Modified NK-92 haNK003 cells for clinical use

Abstract

We provide medicines to treat cancer. [Solution] A pharmaceutical product comprising a population of modified NK-92 cells, wherein the modified NK-92 cells contain CD16 and IL-2, contain a specific sequence on chromosome 17, and more than 90% of the cells in the population express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the population express CD3.

Description

Background Art

[0001] Background Cancer treatment using monoclonal antibodies (mAbs) has significantly improved the clinical outcomes in patients with cancer. One of the major mechanisms of action of therapeutic antibodies is through antibody-dependent cell cytotoxicity (ADCC). Natural killer cells are the major effector cells of ADCC and can be used as cytotoxic effector cells for cell-based immunotherapy.

[0002] NK-92 is a cytolytic cancer cell line that was discovered in the blood of subjects with non-Hodgkin lymphoma and subsequently immortalized ex vivo. NK-92 cells are derived from NK cells, retain the majority of activating receptors, but lack the major inhibitory receptors presented by normal NK cells. However, NK-92 cells do not attack normal cells and do not induce unwanted immune rejection responses in humans. The characterization of the NK-92 cell line is disclosed in International Publication No. WO 1998 / 49268 (Patent Document 1) and US Patent Application Publication No. US 2002-0068044 (Patent Document 2). NK-92 cells have also been evaluated as potential therapeutic agents in the treatment of certain cancers.

[0003] NK-92 cells retain almost all of the activating receptors and cell lysis pathways associated with NK cells, but do not express CD16 on their cell surface. CD16 is an Fc receptor that recognizes and binds to the Fc portion of antibodies, activating NK cells for the ADCC effector mechanism. Unmodified NK-92 cells lack the CD16 receptor and thus cannot lyse target cells via the ADCC mechanism.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

[0005] overview This specification provides a population of modified NK-92 cells, compositions and kits containing the cells, and methods for preparing and using the population of the cells. [Invention 1001] A population of modified NK-92 cells exhibiting antibody-dependent cell-mediated cytotoxicity (ADCC), containing heterogeneous nucleic acid molecules including both CD16 (SEQ ID NO:3) and IL-2 (SEQ ID NO:5), More than 90% of the cells in the population express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the population express CD3. A population of modified NK-92 cells. [Invention 1002] A cell according to the present invention 1001, wherein the nucleic acid molecule is a DNA molecule. [Invention 1003] A cell according to the present invention 1001, wherein the nucleic acid molecule contains, from 5' to 3', a sequence encoding CD16, an IRES sequence, and a sequence encoding IL-2. [Invention 1004] A cell according to any of the inventions 1001 to 1003, wherein the cell contains SEQ ID NO:1 on chromosome 17. [Invention 1005] A cell according to any of the present invention 1001 to 1004, wherein the average doubling time of the cell is 55 to 70 hours. [Invention 1006] Cells according to any of the invention 1001 to 1005, wherein the cell population maintains an average doubling time of 1 to 2, 3, 4, 5, 10, 15, 20, or 25 days. [Invention 1007] A cell according to any of the invention 1001 to 1006, wherein the cell population can be passaged every 1, 2, 3, or 4 days. [Invention 1008] A cell according to any of the invention's 1001-1007, wherein the cell secretes IL-2 at a concentration of 10-60 pg / hour per 1 million cells. [Invention 1009] The cells are irradiated cells, and are any of the cells described in items 1001 to 1007 of this invention. [Invention 1010] Cells according to any of the invention 1001 to 1009, wherein the cells exhibit reduced downregulation of CD16 expression compared to a control. [Invention 1011] Cells according to any of the invention 1001-1009, wherein the cells maintain higher levels of CD16 after ADCC compared to the control. [Invention 1012] A kit comprising a population of any of the cells described in invention 1001 to 1011. [Invention 1013] A kit according to the present invention 1012, further containing antibodies. [Invention 1014] A pharmaceutical composition comprising a population of any of the cells described in invention 1001 to 1011, and a pharmaceutically acceptable excipient. [Invention 1015] A method for treating cancer in a subject, comprising the step of administering the pharmaceutical composition of the present invention 1014 to the subject. [Brief explanation of the drawing]

[0006] [Figure 1] Figure 1A is a graph showing the expansion of haNK003 cells. Figure 1B is a graph showing the population doubling level (PDL) of haNK003 cells. Survival rate (%), cell density (cells / mL), and cumulative PDL were monitored during the expansion period. [Figure 2-1] Representative histograms of surface marker expression in aNK cells and haNK003 cells are shown. [Figure 2-2] Representative histograms of surface marker expression in aNK cells and haNK003 cells are shown. [Figure 3A] This graph shows the spontaneous cytotoxicity of haNK003 cells against K562 cells. [Figure 3B] This graph shows the spontaneous cytotoxicity of haNK003 cells against Raji cells. [Figure 3C] This graph shows the spontaneous cytotoxicity of haNK003 cells against SKOV3 cells. [Figure 3D] This graph shows the spontaneous cytotoxicity of haNK003 cells against SKBR3 cells. [Figure 4] Figure 4A is a graph showing ADCC of haNK003 cells against Raji cells. Figure 4B is a graph showing ADCC of haNK003 cells against SKOV3 cells. Figure 4C is a graph showing ADCC of haNK003 cells against SKBR3 cells. [Figure 5] This graph shows the innate cytotoxic activity of irradiated haNK003 cells versus unirradiated haNK003 cells against K562 cells. haNK003 cells were either sham-irradiated (solid line) or irradiated with 10 Gy. The cytotoxic activity of irradiated haNK003 against K562 cells was assayed at 6 hours (dashed line) or 24 hours (dotted line) post-irradiation. [Figure 6] This graph shows the innate cytotoxic activity of irradiated haNK003 cells versus unirradiated haNK003 cells against DOHH2 cells. haNK003 cells were either sham-irradiated (solid line) or irradiated with 10 Gy. The cytotoxic activity of irradiated haNK003 against DOHH2 cells was assayed at 6 hours (dashed line) or 24 hours (dotted line) post-irradiation. Note that due to insufficient haNK003 cell counts resulting from cell death, data points for the 20:1 E:T ratio were not obtained for irradiated cells. [Figure 7]This graph shows the ADCC activity of irradiated haNK003 cells versus unirradiated haNK003 cells against DOHH2 cells. haNK003 cells were either sham-irradiated (black symbols) or irradiated with 10 Gy (white symbols). The ADCC activity of irradiated and unirradiated haNK003 cells against DOHH2 cells was assayed at 6 hours (dashed line) or 24 hours (dotted line) post-irradiation, combined with Rituxan (square) or Herceptin (triangle), which does not react with DOHH2 cells. Note that due to insufficient haNK003 cell numbers resulting from cell death, data points for the 20:1 E:T ratio were not obtained for irradiated cells at 24 hours. [Figure 8A] This graph shows the amount of IL-2 (pg / mL) released per 1 x 10⁶ cells in irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by sandwich ELISA experiment number 1. [Figure 8B] This graph shows the amount of IL-2 (pg / mL) released per 1 x 10⁶ cells in irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by sandwich ELISA experiment number 2. [Figure 8C] This graph shows the amount of IL-2 (pg / mL) released per 1 x 10⁶ cells in irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by experiment number 1 of the multiplex ELISA. [Figure 8D] This graph shows the amount of IL-2 (pg / mL) released per 1 x 10⁶ cells in irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by experiment number 2 of the multiplex ELISA. [Figure 9A]This graph shows the total intracellular IL-2 content (pg) per cell (1 x 10⁶) of irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by sandwich ELISA experiment number 1. [Figure 9B] This graph shows the total intracellular IL-2 content (pg) per cell (1 x 10⁶) of irradiated versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by sandwich ELISA experiment number 2. [Figure 9C] This graph shows the total intracellular IL-2 content (pg) per cell (1 x 10⁶) of irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by experiment number 1 of the multiplex ELISA. [Figure 9D] This graph shows the total intracellular IL-2 content (pg) per cell (1 x 10⁶) of irradiated cells versus unirradiated cells at 6, 12, 24, and 48 hours (h), as determined by experiment number 2 of the multiplex ELISA. [Figure 10A] This graph shows the amount of soluble IL-2(pg) per 1 x 10⁶ cells in experiment number 1, as determined by multiplex ELISA and sandwich ELISA. [Figure 10B] This graph shows the amount of soluble IL-2(pg) per 1 x 10⁶ cells in experiment number 2, as determined by multiplex ELISA and sandwich ELISA. [Figure 11] This graph shows the effect of intravenously administered haNK003 on animal body weight in NOD / SCID mice. NOD / SCID mice (3 males and 3 females per group) were treated with a single intravenous infusion of either PBS or unirradiated or irradiated haNK003 cells at a dose of 1 x 10⁷ cells, respectively. Animal body weight was monitored twice weekly for 5 weeks. Values ​​are mean ± SEM, and n = 6. [Figure 12]This graph shows the effect of haNK003 cells on animal body weight in NOD / SCID mice. NOD / SCID mice (3 males and 3 females per group) were treated with PBS or 1 x 10⁷ unirradiated or irradiated haNK003 cells once a week for 4 weeks, and animal body weight was monitored twice a week for 5 weeks. Values ​​are mean ± SEM, and n = 6. [Figure 13] This graph shows a comparison of spontaneous cytotoxicity between aNK cells and haNK003 cells in relation to K562 cells. [Figure 14] This graph shows a comparison of innate cytotoxicity between NK cells and haNK003 cells against Daudi cells. [Figure 15] This graph shows a comparison of spontaneous cell damage between aNK cells and haNK003 cells against DOHH2 cells. [Figure 16] This graph shows a comparison of spontaneous cytotoxicity between aNK cells and haNK003 cells against SKOV-3 cells. [Figure 17] This graph shows a comparison of spontaneous cytotoxicity between aNK cells and haNK003 cells in relation to HL-60 cells. [Figure 18] This graph shows a comparison of spontaneous cell damage to SR-91 cells between aNK cells and haNK003 cells. [Figure 19] This graph shows the antitumor activity of haNK003 cells in a subcutaneous xenograft model of MDA-MB-453 in female NSG mice. Female NSG mice carrying MDA-MB-453 human breast cancer tumors were treated twice weekly for 4 weeks with intravenous infusion of irradiated haNK003 cells at doses of PBS or 2.5 x 10⁶ cells or 1 x 10⁷ cells, respectively. Tumor volume was monitored twice weekly. Values ​​are mean ± SEM; n = 8. [Figure 20]This graph shows the effect of haNK003 cells on animal body weight in female NSG mice. Female NSG mice with MDA-MB-453 human breast cancer tumors were treated twice a week for 4 weeks by intravenous infusion of irradiated haNK003 cells at doses of PBS or 2.5 x 10⁶ cells or 1.0 x 10⁷ cells, respectively. Mouse body weight was monitored twice a week. Values ​​are mean ± SEM; n = 4. [Figure 21] This table shows the distance between sample pairs for gene expression in haNK cells, as well as in normal NK cells 950, 962, and 996, under 20% oxygen and 0% oxygen (hypoxic) conditions. [Figure 22] This table shows the genes exhibiting the greatest variability in expression between 20% oxygen and 0% oxygen conditions in 950, 962, 996, and haNK cells. [Figure 23] This table shows the changes in gene expression for the genes that show the greatest change between 20% oxygen and 0% oxygen conditions in 950, 962, 996, and haNK cells. [Figure 24] This table shows the changes in the expression of hypoxia-related genes between 20% oxygen conditions and 0% oxygen conditions in haNK cells, as well as NK cells 950, 962, and 996. [Figure 25] This graph shows flow cytometry analysis of CD16 expression in haNK003 cells and donor NK cells before and after PMA treatment. Downregulation of CD16 expression was 94.36% ± 3 in donor NK cells and 30% ± 0.04 in haNK003 cells. aNK (NK-92 cells lacking CD16) was used as a control. [Figure 26]This graph shows flow cytometry analysis of CD16 expression levels in haNK003 cells and donor NK cells (E:T = 1:1) co-cultured with K562 cells. After 4 hours, haNK003 cells showed stable CD16 expression compared to donor NK cells. CD16 downregulation in donor NK cells after 4 hours of co-culture with K562 was 60.25% ± 0.9, while in haNK003 cells it was 4.9% ± 2.57. After overnight recovery, donor NK cells still showed downregulation of CD16 expression at 57.54% ± 2.82, while in haNK003 cells, CD16 levels recovered to near normal levels with only a downregulation of 2.78% ± 3.5. aNK (NK-92 cells lacking CD16) was used as a control. [Figure 27] Figures 27A and 27B are graphs showing CD16 expression levels in haNK003 cells after ADCC. ADCC was performed by co-culturing haNK003 cells and DoHH in the presence of 1 μg / ml rituximab for 4 hours at E:T ratios ranging from 1:0 (effector alone) to 1:4. CD16 expression levels were measured by flow cytometry at 4 hours and 24 hours. Figure 27A shows the flow cytometry analysis of CD16 expression levels in haNK-003 after ADCC, along with the control (E:T = 1:0). Figure 27B shows the median fluorescence intensity (MFI) of CD16 expression after ADCC and 24 hours after ADCC. [Modes for carrying out the invention]

[0007] Detailed explanation This specification provides modified NK-92 haNK003 cells, which express the Fc receptor CD16 and endoplasmic reticulum-bound IL-2. Therefore, these cells are not dependent on external IL-2 for proliferation. Furthermore, the modified NK-92 cells possess enhanced cytotoxicity through the insertion of a high-affinity variant of the CD16 receptor, and thus possess the ability to perform CD16-targeted antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is mediated by the recognition of the Fc fragment of a target-bound antibody (IgG) via the CD16 Fc receptor. Therefore, with respect to oncological applications, ADCC by the modified cells is induced by the binding of the CD16 receptor to the Fc fragment of IgG bound to tumor cells, and thus the modified NK-92 cells are activated in relation to target death. As described herein, the modified NK-92 cells provided were prepared by stable transfection of a bisistronic plasmid-based vector containing a sequence of CD16 high-affinity Fc-gamma receptor (FcγRIIIa / CD16a) along with a sequence of IL-2 directed to the endoplasmic reticulum. The cells contain a plasmid sequence inserted at a single position of position 15,654,977 on the + strand of chromosome 17. The modified NK-92 cells produce endogenous IL-2 and are phenotypic CD56+, CD3-, and CD16+. The modified NK-92 haNK003 cells provided herein are sometimes simply referred to as haNK003 cells.

