company Reference U.Penn. patent WO2014130657 nti-EGFRIII EGFR UP nn A1 - - 3 7 -
Docket No.49755-0045WO1 Name Internal Name Antigen(s) Company Reference duligotumab, Hill et al., Clin RG7597 Anti- Cn r R 2018 - 9 7- 3 ., 5 4 - t 7 -
Docket No.49755-0045WO1 Table 7. Exemplary Cryopreservation Composition #2 Exemplary v/v% Final Concentration in Excipient in 0;
Exemplary v/v% Final Concentration in 0;
Docket No.49755-0045WO1 [0126] In some embodiments, the immune checkpoint inhibitor is an antibody. [0127] In some embodiments, the PD-1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, toripalimab, cemiplimab-rwlc, sintilimab, and combinations thereof. [0128] In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, durvalumab, avelumab, and combinations thereof. [0129] In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some embodiments, the PD-1 inhibitor is selected from the group of inhibitors shown in Table 11. Table 11. Exemplary PD-1 Inhibitor Antibodies Name Internal Name Antigen Company nivolumab Opdivo, ONO-4538, PD-1 BMS, Medarex, Ono n
Docket No.49755-0045WO1 Name Internal Name Antigen Company F520 PD-1 Shandong New Time e
Docket No.49755-0045WO1 Name Internal Name Antigen Company camrelizumab SHR-1210 PD-1 Incyte, Jiangsu u
, ors shown in Table 12. Table 12. Exemplary PD-L1 Inhibitor Antibodies Name Internal Name Antigen Company Imfinzi, MEDI-4736, AstraZeneca, Celgene, Me i
Docket No.49755-0045WO1 Name Internal Name Antigen Company IBI322 IBI-322 CD47, PD-L1 Innovent ai
[0131] In some embodiments, the CTLA-4 inhibitor is selected from the group of inhibitors shown in Table 13. Exemplary CTLA4 Inhibitor Antibodies Name Internal Name Antigen Company
Docket No.49755-0045WO1 Name Internal Name Antigen Company vudalimab XmAb20717 CTLA4, PD-1 Xencor n i