[0008] As described in more detail in the following examples, NK-92 cells were transformed with the pNEUKv1_FcRIL2 plasmid (SEQ ID NO:1). The pNEUKv1_FcRIL2 plasmid is a bicistronic construct expressing a modified CD16 containing valine at amino acid 176 (referring to the full-length CD16 peptide) and IL-2 with an endoplasmic reticulum-retained signal. Whole-genome sequencing (WGS) of the cells was performed, and a single plasmid insertion site on chromosome 17 was identified. WGS confirmed that the integration of the bicistronic plasmid in the haNK003 cell line was located in a region of the genome, far from any genes with oncogenic potential. The nearest gene at 5', TBC1D26, is 10,722 bp upstream, and the nearest gene at 3', ADORA2B, is 186,828 bp downstream. The cells were passaged every 3–4 days, with cells approximately 0.3–0.5 x 10⁶. 6 When seeded at a density of cells / mL, modified NK-92 cells proliferate stably. The mean doubling time was 65 (48-95) hours from day 3 to day 29. Analysis of flow cytometry data shows that modified NK-92 cells express CD54, CD56, NKG2D, NKp30, and CD16 surface marker proteins and lack CD3. Modified NK-92 cells can proliferate without the addition of IL-2 to the culture medium. Furthermore, modified NK-92 cells expressing IL-2 were confirmed to release low levels of IL-2 into the culture medium. Unirradiated haNK003 cells alone secreted an average of approximately 276.1 pg / mL per 1,000,000 cells at 6 hours of culture and a maximum of 1403.3 pg / mL per 1,000,000 cells at 48 hours of culture. The modified NK-92 cells provided are innately cytotoxic to several cancer cell lines and, when combined with antibodies, possess enhanced specific lysis capabilities via ADCC.

[0009] The NK-92 cell line was found to proliferate in the presence of interleukin-2 (IL-2). Gong et al., Leukemia 8:652-658 (1994). These cells exhibit high cytolytic activity against a variety of cancers. The NK-92 cell line is a homogeneous population of NK cells with broad antitumor cytotoxicity, and its expanded yield is predictable. Its safety profile has been confirmed in a Phase I clinical trial. NK-92 was discovered in the blood of subjects with non-Hodgkin lymphoma and subsequently immortalized ex vivo. Although NK-92 cells are derived from NK cells, they possess the majority of activating receptors while lacking the major inhibitory receptors presented by normal NK cells. NK-92 cells, however, do not attack normal cells and do not induce undesirable immune rejection responses in humans. The characterization of the NK-92 cell line is disclosed in International Publication No. 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044.

[0010] NK-92 cells are known and, though not limited thereto, include, for example, those described in U.S. Patent Nos. 7,618,817, 8,034,332, and 8,313,943, U.S. Patent Application Publication No. 2013 / 0040386, all of which are incorporated herein by reference in their entirety, such as wild-type NK-92, NK-92-CD16, NK-92-CD16-γ, NK-92-CD16-ζ, NK-92-CD16(F176V), NK-92MI, and NK-92CI.

[0011] This specification provides a population of modified NK-92 haNK003 cells that possess antibody-dependent cell-mediated cytotoxicity (ADCC) and contain nucleic acid molecules comprising both CD16 (SEQ ID NO:3) and IL-2 (SEQ ID NO:5), wherein more than 90% of the cells in the population express CD56, CD16, CD54, and NKp30, and less than 5% of the cells express CD3. Optionally, the nucleic acid molecule is an mRNA molecule. Optionally, the mRNA molecule contains a sequence encoding CD16, an IRES sequence, and an IL-2 sequence from 5' to 3'. Optionally, the cells contain SEQ ID NO:1 on chromosome 17. Optionally, the mean doubling time of the cells is 55–70 hours. Optionally, the cell population maintains a mean doubling time of 1 to 2, 3, 4, 5, 10, 15, 20, 25 days, or more. Optionally, a population of cells may be passaged for 1, 2, 3, 4 days, or more. Optionally, the cells secrete IL-2 at a concentration of 10–40 pg / hour per million cells. Optionally, the cells are irradiated cells.

[0012] In response to certain stimuli, CD16 cleaves near the cell membrane, resulting in the release of the extracellular portion of the receptor and subsequent downregulation of its expression (see Jing, et al., PLOS one, 10(3):e0121788 DOI:10.1371 / journal.pone.0121788 (2015)). Under normal conditions, this mechanism helps control NK cell cytotoxicity, but in a tumor environment, it can reduce the potency of ADCC and cancer cell death. Advantageously, the provided haNK003 cells exhibit enhanced ADCC activity against cancer cells. While not theoretically bound, this is thought to be due to the event of stable CD16 expression in haNK003 cells after ADCC. As shown in the following examples, CD16 expression remained high compared to control cells after activation with phorbol-12-myrisstart-13-acetate or stimulation in K562 cells. Furthermore, even after ADCC, CD16 expression remained high in haNK003 cells. Therefore, the provided haNK003 cells exhibit reduced downregulation of CD16 expression compared to controls. Moreover, haNK003 cells have increased levels of CD16 after ADCC compared to controls. In other words, these cells maintain higher levels of CD16 after ADCC compared to controls. Therefore, haNK003 cells have more stable CD16 expression compared to controls, such as normal NK cells.

[0013] The lytic activity of natural killer (NK) cells is suppressed in vitro under hypoxic conditions (1% O2) and is associated with the downregulation of NKG2D, perforin, and granzymes. There is some variability in the sensitivity of NK cells from normal donors to hypoxia (1% O2). However, the lytic activity of NK cells can be partially restored in vitro by activation with exogenous IL-2 (16 hours, 1000 IU / ml). Furthermore, NK cells retain their ADCC capacity under 1% oxygen conditions. As described in more detail in the following examples, hypoxia-related genes show no change in expression in haNK cells between 20% oxygen and 0% oxygen (hypoxic) conditions. However, these same hypoxia-related genes are shown to be reduced in expression in normal NK cells.

[0014] As described above, modified NK-92 cells express the Fc receptor CD16. As used herein, the term “Fc receptor” refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to a cell’s Fc receptor (FcR) stimulates the cell’s phagocytic or cytotoxic activity via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified according to the type of antibody they recognize. For example, Fc-gamma receptors (FCγR) bind to IgG class antibodies. FCγRIII-A (also referred to as CD16) is a low-affinity Fc receptor that binds to IgG antibodies and activates ADCC. FCγRIII-A is typically found on NK cells. A representative amino acid sequence encoding CD16 is shown in SEQ ID NO:3. A representative polynucleotide sequence encoding CD16 is shown in SEQ ID NO:4. The complete sequence of CD16 can be found in the SwissProt database as entry P08637.

[0015] Optionally, modified NK-92 cells may contain nucleic acid sequences having 70%, 80%, 90%, or 95% identity with SEQ ID NO:3. Optionally, modified NK-92 cells may contain nucleic acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3. Optionally, modified NK-92 cells may contain polypeptides having 70%, 80%, 90%, or 95% identity with SEQ ID NO:4. Optionally, modified NK-92 cells may contain polypeptides having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4.

[0016] NK-92 cell cytotoxicity depends on the presence of cytokines (e.g., interleukin-2 (IL-2)). In commercial-scale culture, the cost of using externally added IL-2 to maintain and expand NK-92 cells is significant. Administering sufficient amounts of IL-2 to human subjects to sustain NK-92 cell activation can cause adverse side effects. Optionally, IL-2 is expressed with a signaling sequence that directs IL-2 to the endoplasmic reticulum. Directing IL-2 to the endoplasmic reticulum allows for IL-2 expression at levels sufficient for autocrine activation without releasing substantial amounts of IL-2 extracellularly. See Konstantinidis et al “Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells” Exp Hematol. 2005 Feb;33(2):159-64. Representative nucleic acids encoding IL-2 are shown in SEQ ID NO:5, and representative polypeptides of IL-2 are shown in SEQ ID NO:6.

[0017] Optionally, modified NK-92 cells may contain nucleic acid sequences having 70%, 80%, 90%, or 95% identity with SEQ ID NO:5. Optionally, modified NK-92 cells may contain nucleic acid sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:5. Optionally, modified NK-92 cells may contain polypeptides having 70%, 80%, 90%, or 95% identity with SEQ ID NO:6. Optionally, modified NK-92 cells may contain polypeptides having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:6. The modified NK-92 cells provided can, advantageously, be maintained in the absence of IL-2 without secreting amounts of IL-2 that would cause clinically adverse effects.

[0018] As used herein, "nucleic acid" refers to deoxyribonucleotides or ribonucleotides, as well as their polymers and complements. The term includes single-chain or double-chain deoxyribonucleotides or ribonucleotides. The term encompasses known nucleotide analogs or modified backbone residues or bindings, including synthetic, natural, and unnatural nucleotides, that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, and peptide-nucleic acid (PNA). Unless otherwise indicated, conservatively modified variants of nucleic acid sequences (e.g., degenerate codon substitutions) and complementary sequences may be used in place of specific nucleic acid sequences described herein. Specifically, degenerate codon substitution can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0019] Nucleic acids are functionally ligated when they are placed in a functional relationship with another nucleic acid sequence. For example, DNA encoding a pre-sequence or secretion leader is functionally ligated to the polypeptide-encoding DNA if it is expressed as a preprotein involved in polypeptide secretion; a promoter or enhancer is functionally ligated to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is functionally ligated to a coding sequence if it is positioned to facilitate translation. Generally, functional ligation means that the ligated DNA sequences are close to each other and, in the case of a secretion leader, are contiguous and in the reading phase. Enhancers, however, do not have to be contiguous. For example, a nucleic acid sequence functionally ligated to a second nucleic acid sequence is covalently bonded to such a second sequence, either directly or indirectly, although any valid three-dimensional association is permitted. A single nucleic acid sequence can be functionally ligated to multiple other sequences. For example, a single promoter may act on the transcription of multiple RNA species. Ligation can be achieved by ligation at a convenient restriction site. If such a site is not present, a synthetic oligonucleotide adapter or linker is used according to conventional practice.

[0020] The terms identical, or percent identical, in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical or have a specific percentage of the same amino acid residues or nucleotides (i.e., approximately 60% identity across a specific region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity, when compared and aligned to the maximum match across a comparison window or specified region). Such sequences are therefore said to be substantially identical. This definition may also refer to or apply to complements of test sequences. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, a preferred algorithm can reveal gaps, etc. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50 to 100 amino acids or nucleotides in length.

[0021] For sequence comparison, typically one sequence acts as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer; the coordinates of subsequences are specified if necessary; and the program parameters for the sequence algorithm are specified. Preferably, default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters.

[0022] A comparison window, as used herein, includes references to a number of consecutive positional segments selected from the group consisting of 20 to 600, typically about 50 to about 200, and more commonly about 100 to about 150, where the sequences may be compared with reference sequences of the same number of consecutive positions after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art. For comparison, optimal alignment of sequences can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970); by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); by computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0023] Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used to determine the percent sequence identity of nucleic acids or proteins using the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information, as is well known in the art. The algorithm first includes identifying high-scoring sequence pairs (HSPs) by identifying short words of a selected length (W) in a query sequence that, when aligned with a word of the same length in a database sequence, either match or satisfy a threshold score T of some positive value. T is called the neighbor word score threshold (Altschul et al., see above). These initial neighbor word hits act as seeds to initiate a search for longer HSPs that contain them. Word hits are extended bidirectionally along each sequence as long as the cumulative alignment score can increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.The expansion of word hits in each direction stops when the cumulative alignment score decreases by an amount of X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or when the end of any sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The expected value (E) represents the number of different alignments that have a score equal to or better than what is expected to occur by chance in a database search. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected value (E) of 10, M = 5, N = -4, and a comparison of both strands by default. The BLASTP program for amino acid sequences uses, by default, a word length of 3, an expected value of 10 (E), and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), an alignment of 50 (B), an expected value of 10 (E), M = 5, N = -4, and a comparison of both strands.

[0024] When used herein, the term polypeptide, as it is understood in the art, typically refers to a polymer of at least three amino acids and is intended to include peptides and proteins. However, the term is also used to refer to a specific functional class of polypeptides, such as desaturases, elongases, etc. For each such class, this disclosure provides some examples of known sequences of such polypeptides. Those skilled in the art, however, will understand that the term polypeptide is intended to be so general as to include not only polypeptides having complete sequences as referred herein (or in reference to or in databases specifically mentioned herein), but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments retaining at least one activity). Furthermore, those skilled in the art will understand that protein sequences generally tolerate some substitutions without disrupting activity. Therefore, any polypeptide that retains activity and shares at least about 30–40%, often more than about 50%, 60%, 70%, or 80% overall sequence identity with another polypeptide of the same class, and further, usually contains at least one region of higher identity, often more than 90%, or even more than 95%, 96%, 97%, 98%, or even more than 99%, in one or more highly conserved regions that typically contain at least 3–4 amino acids, and often up to 20 or more amino acids, is included in the appropriate term "polypeptide" as used herein. Those skilled in the art can determine the similarity and / or identity of other regions by analyzing the sequences of the various polypeptides described herein. As is known to those skilled in the art, a variety of strategies are known and tools are available for performing amino acid or nucleotide sequence comparisons to assess the degree of identity and / or similarity. These strategies include, for example, manual alignment, computer-assisted sequence alignment, and combinations thereof.Numerous algorithms for performing sequence alignment (generally performed on computers) are widely available, or can be created by those skilled in the art. Representative algorithms include, for example, the local homology algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482); the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443); the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. (USA), 1988, 85: 2444); and / or computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). Easily available computer programs incorporating such algorithms include, for example, BLASTN, BLASTP, Gapped BLAST, PILEUP, and CLUSTALW. When using the BLAST and Gapped BLAST programs, the default parameters of each program may be used. Alternatively, practitioners may use non-default parameters depending on experimental requirements and / or other requirements (see, for example, the website with the URL www.ncbi.nlm.nih.gov).

[0025] As used herein, the terms promoter, promoter element, and regulatory sequence refer to a polynucleotide functionally ligated to a promoter that controls the expression of a selected polynucleotide sequence and results in the expression of the selected polynucleotide sequence in a cell.

[0026] When used herein, the term transformation refers to the process by which a foreign or heterologous nucleic acid molecule (e.g., a vector or recombinant nucleic acid molecule) is introduced into a recipient cell or microorganism. The foreign or heterologous nucleic acid molecule may or may not be incorporated into (i.e., covalently bonded to) the chromosomal DNA that constitutes the genome of the host cell or microorganism. For example, a foreign or heterologous polynucleotide may be maintained on an episomal element such as a plasmid. Alternatively, or in addition, the foreign or heterologous polynucleotide may be incorporated into a chromosome so that it is inherited by daughter cells through chromosomal replication. Methods for transformation include, but are not limited to, calcium phosphate precipitation; fusion of recipient cells with bacterial protoplasts containing recombinant nucleic acid; treatment of recipient cells with liposomes containing recombinant nucleic acid; DEAE dextran; fusion using polyethylene glycol (PEG); electroporation; magnetoporation; bioristic delivery; retroviral infection; lipofection; and direct microinjection of DNA into cells.

[0027] When used in reference to cells, the term "transformed" refers to cells that have been transformed as described herein to possess foreign or heterologous genetic material (e.g., recombinant nucleic acid). The term "transformed" may also be used to refer to microorganisms, microbial strains, tissues, organisms, etc., that contain foreign or heterologous genetic material.

[0028] The terms "modified" and "recombinant," when used in relation to cells, nucleic acids, polypeptides, vectors, etc., indicate that the cells, nucleic acids, polypeptides, vectors, etc., have been modified by laboratory methods or are the result of laboratory methods and are non-natural. Therefore, for example, a modified cell includes cells produced or modified by laboratory methods, such as transformation methods for introducing nucleic acids into cells. A modified cell may contain nucleic acid sequences not found in natural (non-recombinant) cells, or may contain altered nucleic acid sequences, such as nucleic acid sequences bound to non-natural promoters.

[0029] As described herein, a control or standard control refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison with a test sample, test measurement, or test value. For example, test cells, such as cells transformed with a nucleic acid sequence encoding the gene for the Fc receptor, may be compared to known normal (wild-type) cells (e.g., standard control cells). A standard control may also represent an average measurement or mean obtained from a population of cells that do not express the Fc receptor, or have no Fc receptor activity, or have the lowest level of Fc receptor activity (e.g., a standard control microorganism). Those skilled in the art will understand that standard controls can be designed for the evaluation of any number of parameters (e.g., RNA levels, polypeptide levels, specific cell types, etc.).

[0030] As used herein, the term “antibody” refers to an immunoglobulin or a fragment thereof. The antibody may be of any type (e.g., IgG, IgA, IgM, IgE, or IgD). Preferably, the antibody is IgG. The antibody may be a non-human antibody (e.g., derived from mouse, goat, or any other animal), a fully human antibody, a humanized antibody, or a chimeric antibody. The antibody may be polyclonal or monoclonal. Optionally, the antibody is monoclonal.

[0031] As used herein, the term "monoclonal antibody" refers to a pure, target-specific antibody produced from a single clone of a cell that can grow in culture and proliferate indefinitely. Possible monoclonal antibodies include naked antibodies that bind to and inhibit antigens on cancer cells. Optionally, a naked monoclonal antibody is alemtuzumab that binds to the CD52 antigen on lymphocytes. Possible monoclonal antibodies also include conjugated monoclonal antibodies, such as tagged, labeled, or appended antibodies. Specifically, antibodies may be tagged with a drug or toxin, or these may be appended, or they may be radiolabeled. Examples of such antibodies, but not limited to, include ibritumomab targeting the CD20 antigen; brentuximab targeting the CD30 antigen; and trastuzumab targeting the HER2 protein. Other monoclonal antibodies that may be used include bispecific monoclonal antibodies such as blinatumomab, which targets CD19 in lymphoma cells and CD3 in T cells.

[0032] As used herein, the term “antibody fragment” refers to any portion of an antibody that recognizes an epitope. Antibody fragments may be glycosylated. Non-limiting examples include Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, rIgG fragments, functional antibody fragments, and single-chain recombinants of the aforementioned. F(ab')2, Fab, Fab', and Fv are antigen-binding fragments that can be prepared from the variable regions of IgG and IgM. They differ in size, titer, and Fc content. Fragments may be prepared by any method, including the expression of components (e.g., heavy and light chain portions) by one cell or cell line or by multiple cells or cell lines. Preferably, the antibody fragment contains a sufficient portion of the Fc region so that it can recognize an epitope and bind to the Fc receptor.

[0033] As used herein, the term "cancer" refers to all types of cancer, neoplasm, or malignant tumor found in mammals, including leukemia, carcinoma, and sarcoma. Exemplary cancers include cancer of the brain, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer, and medulloblastoma. Additional examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, Waldenström's macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, pre-cancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, urogenital cancer, hypercalcemia of malignancy, endometrial cancer, adrenocortical cancer, pancreatic endocrine and exocrine neoplasms, and prostate cancer.

[0034] As described herein, methods of treating a subject with modified NK-92 cells are also provided. Optionally, the subject is treated with modified NK-92 cells and an antibody.

[0035] Modified NK-92 cells can be administered to a subject by the absolute number of cells, e.g., the subject can be administered about, at least about, or up to about 1 x 10 10 、1 x 10 9 、1 x 10 8 、1 x 10 7 、5 x 10 7 、1 x 10 6 、5 x 10 6 、1 x 10 5 、5 x 10 5 、1 x 10 4 、5 x 10 4 、1 x 10 3 、5 x 10 3 (etc.) NK-92 cells, or any range between any two including the endpoints of these numbers, such as from about 1000 cells / injection to about 10 billion cells / injection of cells, can be administered. Optionally, 1 x 10 8 ~1 x 10 10Individual cells are administered to the target. Optionally, cells are administered once or multiple times per week for one or more weeks. Optionally, cells are administered once or twice per week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more weeks.

[0036] Optionally, the target is approximately, at least approximately, or at most approximately 1 x 10 per injection. 8 pieces / m 2 , 1 x 10 7 pieces / m 2 , 5 x 10 7 pieces / m 2 , 1 x 10 6 pieces / m 2 , 5 x 10 6 pieces / m 2 , 1 x 10 5 pieces / m 2 , 5 x 10 5 pieces / m 2 , 1 x 10 4 pieces / m 2 , 5 x 10 4 pieces / m 2 , 1 x 10 3 pieces / m 2 , 5 x 10 3 pieces / m 2 Approximately 1000 cells / injection / m² of NK-92 cells (etc.), or any range between any two of these endpoints. 2 Approximately 10 billion cells per injection / m 2 It is administered up to this point.

[0037] Optionally, NK-92 cells may be administered to such an individual by the relative number of cells, for example, the individual may receive approximately, at least approximately, or at most approximately 1 x 10⁶ cells per kilogram of the individual. 8 , 1 x 10 7 , 5 x 10 7 , 1 x 10 6 , 5 x 10 6 , 1 x 10 5 , 5 x 10 5 , 1 x 10 4 , 5 x 10 4 , 1 x 10 3 , 5 x 103 Individuals may be administered with approximately 1,000 to 10 billion cells per kilogram, such as (etc.) NK-92 cells, or any range between any two of these endpoints.

[0038] Optionally, the total dose is calculated based on the body surface area m². 2 It may be calculated by, 1 m 2 Approximately 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 This includes 1,0 2 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 Includes one.

[0039] NK-92 cells, and optionally other anticancer agents, may be administered once to a patient with cancer, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours during treatment, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more, or once every any range between any two of these endpoints.

[0040] Optionally, NK-92 cells are administered in a composition comprising NK-92 cells and a medium such as human serum or its equivalent. Optionally, the medium comprises human serum albumin. Optionally, the medium comprises human plasma. Optionally, the medium comprises about 1% to about 15% human serum or human serum equivalent. Optionally, the medium comprises about 1% to about 10% human serum or human serum equivalent. Optionally, the medium comprises about 1% to about 5% human serum or human serum equivalent. Optionally, the medium comprises about 2.5% human serum or human serum equivalent. Optionally, the serum is human AB serum. Optionally, a serum substitute acceptable for use in human therapeutics is used instead of human serum. Such serum substitutes may be known in the art. Optionally, NK-92 cells are administered in a composition comprising a solution of NK-92 cells and an isotonic liquid that sustains cell viability. Optionally, NK-92 cells are administered in a composition reconstituted from cryopreserved samples.

[0041] An effective amount of one or more active substances provided herein is administered to a subject according to the methods provided herein. The terms effective amount and effective dose are interchangeable. The term effective amount is defined as any amount necessary to produce a desired physiological response (e.g., a reduction in inflammation). The effective amount and schedule for administration of the active substance may be determined empirically by those skilled in the art. The range of doses for administration is large enough to produce the desired effect of affecting (e.g., reducing or delaying) one or more symptoms of a disease or disorder. The dose should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions or anaphylactic reactions. Generally, the dose may vary depending on age, health status, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the regimen, and may be determined by those skilled in the art. The dose may be adjusted by the individual physician if there are any contraindications. The dose may be varied and may be administered once daily for one day or for several days, in one or more doses. Guidelines can be found in the literature regarding appropriate doses for a given class of pharmaceutical products. For example, an effective dose may show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100% with respect to a given parameter. Efficacy may also be expressed as a "double" increase or decrease. For example, a therapeutically effective dose may have an effect at least 1.2 times, 1.5 times, 2 times, 5 times, or more than that of the control.The exact dosage and dosage form are determined by the purpose of the treatment and can be verified by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Editor (2012); and Pickar, Dosage Calculations (1999)).

[0042] Pharmacovigilantly acceptable compositions may contain a variety of carriers and excipients. A variety of aqueous carriers, such as buffered saline, may be used. These solutions are sterile and generally free of undesirable substances. Suitable carriers and excipients, as well as their formulations, are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). A pharmaceutically acceptable carrier means a material that is not biologically or otherwise harmful. That is, the material is administered to a subject without causing harmful biological effects or interacting in a harmful manner with other components of the pharmaceutical composition in which it is contained. When administered to a subject, the carrier is optionally selected to minimize the degradation of the active ingredient and to minimize adverse side effects in the subject. As used herein, the term pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable. Pharmaceutical compositions generally contain buffers and preservatives for storage, and may also contain appropriate buffers and carriers for delivery depending on the route of administration.

[0043] The composition may contain acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., including sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentrations of cells and / or other active ingredients in these formulations can be varied and selected primarily based on the volume, viscosity, body weight, etc., according to the chosen specific administration method and the needs of the subject.

[0044] Optionally, NK-92 cells are administered to the subject in conjunction with one or more other therapies for the cancer being treated. While not bound by theory, the co-treatment of a subject with NK-92 cells and another therapy for cancer is thought to enable NK-92 cells and the other therapy to give the endogenous immune system an opportunity to eliminate the cancer that had previously overwhelmed such endogenous action. Optionally, two or more other therapies for the cancer being treated include, for example, antibody therapy, radiotherapy, chemotherapy, stem cell transplantation, or hormone therapy.

[0045] Optionally, the antibody is administered to the patient together with NK-92 cells. Optionally, NK-92 cells and the antibody may be administered together to the subject, for example, in the same formulation; separately, for example, simultaneously in separate formulations; or separately, for example, on different dosing schedules or at different times of the day. If administered separately, the antibody may be administered by any suitable route, such as intravenous or oral administration.

[0046] Optionally, antibodies may be used to target cancer cells or cells expressing cancer-related markers. Numerous antibodies are approved solely for the treatment of cancer.

[0047] (Table 2) Examples of therapeutic monoclonal antibodies approved by the FDA TIFF2026063125000002.tif42142TIFF2026063125000003.tif163142

[0048] Antibodies can treat cancer through several mechanisms. ADCC occurs when immune cells, such as NK cells, bind to antibodies that bind to target cells via Fc receptors such as CD16.

[0049] Therefore, NK-92 cells expressing CD16 are administered to the subject along with an effective dose of at least one monoclonal antibody against a specific cancer-associated protein, such as alemtuzumab, bevacizumab, ibritumomab tiuxetan, ofatumumab, rituximab, and trastuzumab. Optionally, the monoclonal antibody may be a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. Optionally, a bispecific antibody that binds to cancer cells and also to cell surface proteins present on the surface of NK-92 cells may be used.

[0050] Cancer-specific antibodies bind to specific protein antigens expressed on the surface of cancer cells. NK-92 cells can be modified so that antibodies bind to the surface of NK-92 cells. Optionally, the antibodies are cancer-specific. In this way, NK-92 cells can specifically target cancer. Neutralizing antibodies can also be isolated. For example, the secreted glycoprotein, YKL-40, is elevated in several types of advanced human cancer. Antibodies against YKL-40 may be used to suppress tumor growth, angiogenesis, and / or metastasis. See Faibish et al., (2011) Mol. Cancer Ther. 10(5):742-751.

[0051] Antibodies against cancer can be purchased from commercially available sources or manufactured by any method known in the art. For example, antibodies can be manufactured by obtaining B cells, bone marrow, or other samples from one or more patients who have previously had and recovered from cancer, or who were recovering at the time the samples were taken. Methods for identifying, screening, and amplifying antibodies (e.g., monoclonal antibodies) from these samples are known. For example, a phage display library can be manufactured by isolating RNA from a sample or cells of interest, preparing cDNA from the isolated RNA, enriching the cDNA for heavy and / or light chain cDNA, and constructing a library using a phage display vector. The library can be prepared and screened, for example, as described by Maruyama et al., which are incorporated in their entirety herein by reference. Antibodies can be manufactured by recombinant techniques or any other method. The isolation, screening, characterization, and production of human monoclonal antibodies are also described in Beerli, et al., PNAS (2008) 105(38):14336-14341, which is incorporated herein by reference in its entirety.

[0052] A combination of active substances or compositions may be administered together (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous routes), or sequentially (e.g., one active substance is administered first, followed by the administration of a second active substance). Therefore, the term "combination" is used to refer to the combined, simultaneous, or sequential administration of two or more active substances or compositions. A series of treatments is best determined individually, depending on the specific characteristics of the subject and the type of treatment chosen. Treatments such as those disclosed herein may be administered to a subject once daily, twice daily, once every two weeks, once a month, or to any appropriate therapeutically effective standard. Treatments may be administered alone or in combination with any other treatments disclosed herein or known in the art. Additional treatments may be administered concurrently with the first treatment, at different times, or on a completely different treatment schedule (e.g., the first treatment may be once daily, while the additional treatment is once weekly).

[0053] A kit containing the provided modified NK-92 cells is also disclosed. Optionally, the kit further comprises one or more additional active ingredients, such as antibodies. The components of the kit may be contained in one or more different containers, such as one or more vials. The antibodies may be in liquid or solid form (e.g., after lyophilization) to increase their shelf life. If in liquid form, the components may contain additives such as stabilizers and / or preservatives, such as proline, glycine, or sucrose, or other additives that increase shelf life.

[0054] Optionally, the kit may include additional compounds, such as therapeutically effective compounds or drugs, to be administered before, simultaneously with, or after the administration of modified NK-92 cells or NK-92 cells and antibodies. Examples of such compounds include vitamins, minerals, fludrocortisone, ibuprofen, lidocaine, quinidine, and chemotherapeutic agents.

[0055] Optionally, the instructions for use of the kit may include instructions for using the kit's components in the treatment of cancer. The instructions may further include information on how to prepare (e.g., dilution or reconstitution in the case of lyophilized proteins) antibodies and NK-92 cells (e.g., thawing and / or culturing). The instructions may further include guidance on dosage and frequency of administration.

[0056] Materials, compositions, and components that may be used for, in conjunction with, or for the preparation thereof, or are products thereof, are disclosed. Where these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, and specific references to each of the various individual and collective combinations and modifications of these compounds may not be expressly disclosed, it is understood that each is specifically intended and described herein. For example, where a method is disclosed and considered, and several modifications that may be made to some molecules including the method are considered, each and every combination and modification of the method, and possible modifications, are specifically intended unless it is specifically indicated that they are not. Similarly, any subset or combination of these is also specifically intended and disclosed. This concept applies to all aspects of this disclosure, including, but not limited to, steps in methods using the disclosed compositions. Therefore, where there are various additional steps that can be performed, it should be understood that each of these additional steps may be performed in any particular step of the disclosed method or in a combination of steps of the method, and that each of such combinations or subsets of combinations should be considered to be specifically intended and disclosed.

[0057] Publications and materials cited herein are, as expressly, incorporated herein by reference in their entirety.

[0058] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein and are not intended to limit the scope of the claims. [Examples]

[0059] Example 1: Structural and Functional Characteristics of haNK003 NK-92 [CD16.176V, ER IL-2](haNK003) was created by modifying NK-92 cells. NK-92 cells were initially isolated in 1992 from a 50-year-old male patient with rapidly progressive non-Hodgkin lymphoma (Gong, et al., Leukemia, 8(4):652-8 (1994)). The NK-92 cell line was subsequently characterized and shown to be IL-2 dependent, and phenotypically CD56+, CD3-, and CD16-. haNK003 is an allogeneic cell line created by stable electroporation transfection of NK-92 cells with a bicistronic plasmid-based vector containing the sequences of CD16 and IL-2. The transfected plasmid is shown in Figure 1 and was constructed using GeneArt AG. The CD16 sequence encodes valine at amino acid position 176 (176V), which enables enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) potential. The IL-2 sequence is tagged with the endoplasmic reticulum retention signal, KDEL, to inhibit IL-2 protein secretion from the endoplasmic reticulum (ER). Inclusion of the IL-2 sequence makes haNK™ IL-2 independent.

[0060] EUFETS GmbH (Regensburg, Germany) performed transfection by electroporation and selected multiple clones through one round of limiting dilution. One clone from EUFETS was sent to BioReliance to establish the GMP master cell bank, haNK003. Whole-genome sequencing of the selected clone confirmed that the plasmid insertion site was a single location on chromosome 17, at positions 15,654,977–15,661,403.

[0061] Plasmid transfection The plasmid was constructed by GeneArt AG based on the provided specifications. The synthetic gene pNEUKv1_FcRIL2 was assembled from synthetic oligonucleotides and PCR products. The fragment was cloned into the pNEUKv1_O059 vector backbone using EcoRI and NotI restriction sites. pNEUKv1_O059 is a synthetic vector containing an ampicillin-resistant cassette. The promoter used for transgene expression is EF-1alpha with an SV40 polyadenylated sequence. The resulting plasmid is 5,491 base pairs (bp) long and contains human-derived sequences for CD16 and IL-2. Neither CD16 nor IL-2 possesses any transformative properties. Plasmid DNA was purified from transformed bacteria, and its concentration was determined by UV spectroscopy. The final construct was confirmed by sequencing. Sequence matching within the restriction sites used was 100%. The plasmid was produced under TSE-free production conditions.

[0062] The complete nucleotide sequence (SEQ ID NO:1) of the pNEUKv1_FcRIL2 plasmid is shown below. TIFF2026063125000004.tif89128TIFF2026063125000005.tif216128

[0063] To construct the haNK003 cell line, vials of the NK-92(aNK) master cell bank (MCB) (aNK COA) and 250 mg of the pNEUKv1_FcRIL2 plasmid were sent to EUFETS GmbH. EUFETS thawed the MCB vials and cultured NK-92 cells to a suitable number for plasmid transfection. Transfected cells were grown in medium supplemented with IL-2, X-Vivo 10, and 5% heat-inactivated human AB serum for the first two days after transfection. After two days, IL-2 was no longer added to the growth medium, and the transfected cells producing a suitable amount of IL-2 continued to grow. Multiple clones were isolated by limiting dilution and preliminaryly screened for phenotype and Fc receptor expression. Six clones exhibiting excellent viability (>70%), acceptable doubling time, expected phenotype, and positive Fc receptor expression were sent to the German Red Cross GMP Testing Laboratory (GRC) for further screening and final selection of a single clone. At the GRC, all clones were tested for phenotype (including Fc receptor expression), ADCC, cytokine profile, proliferation characteristics, and radiosensitivity. The selected cell line, haNK003, was used to create a master cell bank.

[0064] The NantKwest Master Cell Bank (MCB haNK003) was prepared from selected cell lines and tested by BioReliance. The MCB was tested for purity, potency, identity, sterility, and viral / external active agents. The MCB was cryopreserved in aliquots of 1 x 10⁷ cells / vial in a preparation of 10% DMSO, 40% X-Vivo 10, and 50% human AB serum. A total of 218 vials were prepared from cryopreservations for the MCB.

[0065] Built-in parts DNA extracts from haNK003 were provided to the CLIA / CAP-certified NantOmics Sequencing Lab (Culver City, CA) for whole-genome sequencing. The whole-genome library was prepared for the cell line sample using the KAPA Hyperprep kit and sequenced on an Illumina HiSeq instrument to provide a minimum 25x coverage encompassing haNK003. The DNA sequencing data, including plasma sequences reported by bwa-mem, were aligned to an improved Genome Reference Consortium Human Build 37 (GRCh37, also known as hg19, originally obtained from the University of California, Santa Cruz Genome Browser - http: / / genome.ucsc.edu), with duplicates marked by samblaster, indels realigned and base quality recalibrated by the Genome Analysis Toolkit (GATK). Mutant analysis was performed using the NantOmics Contraster analysis pipeline to identify mutants containing single nucleotide changes, small insertions or deletions (indels), copy number changes, transpositions, and integration sites. The integrated plasmids and resulting integration sites were visualized using the NantOmics Genome Browser, and further comparison and visualization were performed on the UCSC Genome Browser to identify any potential interactions with existing genomic elements.

[0066] haNK003 showed signs of inconsistent readings in its mapping to chromosome 17:15654977~15661403. The nearest gene at 5', TBC1D26 (chromosome 17:15,635,591~15,644,255), was 10,722 bp upstream, while the nearest gene at 3', ADORA2B (chromosome 17:15,848,231~15,871,210), was 186,828 bp downstream. Little is known about TBC1D26 other than its annotation in UniProt as a GTPase activating protein for Rab family proteins. ADORA2B has been annotated as a membrane protein that stimulates adenylate cyclase activity in the presence of adenosine (Strohmeier, et al., J. Biol. Chem. 270(5):2387-2394 (1995)). No coding variants were found in the two annotated ORFs with respect to the coding sequence named pNEUKv1_FcRIL. The UCSC Encode track and lincRNA show traces of a lincRNA transcript downstream of the insertion site (TCONS_12_00011108), however, it is approximately 2,450 bp downstream of the 3' integration site, suggesting that this transcript is likely still intact. A study of 100 different alignments across vertebrate species showed that the negative logarithmic p-values ​​ranged from -3.874 to 1.507, with a mean conservation of 0.01 and a standard deviation of 0.58, indicating little to no base-level conservation across integration sites.

[0067] haNK003 showed no signs of gene corruption, transcript corruption, or regulatory corruption at human genome integration sites. The integration of cell line haNK003 was at least 10 kbp away from any gene. This cell line is acceptable in that it shows no signs of disruption to any known genomic features in the target cell line's human genome.

[0068] Characteristics of proliferation The growth characteristics of the clonal cell line haNK003 used to produce MCB haNK003 are shown in Figures 1A and 1B. Data were analyzed from the cell culture history when the cell line haNK003 was grown for cryopreservation in the master cell bank. The mean doubling time was 65 (48–95) hours from day 3 to day 29. Equivalent cell densities were achieved throughout the passage, with passages every 3–4 days and cells approximately 0.3–0.5 x 10⁶. 6 This study demonstrates that haNK003 cells proliferate stably when seeded at a density of cells / mL.

[0069] Phenotype A study was conducted to quantify the expression of a panel of six protein markers on the surface of haNK003 cells and to compare the haNK003 profile with that of the parental cell line, NK-92(aNK). The panel of surface markers was selected to be representative of natural killer (NK) cells.

[0070] aNK cells express surface markers typical of NK cells in the early stages of differentiation, including numerous activating receptors such as NKG2D and NKp30, but lacking FcγRIIIa (CD16) and inhibitory KIR (killer immunoglobulin-like receptor). This specific surface marker expression profile of aNK cells confers unique cytotoxic properties to them. Therefore, it was important to establish that the creation of the haNK003 cell line by stable transfection with plasmids encoding high-affinity FcγRIIIa and intracellularly retained IL-2 (ERIL-2) does not alter the expression profile of key surface markers in the parental aNK cell line. Surface markers CD54, CD56, NKG2D, NKp30, CD3, and CD16 were analyzed, and marker expression was determined by cell staining with specific antibodies conjugated with fluorescent dyes and detection of the conjugated antibodies by flow cytometry.

[0071] The results of the flow cytometry analysis are summarized in Table 1, and a representative histogram is provided in Figure 2.

[0072] (Table 1) Expression of surface markers TIFF2026063125000006.tif32142% = Percentage of cells positive for expression ± standard deviation

[0073] haNK003 and aNK express equivalent amounts of CD54, CD56, NKG2D, and NKp30, as determined by the median fluorescence intensity. In addition, the percentage of cells expressing these markers is equivalent. Neither haNK003 nor aNK expresses the T cell marker CD3. As expected, haNK003 expresses the CD16 marker, while aNK does not.

[0074] The creation of the haNK003 cell line did not alter the expression of important surface markers in the parent aNK cell line, but only added the additional functionality in the form of CD16 expression.

[0075] Cell damage Spontaneous cytotoxicity of the haNK003 cell line was evaluated against the K562, Raji, SKOV3, and SKBR3 cell lines at various effector-to-target ratios (E:T). ADCC activity of haNK003 was also evaluated against the Raji, SKOV3, and SKBR3 cell lines at various E:T ratios. Susceptibility to spontaneous cytotoxicity-induced death in haNK cells varied across different target cell lines, with the K562 cell line being the most sensitive and solid tumor cell lines (SKOV3 and SKBR3) being less sensitive (Figures 3A, 3B, 3C, and 3D). Some differences in ADCC activity of haNK003 across different target cell lines were also observed, with the greatest specific lysis observed in Raji cells in combination with rituximab (Figures 4A, 4B, and 4C).

[0076] The results demonstrate that haNK003 cells exhibit innate cytotoxicity in the presence of several cancer cells and possess enhanced specific lysis capabilities via antibody-mediated ADCC. Specifications and results for haNK(trademark)MCB(haNK003) are provided in Table 2.

[0077] (Table 2) Specifications of haNK003 cells TIFF2026063125000007.tif90142TIFF2026063125000008.tif159142 a - Although the EBV virus genome was detected, testing with NK-92 (aNK) confirmed that the cells did not cause EBV infection. Infectivity studies were conducted by co-culturing aNK cells (irradiated and unirradiated) with B lymphocytes to determine whether aNK cells release viral particles that can infect normal cells. The results showed no signs of B lymphocyte proliferation or growth, indicating that aNK cells do not pose a risk of EBV infection.

[0078] Example 2: Effects of irradiation on proliferative capacity and functionality in vitro haNK® cells are irradiated to mitigate the risk of uncontrolled proliferation. The effects of irradiation on proliferative capacity and functionality in vitro were evaluated. These studies demonstrate that irradiation at 10 Gy suppresses the proliferative capacity of at least 99.9% of haNK cells, while functional activity is still maintained for at least 6 hours after irradiation.

[0079] haNK003 cells exhibit both innate (direct) cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). In both cases, the antigens of the target cells are recognized by activating receptors on NK cells. With respect to innate cytotoxicity, the antigens of these target cells are stress antigens characteristic of virus-infected or transformed cells. With respect to ADCC, the antigens are tumor-specific antigens recognized by antibodies, which then bind to the NK cell activating receptor, FcγRIIIa(CD16), via its constant (Fc) region.

[0080] The interaction between NK cells and target cell ligands (either through direct interaction or antibody-mediated interaction) leads to the formation of cell junctions, followed by the release of perforin and granzyme. This then induces apoptosis within the target cells, resulting in cell membrane breakdown and target cell death.

[0081] One study was conducted to determine the levels of innate cytotoxicity and ADCC activity in preliminaryly formulated cells, as well as the duration of that activity after gamma irradiation at 10 Gy. For this study, 1.5 x 10⁷ cells per flask were irradiated. Although the number of irradiated cells was not equivalent to that used in the production of clinical doses, the post-irradiation functional assay provides insights into the potential for commercialization.

[0082] For the purposes of these experiments, haNK003 cells were irradiated with 10 Gy using an X-ray irradiator. Unirradiated cells underwent the same treatment without irradiation. Target cells were selected to represent sensitivity to different mechanisms of NK cell death. For example, K562 cells are highly sensitive to death by innate cytotoxicity, while DOHH cells are incompletely sensitive to death via innate cytotoxicity but are sensitive to ADCC using appropriate antibodies. Irradiated and unirradiated cells were assayed in parallel for specific cytotoxicity using a proprietary flow cytometry-based assay. Target cells were labeled with a green fluorescent dye (PKH67) with a long aliphatic tail, ensuring stable integration of the dye into the lipid region of the cell membrane. Cell lysis was monitored by propidium iodide staining.

[0083] Analysis of innate cytotoxic activity demonstrates that the effects of irradiation differ depending on the target cell and the time elapsed after irradiation. For sensitive cells such as K562, innate cytotoxic activity was maintained within 6 hours of irradiation, but decreased by more than 40% after 24 hours across all effector-to-target ratios (E:T) (Figure 5). For DOHH2, innate cytotoxic activity was also maintained at 6 hours post-irradiation, but decreased by more than 14% after 24 hours across all effector-to-target ratios, and by as much as 50% at an E:T ratio of 10:1 (Figure 6).

[0084] The level of rituximab-mediated ADCC activity for the DOHH2 target was also maintained in irradiated cells at 6 hours. However, rituximab-mediated ADCC activity in irradiated cells at 24 hours was reduced by more than 16% from the activity observed in cells at 6 hours and in unirradiated cells at 24 hours (Figure 7). As expected, Herceptin (trastuzumab) in combination with haNK003 did not induce any ADCC death in DOHH2 target cells (Figure 4), and the antibody alone (rituximab or trastuzumab) also did not induce any death in DOHH2 target cells (data not shown).

[0085] Adequate levels of cytotoxic activity and ADCC activity were maintained for at least 6 hours after irradiation of pre-formulated haNK003 cells.

[0086] Example 3: Characterization of IL-2 release A study was conducted to analyze the amount of IL-2 released into the culture medium by haNK003 cells, as well as the amount of IL-2 retained intracellularly in haNK003 cells, at various time points. IL-2 levels were measured in the supernatant to determine IL-2 release from haNK003 cells. IL-2 levels were also measured in the cell pellet lysate to determine the total intracellular IL-2 levels in haNK003 cells. Samples were analyzed before and after irradiation to determine the effect of irradiation on IL-2 release and intracellular IL-2 levels.

[0087] Two separate assays were performed (experiment number 1 and experiment number 2), in which haNK003 cells were cultured in T-75 flasks and irradiated with an X-ray irradiator at 0 Gy (unirradiated) or 10 Gy (irradiated). The haNK003 cells (unirradiated and irradiated) were then cultured for up to 48 hours in X-Vivo 10 supplemented with 5% thermally inactivated human AB serum. For analysis, samples were collected at various time points from both the cell pellet and the culture supernatant. The cell pellet was lysed using a surfactant-based solution to quantify total intracellular IL-2. IL-2 concentrations in the culture supernatant and cell lysate samples were independently measured by NantKwest or AllCells, LLC using two different detection methods (sandwich ELISA or multiplex ELISA).

[0088] IL-2 concentrations measured by two different methods differed by an average of five-fold for the same sample. Both methods showed a linear increase in IL-2 release over time for both irradiated and unirradiated cells (Table 3, as well as Figures 8A, 8B, 8C, and 8D). Although the differences between assays #1 and #2 varied, IL-2 concentrations in the culture supernatant of irradiated cells tended to be higher than those of unirradiated cells at all time points.

[0089] (Table 3) Cell size 1 x 10⁶ of irradiated haNK003 vs. unirradiated haNK003 at different time points. 6 IL-2 released per cell (pg / mL) (mean and standard deviation of two readings [StDev]) TIFF2026063125000009.tif76142h-time

[0090] Total intracellular IL-2 concentrations measured by two different methods differed by an average of five-fold for the same sample (Table 4, and Figures 9A, 9B, 9C, and 9D). Both methods showed a time-dependent increase in total intracellular IL-2 in unirradiated cell lysates, while the amount of total intracellular IL-2 in irradiated cell lysates decreased over time. At 6 hours of culture, the levels of total intracellular IL-2 were similar between irradiated and unirradiated cells when measured by both methods. In irradiated cells, these levels decreased after 48 hours of culture.

[0091] (Table 4) Cell size 1 x 10⁶ of irradiated haNK003 vs. unirradiated haNK003 at different time points. 6 Total intracellular IL-2 content per cell (pg) (mean and standard deviation of two readings [StDev]) TIFF2026063125000010.tif118142h-time

[0092] Cells were subjected to hypotonic shock to stimulate IL-2 release during cellular necrosis. This level was compared to the total intracellular IL-2 concentration measured in cell lysates prepared using surfactant-based methods. The solubilized IL-2 concentrations measured by the two methods (sandwich ELISA or multiplex ELISA) differed by approximately 10-fold for the same sample (Table 5, and Figures 10A and 10B). The IL-2 concentrations in lysates from hypotonic shock were either 181.93–289.54 pg / cell⁶⁶ (sandwich ELISA) or 2619.15–3301.02 pg / cell⁶⁶ (multiplex ELISA), which correspond to an average of 14% (sandwich ELISA) and 27% (multiplex ELISA) of the total intracellular IL-2 concentration measured in cell lysates prepared using surfactant-based lysis.

[0093] (Table 5) Amount of soluble IL-2 per 1 x 10⁶ cells (pg) of haNK003 cells (mean and standard deviation of two readings [StDev]) TIFF2026063125000011.tif81142

[0094] The IL-2 quantification values ​​from multiplex ELISA were 5–10 times higher than those from sandwich ELISA. While the absolute values ​​from these two methods varied, the trends were consistent between both datasets and are summarized below. This data will be useful in further developing the product, enabling NantKwest to continue characterizing IL-2 secretion and intracellular IL-2 levels from haNK003 cells.

[0095] In summary, haNK003 cells release detectable amounts of IL-2 into the culture medium (10–40 pg / hour per million cells), and the amount of IL-2 released by viable cells under steady-state culture conditions corresponds to less than 10% of the average total intracellular IL-2 storage.

[0096] Irradiation of haNK003 cells with a dose of 10 Gy increased the amount of IL-2 released over a 48-hour period, which likely reflects the presence of dead cells. Furthermore, the irradiation did not cause a sudden release of IL-2, but rather a gradual, time-dependent release.

[0097] To stimulate IL-2 release during necrotic cell death, haNK003 cells were subjected to hypotonic shock. The amount of IL-2 released under these conditions corresponds to either 14% or 27% of the total intracellular IL-2 determined in Triton X-100 lysate (which solubilizes proteins from all intracellular compartments).

[0098] Overall, haNK003 cells secrete low levels of IL-2 (493.8 pg / mL in irradiated cells and 276.1 pg / mL in unirradiated cells over 6 hours, averaged across all experiments from both methods). Taken together, the low levels of IL-2 secreted by haNK003 cells, the extremely short half-life of IL-2 in plasma, and the lack of persistence in vivo from irradiated haNK003 cells suggest that IL-2 release by injected haNK003 is unlikely to cause clinical adverse effects.

[0099] The effects of irradiation on in vitro proliferation capacity and functionality were tested in preliminary development studies using formulated cells, demonstrating that haNK003 cells exhibit limited proliferation in vitro (less than 0.1% of cells), and that levels of cytotoxic activity and ADCC activity are maintained for at least 6 hours after irradiation.

[0100] Pre- and post-irradiation IL-2 secretion and intracellular IL-2 levels of haNK003 cells demonstrate that haNK003 cells secrete low levels of IL-2. HaNK003 cells, whether irradiated or unirradiated, do not release amounts of IL-2 that are expected to have adverse effects in humans.

[0101] Example 4 Tolerance and tumorigenicity of single-dose haNK003 cells administered intravenously Natural killer (NK) cells are effective cytotoxic effector cells for cancer therapy and are potentially effective against viral infections. NantKwest has successfully established a proprietary NK cell-based platform for producing GMP-grade activated NK (aNK cells). aNK cells are being actively pursued clinically for cell therapy in patients with a variety of advanced hematological malignancies and solid tumors. Recently, a GMP-grade, plasmid-transfected, high-affinity CD16 receptor-expressing NK-92 variant has been developed using a novel transfection vector containing the ER IL-2 gene, which enables the resulting haNK003 cells to proliferate independently of IL-2. High-affinity CD16 receptor expression allows haNK003 cells to exhibit high antibody-dependent cell-mediated cytotoxicity (ADCC) against target cell lines that could not be killed by parental NK-92 cells in combination with rituximab, trastuzumab, and daratumumab. One cell clone was selected to create the master cell bank haNK003, which is currently in clinical development.

[0102] material and method Eighteen (18) NOD.CB17-Prkdc scid / J(NOD / SCID) mice (9 males and 9 females) were used to investigate the tolerance and tumorigenicity of single-dose haNK003 cells administered intravenously in NOD / SCID mice. The mice were obtained from Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US).

[0103] Eighteen NOD / SCID mice were selected and randomly assigned to three groups of six mice each (three males and three females) based on animal weight. The mice were administered intravenously as a single dose of PBS, unirradiated haNK003 cells, or haNK003 cells irradiated to 10 Gy, as shown in Table 6. The animals were then monitored by daily observation and twice-weekly weight measurements. After five weeks, the animals were euthanized, major organs were collected, and processed for further histopathological examination and immunohistochemical analysis using anti-CD56 antibodies.

[0104] (Table 6) Study Design TIFF2026063125000012.tif56142

[0105] For cell culture, haNK003 cells were cultured in X-Vivo 10 medium (catalog number BE02-055Q) supplemented with 5% thermo-inactivated human AB serum (catalog number IPLA-SERAB-HI, Innovative Research), 100 U penicillin / ml, and 100 μg / ml streptomycin (Corning, catalog number 30-002-CI).

[0106] Regarding irradiation, haNK003 cells in the exponential growth phase were harvested, and the number of viable cells and viability were calculated. On the appropriate day, half of the haNK003 cells were treated with JL Shephard Mark 1 Model 68. 137 The sample was irradiated with a dose of 1000 cGy using a Cs irradiator (service provided by the Department of Radiation Oncology, the University of California, Irvine, CA 92697).

[0107] For cell preparations for administration, unirradiated or irradiated haNK003 cells were maintained on ice during transport to the animal facility (1124 W. Carson Street, Torrance, CA, 90502). The cells were washed twice with cold PBS, then resuspended in an appropriate amount of cold PBS, and passed through a 40 μm cell strainer to produce single-cell preparations with a final cell density of 5 x 10⁷ cells / ml. Cell viability was determined using a Vi-CELL cell viability analyzer, and only cells with a viability of over 85% were used for this study. These unirradiated or irradiated haNK003 cells were then stored at room temperature for intravenous administration to appropriate animal groups.

[0108] Eighteen NOD / SCID mice were selected and randomly assigned to three groups of six mice each (three males and three females), based on the animals' body weight.

[0109] On the appropriate day, each animal in group A received a specific amount of PBS. The dose was 200 μl regardless of the individual animal's body weight. As indicated in the study protocol, the route of administration was intravenous infusion via the tail vein, and the administration schedule was a single dose. On the appropriate day, each animal in groups B and C received 1 x 10¹⁶ μl of PBS in 200 μl. 7 Each patient received one unirradiated haNK003 cell and one irradiated haNK003 cell, respectively. The dose was 200 μl, administered intravenously via the tail vein; the administration schedule was a single dose.

[0110] The animals were observed once a day for their overall appearance. Clinical observations were performed and recorded twice a day. After the procedure, the animals were routinely monitored for effects on normal behavior such as mobility, food and water consumption (by visual estimation), and body weight (gain / loss).

[0111] Summary statistics, including the mean and standard error of the mean (SEM), were provided for the body weight of animals in each group at each time point. Statistical analysis of differences in animal body weight change between groups was evaluated using a two-way repeated measures ANOVA followed by a Bonferroni test. All data were analyzed using GraphPad Prism software version 5. A p < 0.05 was considered statistically significant.

[0112] result The cells were administered intravenously as a monotherapy, 1 x 10⁶ 7 The doses of unirradiated and irradiated haNK003 cells were well tolerated, with maximum mean weight loss of 5.2% and 4.4%, respectively. As shown in Table 7 and Figure 11, irradiated haNK003 cells and unirradiated haNK003 cells (1 x 10⁶) were well tolerated compared to the PBS-treated control group. 7 Regardless of whether the treatment was administered as a single dose, there was no significant weight loss in NOD / SCID mice. As shown in Table 7, no treatment-related deaths occurred in any of the treatment groups during the 5-week observation period.

[0113] Regardless of whether the mice were male or female, no visible tumor masses were found in any of the evaluated tissues and organs, including the brain, heart, liver, lungs, kidneys, spleen, and thymus, in any of the treatment groups. Results from histological analysis and IHC staining with anti-CD56 antibody confirmed the absence of any lymphoid aggregates associated with haNK003 cells in the tissues and organs, including the brain, bone marrow, heart, liver, lungs, kidneys, spleen, and thymus, suggesting that neither unirradiated nor irradiated haNK003 cells possess tumorigenic potential in NOD / SCID mice.

[0114] Pathological examination of all specimens obtained in this study, including the brain, bone marrow, heart, liver, lungs, kidneys, spleen, and thymus, showed no significant toxicity related to haNK003 treatment in either the group treated with unirradiated haNK003 cells or the group treated with irradiated haNK003 cells, compared to the group treated with PBS.

[0115] (Table 7) Effects of intravenously administered haNK003 on animal body weight, mortality rate, and tumorigenesis in NOD / SCID mice TIFF2026063125000013.tif38142Note: a MWL: Maximum weight loss; b p-value for PBS treatment (repeated measures two-way ANOVA followed by Bonferroni test); c (n / total): The number of animal deaths related to treatment out of the total number of animals in each group.

[0116] The cells were administered intravenously as a monotherapy, 1 x 10⁶ 7 Both irradiated and unirradiated haNK003 cells at doses were well tolerated in both male and female NOD / SCID mice. There was no significant weight loss associated with either treatment with irradiated or unirradiated haNK003. No treatment-related deaths occurred in any of the treatment groups during the 5-week observation period. No significant pathological changes were observed in major organs, including the brain, bone marrow, heart, liver, lungs, kidneys, spleen, and thymus. Most importantly, neither irradiated nor unirradiated haNK003 cells possessed tumorigenic potential in both male and female NOD / SCID mice.

[0117] Example 5 Tolerance and tumorigenicity of repeatedly administered intravenously administered haNK003 cells material and method Eighteen NOD.CB17-Prkdcscid / J (NOD / SCID) mice (9 males and 9 females) were used to investigate the tolerance and tumorigenicity of a single intravenous dose of haNK003 cells in NOD / SCID mice. The mice were obtained from Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US).

[0118] Eighteen NOD / SCID mice were selected and randomly assigned to three groups of six mice each (three males and three females) based on animal weight. The mice were administered intravenously once a week for four weeks, with repeated doses of PBS, unirradiated haNK003 cells, or haNK003 cells irradiated at 10 Gy, as shown in Table 8. The animals were then monitored by daily observation and twice-weekly weight measurements. After five weeks, the animals were euthanized, major organs were collected, and processed for further histopathological examination and immunohistochemical (IHC) analysis using anti-CD56 antibodies.

[0119] (Table 8) Study Design TIFF2026063125000014.tif49128

[0120] For cell culture, haNK003 cells were cultured in X-Vivo 10 medium (catalog number BE02-055Q) supplemented with 5% thermo-inactivated human AB serum (catalog number IPLA-SERAB-HI, Innovative Research), 100 U penicillin / ml, and 100 μg / ml streptomycin (Corning, catalog number 30-002-CI).

[0121] Regarding irradiation, haNK003 cells in the exponential growth phase were harvested, and the number of viable cells and viability were calculated. On the appropriate day, half of the haNK003 cells were treated with JL Shephard Mark 1 Model 68. 137The sample was irradiated with a dose of 1000 cGy using a Cs irradiator (service provided by the Department of Radiation Oncology, the University of California, Irvine, CA 92697).

[0122] For cell preparations for administration, unirradiated or irradiated haNK003 cells were maintained on ice during transport to the animal facility (1124 W. Carson Street, Torrance, CA, 90502). The cells were washed twice with cold PBS, then resuspended in an appropriate volume of cold PBS, and passed through a 40 μm cell strainer to produce single-cell preparations with a final cell density of 5 x 10⁷ cells / ml. Cell viability was determined using a Vi-CELL cell viability analyzer, and only cells with a viability of over 85% were used for this study. These unirradiated or irradiated haNK003 cells were then stored at room temperature for intravenous administration to appropriate animal groups.

[0123] Eighteen NOD / SCID mice were selected and randomly assigned to three groups of six mice each (three males and three females), based on the animals' body weight.

[0124] On appropriate days, each animal in group A received 200 μl of PBS, regardless of the individual animal's body weight. As shown in the study protocol (Appendix 1), the administration route was intravenous infusion via the tail vein, and the administration schedule was once a week for a total of four weeks. Animals in groups B and C each received 1 x 10⁷ cells of unirradiated and irradiated haNK003 cells in 200 μl of PBS, respectively. As shown in the study protocol, the dose was 200 μl, the administration route was intravenous infusion via the tail vein, and the administration schedule was once a week for a total of four weeks.

[0125] The animals were observed once a day for their overall appearance. Clinical observations were performed and recorded twice a day. After the procedure, the animals were routinely monitored for effects on normal behavior such as mobility, food and water consumption (by visual estimation), and body weight (gain / loss).

[0126] Summary statistics, including the mean and standard error of the mean (SEM), were provided for the body weight of animals in each group at each time point. Statistical analysis of differences in animal body weight change between groups was evaluated using a two-way repeated measures ANOVA followed by a Bonferroni test. All data were analyzed using GraphPad Prism software version 5. A p < 0.05 was considered statistically significant.

[0127] result As monotherapy, unirradiated or irradiated haNK003 cells administered intravenously once weekly for 4 weeks were well tolerated, with maximum mean weight loss of 3.4% and 4.9%, respectively. As shown in Table 9 and Figure 12, unirradiated haNK003 cells and irradiated haNK003 cells (1 x 10⁶) were well tolerated compared to the PBS-treated control group, regardless of whether the mice were male or female. 7 Regardless of which of the two drugs was administered, there was no significant weight loss in NOD / SCID mice. As summarized in Table 9, no treatment-related deaths occurred in any of the treatment groups during the 5-week observation period.

[0128] (Table 9) Effects of haNK003 cells on animal body weight, mortality rate, and tumorigenesis in NOD / SCID mice TIFF2026063125000015.tif34142Note: a MWL: Maximum weight loss; b p-value for PBS treatment (repeated measures two-way ANOVA followed by Bonferroni test); c (n / total): The number of animal deaths related to treatment out of the total number of animals in each group.

[0129] Macropathological and histopathological examinations of all specimens obtained in this study, including the brain, bone marrow, heart, liver, lungs, kidneys, spleen, and thymus, showed no significant toxicity related to haNK003 treatment in these organs, in both the groups treated with unirradiated and irradiated haNK003 cells, compared to the PBS-treated control group. Splenomegaly was not observed in any animals. No tumor masses were found macroscopically in any treatment group. Results from IHC using anti-CD56 antibody confirmed the absence of any haNK003 cell-associated lymphoid agglutination in tissues and organs, including bone marrow, brain, liver, lungs, heart, kidneys, spleen, and thymus, during the 5-week follow-up period, suggesting that in NOD / SCID mice, there was no leukemia or tumorigenesis associated with either irradiated or unirradiated haNK003 cells during these 5 weeks.

[0130] No edema, degeneration, or necrosis was present in any of these tissues. Compared to the PBS control group, extremely localized, mild steatosis and minimal steatosis were observed in the liver tissue of the unirradiated and irradiated haNK003 treatment groups, respectively. It is also noteworthy that the liver parenchyma in the PBS treatment group, the unirradiated haNK003 treatment group, and the irradiated haNK003 treatment group showed slight, minute lesions of mixed inflammatory cells, including neutrophils and mononuclear cells. Since these small cluster cells were CD56-negative, it is highly likely that these mixed inflammatory cells were due to repeated treatment (tail vein infusion) and not related to the haNK003 treatment.

[0131] In summary, both unirradiated and irradiated HaNK003 cells were well tolerated. This administration regimen did not result in any significant toxicity or tumorigenic issues associated with haNK003 treatment in NOD / SCID mice.

[0132] Example 6: Comparison of innate cytotoxic activity between aNK and haNK003 The primary mechanism by which natural killer (NK) cells kill target cells is through the formation of cell junctions and the subsequent secretion of perforin and granzyme. This then induces apoptosis within the target cell, leading to plasma membrane breakdown and cell death. Target cells can be recognized by expressing stress antigens that are characteristic of virus-infected and / or transformed cells. The recognition of target cells and subsequent death via the involvement of stress antigens at activating receptors on NK cells is referred to as innate (or direct) cytotoxicity.

[0133] Since spontaneous cell injury is a major functional characteristic of NK cells, analyzing this functionality in cell variants and comparing it with activated NK-92 cell (aNK) activity will help confirm the impact of specific genetic modifications of aNK cells.

[0134] material and method Six representative cell lines (6) of humoral and solid tumors were selected as targets. The targets were also selected to exhibit a range of sensitivity to death in aNK cells; SR-91 was relatively insensitive to death, K562 was highly sensitive to death, and the others fell somewhere in between. Target cells and effector cells (haNK003 or aNK) were co-incubated for 4 hours at 37°C, and target cell death was determined by flow cytometry using a proprietary method for determining specific cytotoxicity of effector cells against target cells stained with PKH67 fluorescent dye. PKH67 Fluorescent Cell Linker Kits use proprietary membrane labeling technology (Sigma-Aldrich) to stably incorporate a green fluorescent dye (PKH67) with a long aliphatic tail into the lipid region of the cell membrane. Due to its longer aliphatic carbon tail, PKH67 exhibits reduced cell-to-cell translocation. PKH67 is suitable for cytotoxicity assays using propidium iodide as a viability probe. Staining with propidium iodide distinguishes dead target cells (double-stained) from dead effector cells (aNK or haNK cells).

[0135] Regarding cell culture, aNK cells were cultured in X-Vivo 10 medium supplemented with 5% thermo-inactivated human AB serum (from CMV-negative test donors) and 500 IU / ml recombinant human IL-2. aNK cultures were passaged every 1–4 days to maintain a cell density of >10e5 cells / mL and <10e6 cells / mL. haNK003 cells were cultured in X-Vivo 10 medium supplemented with 5% thermo-inactivated human AB serum (from CMV-negative test donors) without IL-2. haNK003 cultures were passaged every 1–4 days to maintain a cell density of >10e5 cells / mL and <10e6 cells / mL. K562, Daudi, DOHH2, HL-60, SR-91, and SKOV3 cells were cultured in RPMI-1640 supplemented with 10% thermo-inactivated fetal bovine serum (FBS) and an antibiotic / antifungal mixture. Cells growing in suspension were subculturized by simple dilution, while adherent cells (SKOV3) were subculturized by trypsinizing the culture using TrypLE®. Subculturing was performed every 2–5 days (depending on the cell line-specific doubling time) or when the culture medium turned yellow (acidic), indicating medium consumption.

[0136] For sample preparation, cell lines growing in suspension were resuspended by pipetting the cell culture up and down. Adherent target cell lines (SKOV3) were enzymatically separated from the culture vessel using TrypLE®, and the trypsin-treated cell pellet was resuspended by pipetting up and down. Cell viability was determined by manual measurement (trypan blue exclusion method). Dilution of target and effector cells to the required cell concentration was performed in RPMI-1640 supplemented with 10% heat-inactivated FBS and antibiotics / antifungal agents. Effector and target cells were mixed in 96-well plates in different effector-to-target ratios (E:T of 20:1, 10:1, 5:1, 2.5:1, 1.25:1, 0.62:1, 0.31:1, and 0.15:1) and co-incubated for 4 hours in a 37°C incubator under a 5% CO2 atmosphere.

[0137] Samples were analyzed on a MACSQuant flow cytometer (Miltenyi) using the B1 (FITC) and B3 (PerCP-Vio700 / PI) fluorescence channels. Targets alone, both in the absence of PI and in the presence of PI, were used to determine the B1 / B3 correction parameters.

[0138] The cytotoxicity percentage was calculated using the formula = [(percentage of FITC+ / PI+ cells in the sample) - (percentage of FITC+ / PI+ cells in the target alone in the presence of PI)] / [100 - (percentage of FITC+ / PI+ cells in the target alone in the presence of PI)].

[0139] result The aNK cells and haNK003 cells used in this study were thawed on July 22, 2016. All target cell cultures used in this study were less than 8 weeks old. Target cell and effector cell cultures were passaged no more than 48 hours before the assay.

[0140] The results shown in Figures 13–18 confirm the sensitivity of the target cell lines to aNK-mediated death, with K562 being the most sensitive (75% specific lysis at a low effector-to-target ratio of 1:1). Daudi, DOHH2, and HL-60 showed intermediate sensitivity, requiring higher effector-to-target ratios (10:1) to achieve 65–80% specific lysis. SKOV3 and SR-91 were the most resistant, requiring effector-to-target ratios greater than 10:1 to achieve approximately 40% specific lysis. In each case, the innate cytotoxic activity of haNK003 was comparable to that of aNK and, overall, followed the same activity profile within the experimental margin of error.

[0141] aNK cells and haNK003 cells exhibited equivalent cytotoxic activity against the six cancer cell lines tested, demonstrating that the innate cytotoxic activity of aNK cells is essentially the same despite the genetic modifications used to create haNK003 cells. haNK003 cells and aNK cells are equivalent in terms of innate cytotoxic activity.

[0142] Example 7: Evaluation of the antitumor activity of haNK003 in the MDA-MB-453 human breast cancer subcutaneous mouse model. In this study, the antitumor activity of haNK003 cells as a monotherapy was found in female NOD.Cg-Prkdc scid Il2rg tm1Wjl It was evaluated in a subcutaneous xenograft model of human breast cancer using / SzJ (NOD scid gamma, NSG) mice.

[0143] material and method 12 NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NOD scid gamma, NSG) mice were used to evaluate the antitumor activity of haNK003 in the MDA-MB-453 human breast cancer subcutaneous (sc) xenograft model. Mice were obtained from Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US).

[0144] A subcutaneous xenograft model of MDA-MB-453 HER2-positive human breast cancer was constructed in female NSG mice. The average tumor size was approximately 100 mm. 3 Treatment was initiated once the target was reached, and the antitumor activity of haNK003 cells as a monotherapy was evaluated in this xenograft model. Other test materials were evaluated in parallel under protocol LABC-X01612, but only the results of haNK003 compared to PBS are presented herein. The haNK003 treatment groups and the design of the dosing regimens are described in Table 10.

[0145] (Table 10) Study Design TIFF2026063125000016.tif46142

[0146] Regarding the culture of tumor cells, MDA-MB-453 human breast cancer cells (ATCC, catalog number HTB-131) were cultured in Leibovitz's L-15 medium (ATCC, catalog number 30-2008) prepared by ATCC, supplemented with 10% thermally inactivated FBS (GeneTex, catalog number GTX73252), 100 U penicillin / ml, and 100 μg / ml streptomycin (Corning, catalog number 30-002-CI).

[0147] For tumor cell injection, each animal was weighed, and then 0.1 ml of 1.0 x 10⁸ MDA-MB-453 human breast cancer cells per 1 mL of 50% Matrigel (Corning, catalog number 354234) was subcutaneously injected into the left and right flanks using a 25-gauge needle. Cell viability was determined using a Vi-CELL cell viability analyzer, and only cells with a viability of over 95% were used for this in vivo study.

[0148] Regarding the culture of haNK003 cells, the haNK003 cells were cultured in X-Vivo 10 medium (Lonza, catalog number BE02-055Q) supplemented with 5% thermally inactivated human AB serum (Innovative Research, catalog number IPLA-SERAB-HI), 100 U penicillin / ml, and 100 μg / ml streptomycin.

[0149] Regarding irradiation, haNK003 cells in the exponential growth phase were harvested, and the number of viable cells and viability were calculated. On the appropriate day, haNK003 cells were treated with JL Shephard Mark 1 Model 68. 137The sample was irradiated with a dose of 1000 cGy using a Cs irradiator (service provided by the Department of Radiation Oncology, the University of California, Irvine, CA 92697).

[0150] Regarding the cell preparations for administration, the irradiated haNK003 cells were maintained on ice during transport to the animal facility (1124 W. Carson Street, Torrance, CA, 90502). The cells were washed twice with cold PBS, then resuspended in an appropriate amount of cold PBS, and passed through a 40 μm cell strainer (Corning, catalog no. 431750) to produce single-cell preparations with final cell densities of 1.25 x 10⁷ cells / ml or 5 x 10⁷ cells / ml, respectively. These irradiated haNK003 cells were then stored at room temperature for intravenous administration to the appropriate animal groups.

[0151] Twelve NSG mice were selected and randomly assigned to three study groups, each containing four mice, based on appropriate tumor size. Randomization was based on the total tumor volume and body weight of each animal. For this efficacy study, randomization was performed and treatment was initiated when the mean tumor size reached approximately 100 mm³.

[0152] The dose was 200 μl regardless of the individual animal's body weight. As indicated in the study protocol, the administration route was intravenous infusion via the tail vein, and the administration schedule was twice a week for a total of four weeks. On appropriate days, each animal in groups F and G received 2.5 x 10⁶ and 1 x 10⁷ irradiated haNK003 cells, respectively, in 200 μl of PBS. The dose was 200 μl, the administration route was intravenous infusion via the tail vein, and the administration schedule was twice a week for a total of four weeks.

[0153] Animals were observed once daily for overall appearance. Clinical observations were performed twice daily and recorded. Animals were routinely monitored for normal behavior such as mobility, food and water consumption (by visual observation), and effects on body weight (gain / loss) after treatment.

[0154] Tumor size was measured three-dimensionally twice weekly using a digital handheld caliper before the first administration (if the tumor had appeared), and then twice weekly before euthanasia. The primary endpoint was suppression or reduction of tumor growth. Tumor volume was expressed in mm3 using the following formula: V = 0.5 x L x W x H, where L, W, and H are the length, width, and height of the tumor, respectively. Tumor volume was then used for calculation of the T / C value. T / C (%) = ΔT / ΔC x 100, where ΔT and ΔC are the changes in mean tumor volume between the observation day and the first day of measurement in the treatment group and control group, respectively.

[0155] Summary statistics including mean and standard error of the mean (SEM) were provided for tumor volume or animal body weight of each group at each time point. Statistical analysis of differences in tumor volume or animal body weight changes between groups was evaluated using two-way ANOVA of repeated measures followed by the Bonferroni test. All data were analyzed using GraphPad Prism software version 5. p < 0.05 was considered statistically significant.

[0156] result Data are shown on the antitumor activity of haNK003 as a single agent in subcutaneous MDA-MB-453 HER2-positive human breast cancer xenografts in female NSG mice. A summary of the results is presented in Table 11. As shown in Table 11 and Figure 19, cells 2.5 x 10 6 individuals or 1.0 x 10 7haNK003 cells, administered intravenously as monotherapy twice weekly for 4 weeks at a dose of 10¹¹, significantly suppressed tumor growth, with T / C values ​​of 17.4% and 1.3% (p = 0.043 and p = 0.006, respectively, compared to the PBS group). As shown in Table 11 and Figure 20, cells 2.5 x 10¹¹ 6 1 or 1.0 x 10 7 The individual doses of HaNK003 were well tolerated, with maximum mean weight loss rates of 0.6% and 5.6%, respectively (p = 0.203 and p = 0.085 compared to the PBS group). As summarized in Table 11, no deaths associated with haNK003 treatment occurred in this study.

[0157] (Table 11) Antitumor activity of haNK003 cells in the MDA-MB-453 human breast cancer subcutaneous xenograft model in female NSG mice TIFF2026063125000017.tif30130 Note: a T / C (%) was calculated using the following formula: T / C (%) = ΔT / ΔC x 100, where ΔT and ΔC are the change in mean tumor volume between day 26 and day 1 of measurement for the treatment group and the control group, respectively. b MWL: Maximum body weight loss in animals. c P-value for PBS treatment (two-way repeated measures ANOVA followed by Bonferroni test).

[0158] cells 2.5 x 10 6 1 or 1.0 x 10 7 Irradiated haNK003 cells at doses as monotherapy significantly suppressed tumor growth in a female NSG MDA-MB-453 HER2-positive human breast cancer xenograft model. Both doses of irradiated haNK003 cells were well tolerated. No deaths related to haNK003 treatment occurred.

[0159] Example 8: The expression of hypoxia-related genes is not reduced in haNK cells. The lytic activity of natural killer (NK) cells is suppressed in vitro under hypoxic conditions (1% O2) and is associated with the downregulation of NKG2D, perforin, and granzymes. There is some variability in the sensitivity of NK cells from normal donors to hypoxia (1% O2). However, the lytic activity of NK cells can be partially restored in vitro by activation with exogenous IL-2 (16 hours, 1000 IU / ml). Furthermore, NK cells retain their ADCC capacity under 1% oxygen conditions.

[0160] To determine whether hypoxic conditions alter gene expression in haNK cells, RNA expression was determined in NK cell populations from three normal donors and in haNK cells exposed to 20% or 0% O2 for 5 hours. NK cell populations from three patient donors (950, 962, and 996) were compared to haNK cells under two conditions: 0% oxygen and 20% oxygen. Clustering of samples between pairs revealed distinct clusters that distinguished haNK from donor NK cells (see Figure 21). One patient sample appeared to show little change in expression under hypoxic or pre-hypoxic conditions. Specifically, the expression of 962 under 20% oxygen conditions appeared very similar to that of 996 under 0% oxygen conditions, and to that of 962 under 0% oxygen conditions (see Figure 21). The genes showing the greatest variability across samples are shown in Figure 22. The genes showing the greatest change between 20% and 0% oxygen conditions are shown in Figure 23. In haNK cells, hypoxia-related genes that show no change in expression between 20% oxygen and 0% oxygen conditions are shown in Figure 24. These same hypoxia-related genes are shown to have reduced expression in samples 950, 962, and 996, although not as much in sample 950. See Figure 24.

[0161] Example 9: CD16 expression is more stable in hank003 cells. To confer superior cytotoxicity by sequentially killing target cells during antibody-dependent cell-mediated cytotoxicity (ADCC), stable expression of CD16 is desirable. HaNK-003 (containing ER-IL-2) expresses CD16 at higher levels compared to peripheral blood NK cells (donor NK cells) following activation with PMA (phorbol-12-myristart-13-acetate) or in response to stimulation with K562 target cells. Furthermore, flow cytometry measurements of CD16 levels after ADCC showed that CD16 levels in hank003 cells were largely unaffected during and after ADCC.

[0162] It is known that activation of NK cells with PMA / ionomycin induces activation of CD16-specific proteases and CD16 cleavage, leading to downregulation of CD16 expression levels in NK cells. To determine the effects of PMA / ionomycin, both haNK003 cells and donor NK cells were exposed to 40 nM PMA and 669 nM ionomycin for 1 hour, after which CD16 expression levels were examined. PMA / ionomycin treatment resulted in a 94.36% ± 3.00 downregulation of CD16 expression in donor NK cells, while in haNK003 cells, the treatment resulted in only a 30% ± 0.04 downregulation, or three times less CD16 downregulation compared to donor NK cells (Figure 25).

[0163] It is also known that co-culture of NK cells and K562 cells stimulates CD16 cleavage proteases, which lead to the loss of CD16 surface expression in NK cells. Therefore, both donor NK cells and haNK003 cells were cultured with K562 cells, and then CD16 expression was measured 4 hours after co-culture. Standard co-culture conditions (effector:target = 1:1) for haNK003 cells and K562 cells result in complete cytotoxic death of target cells within 4 hours. CD16 expression was measured again after a further 24 hours to allow for the recovery of CD16 expression in haNK003 cells and donor NK cells, and the percentage of CD16 recovery in haNK003 cells and donor NK cells was determined. CD16 expression levels were observed to be downregulated by 60.25% ± 0.9% in donor NK cells 4 hours after co-culture. However, CD16 expression in haNK003 cells was downregulated by only 4.9% ± 2.57. After 24 hours, CD16 downregulation in donor NK cells was 57.54% ± 26.82, while in haNK003 cells it was only 2.78% ± 3.5, which was close to the original CD16 level (Figure 26).

[0164] CD16 expression levels in haNK003 cells were also measured after antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC was performed by incubating haNK003 cells with DOHH-2 (CD20+ human lymphoma B cell line) in the presence of rituximab (a cytolytic monoclonal antibody against CD20), followed by measurement of CD16 expression. After ADCC, CD16 expression was downregulated to less than 10% in haNK003 cells (Figures 27A and 27B). The presence of high levels of CD16 even after ADCC indicates that CD16 expression in haNK003 cells is highly stable.

[0165] Sequence information SEQUENCE LISTING <110> IMMUNITYBIO, INC. <120> Modified NK-92 haNK003 Cells for the Clinic <150> US 62 / 468,890 <151> 2017-03-08 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 5491 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 1 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 60 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct 120 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt 180 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga 240 atctgcttag ggttaggcgt tttgcgctgc ttcgggatcc gctgaccaaa agagcaccaa 300 aggcgccctg accttcagcc cctacctgcg ctccggtgcc cgtcagtggg cagagcgcac 360 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag 420 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga 480 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg 540 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac 600 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga 660 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc 720 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg 780 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg 840 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct 900 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg 960 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc 1020 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg 1080 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg 1140 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc 1200 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga 1260 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt 1320 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga 1380 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg 1440 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt 1500 gtcgtgataa tacgactcac tatagggaga cccaagctgg aattcgccac catgtggcag 1560 ctgctgctgc ctacagctct cctgctgctg gtgtccgccg gcatgagaac cgaggatctg 1620 cctaaggccg tggtgttcct ggaaccccag tggtacagag tgctggaaaa ggacagcgtg 1680 accctgaagt gccagggcgc ctacagcccc gaggacaata gcacccagtg gttccacaac 1740 gagagcctga tcagcagcca ggccagcagc tacttcatcg acgccgccac cgtggacgac 1800 agcggcgagt atagatgcca gaccaacctg agcaccctga gcgaccccgt gcagctggaa 1860 gtgcacatcg gatggctgct gctgcaggcc cccagatggg tgttcaaaga agaggacccc 1920 atccacctga gatgccactc ttggaagaac accgccctgc acaaagtgac ctacctgcag 1980 aacggcaagg gcagaaagta cttccaccac aacagcgact tctacatccc caaggccacc 2040 ctgaaggact ccggctccta cttctgcaga ggcctcgtgg gcagcaagaa cgtgtccagc 2100 gagacagtga acatcaccat cacccagggc ctggccgtgt ctaccatcag cagctttttc 2160 ccacccggct accaggtgtc cttctgcctc gtgatggtgc tgctgttcgc cgtggacacc 2220 ggcctgtact tcagcgtgaa aacaaacatc agaagcagca cccgggactg gaaggaccac 2280 aagttcaagt ggcggaagga cccccaggac aagtgaaatt ccgcccctct cccccccccc 2340 cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg 2400 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga 2460 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa 2520 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa 2580 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct 2640 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg 2700 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg 2760 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca 2820 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg 2880 acgtggtttt cctttgaaaa acacgataac cgccaccatg taccggatgc agctgctgag 2940 ctgtatcgcc ctgtctctgg ccctcgtgac caacagcgcc cctaccagca gcagcaccaa 3000 gaaaacccag ctgcagctgg aacatctgct gctggacctg cagatgatcc tgaacggcat 3060 caacaactac aagaacccca agctgacccg gatgctgacc ttcaagttct acatgcccaa 3120 gaaggccacc gaactgaaac atctgcagtg cctggaagag gaactgaagc ccctggaaga 3180 agtgctgaac ctggcccaga gcaagaactt ccacctgagg cccagggacc tgatcagcaa 3240 catcaacgtg atcgtgctgg aactgaaagg cagcgagaca accttcatgt gcgagtacgc 3300 cgacgagaca gctaccatcg tggaatttct gaaccggtgg atcaccttct gccagagcat 3360 catcagcacc ctgaccggct ccgagaagga cgagctgtga gcggccgccc gctgatcagc 3420 ctcgaacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 3480 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 3540 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 3600 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 3660 gtatcttatc atgtctgtgc ggtgggctct atggcttctg aggcggaaag aaccagctgg 3720 ggctctaggg ggtatccccg gatcctgagc aaaaggccag caaaaggcca ggaaccgtaa 3780 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 3840 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 3900 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 3960 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 4020 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 4080 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 4140 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 4200 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 4260 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 4320 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 4380 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 4440 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 4500 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 4560 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 4620 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 4680 cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa 4740 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 4800 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 4860 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 4920 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 4980 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 5040 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 5100 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 5160 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 5220 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 5280 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 5340 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 5400 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 5460 ttattgtctc atgagcggat acatatttga a 5491 <210> 2 <211> 1872 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 2 gaattcgcca ccatgtggca gctgctgctg cctacagctc tcctgctgct ggtgtccgcc 60 ggcatgagaa ccgaggatct gcctaaggcc gtggtgttcc tggaacccca gtggtacaga 120 gtgctggaaa aggacagcgt gaccctgaag tgccagggcg cctacagccc cgaggacaat 180 agcacccagt ggttcccacaa cgagagcctg atcagcagcc aggccagcag ctacttcatc 240 gacgccgcca ccgtggacga cagcggcgag tatagatgcc agaccaacct gagcaccctg 300 agcgaccccg tgcagctgga agtgcacatc ggatggctgc tgctgcaggc ccccagatgg 360 gtgttcaaag aagaggaccc catccacctg agatgccact cttggaagaa caccgccctg 420 cacaaagtga cctacctgca gaacggcaag ggcagaaagt acttccacca caacagcgac 480 ttctacatcc ccaaggccac cctgaaggac tccggctcct acttctgcag aggcctcgtg 540 ggcagcaaga acgtgtccag cgagacagtg aacatcacca tcacccaggg cctggccgtg 600 tctaccatca gcagcttttt cccacccggc taccaggtgt ccttctgcct cgtgatggtg 660 ctgctgttcg ccgtggacac cggcctgtac ttcagcgtga aaacaaacat cagaagcagc 720 acccgggact ggaaggacca caagttcaag tggcggaagg acccccagga caagtgaaat 780 tccgcccctc tccccccccc ccctctccct cccccccccc taacgttact ggccgaagcc 840 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt 900 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc 960 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc 1020 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc 1080 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg 1140 cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct 1200 cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat 1260 ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc 1320 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgataa ccgccaccat 1380 gtaccggatg cagctgctga gctgtatcgc cctgtctctg gccctcgtga ccaacagcgc 1440 ccctaccagc agcagcacca agaaaaccca gctgcagctg gaacatctgc tgctggacct 1500 gcagatgatc ctgaacggca tcaacaacta caagaacccc aagctgaccc ggatgctgac 1560 cttcaagttc tacatgccca agaaggccac cgaactgaaa catctgcagt gcctggaaga 1620 ggaactgaag cccctggaag aagtgctgaa cctggcccag agcaagaact tccacctgag 1680 gcccagggac ctgatcagca acatcaacgt gatcgtgctg gaactgaaag gcagcgagac 1740 aaccttcatg tgcgagtacg ccgacgagac agctaccatc gtggaatttc tgaaccggtg 1800 gatcaccttc tgccagagca tcatcagcac cctgaccggc tccgagaagg acgagctgtg 1860 agcggccgcc cg 1872 <210> 3 <211> 765 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 3 atgtggcagc tgctgctgcc tacagctctc ctgctgctgg tgtccgccgg catgagaacc 60 gaggatctgc ctaaggccgt ggtgttcctg gaaccccagt ggtacagagt gctggaaaag 120 gacagcgtga ccctgaagtg ccagggcgcc tacagccccg aggacaatag cacccagtgg 180 ttccacaacg agagcctgat cagcagccag gccagcagct acttcatcga cgccgccacc 240 gtggacgaca gcggcgagta tagatgccag accaacctga gcaccctgag cgaccccgtg 300 cagctggaag tgcacatcgg atggctgctg ctgcaggccc ccagatgggt gttcaaagaa 360 gaggacccca tccacctgag atgccactct tggaagaaca ccgccctgca caaagtgacc 420 tacctgcaga acggcaaggg cagaaagtac ttccaccaca acagcgactt ctacatcccc 480 aaggccaccc tgaaggactc cggctcctac ttctgcagag gcctcgtggg cagcaagaac 540 gtgtccagcg agacagtgaa catcaccatc acccagggcc tggccgtgtc taccatcagc 600 agctttttcc cacccggcta ccaggtgtcc ttctgcctcg tgatggtgct gctgttcgcc 660 gtggacaccg gcctgtactt cagcgtgaaa acaaacatca gaagcagcac ccgggactgg 720 aaggaccaca agttcaagtg gcggaaggac ccccaggaca agtga 765 <210> 4 <211> 254 <212> PRT <213> artificial sequence <220> <223> synthetic construct <400> 4 Put Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Dear Leu Ile Dear Dear Gln Ala Dear Dear Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 5 <211> 483 <212> DNA <213> artificial sequence <220> <223> synthetic construct <400> 5 atgtaccgga tgcagctgct gagctgtatc gccctgtctc tggccctcgt gaccaacagc 60 gcccctacca gcagcagcac caagaaaacc cagctgcagc tggaacatct gctgctggac 120 ctgcagatga tcctgaacgg catcaacaac tacaagaacc ccaagctgac ccggatgctg 180 accttcaagt tctacatgcc caagaaggcc accgaactga aacatctgca gtgcctggaa 240 gaggaactga agcccctgga agaagtgctg aacctggccc agagcaagaa cttccacctg 300 aggcccaggg acctgatcag caacatcaac gtgatcgtgc tggaactgaa aggcagcgag 360 acaaccttca tgtgcgagta cgccgacgag acagctacca tcgtggaatt tctgaaccgg 420 tggatcacct tctgccagag catcatcagc accctgaccg gctccgagaa ggacgagctg 480 tga 483 <210> 6 <211> 160 <212> PRT <213> artificial sequence <220> <223> synthetic construct <400> 6 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr Gly Ser Glu Lys Asp Glu Leu 145 150 155 160

Claims

1. A population of modified NK-92 cells exhibiting antibody-dependent cell-mediated cytotoxicity (ADCC), containing heterogeneous nucleic acid molecules including both CD16 (SEQ ID NO:3) and IL-2 (SEQ ID NO:5), More than 90% of the cells in the population express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the population express CD3. A population of modified NK-92 cells.

2. The cell according to claim 1, wherein the nucleic acid molecule is a DNA molecule.

3. The cell according to claim 1, wherein the nucleic acid molecule comprises, from 5' to 3', a sequence encoding CD16, an IRES sequence, and a sequence encoding IL-2.

4. A cell according to any one of claims 1 to 3, wherein the cell contains SEQ ID NO:1 on chromosome 17.

5. A cell according to any one of claims 1 to 4, wherein the average doubling time of the cell is 55 to 70 hours.

6. The cells according to any one of claims 1 to 5, wherein the population of cells maintains an average doubling time of 1 to 2, 3, 4, 5, 10, 15, 20, or 25 days.

7. The cell according to any one of claims 1 to 6, wherein the population of cells can be passaged every 1, 2, 3, or 4 days.

8. A cell according to any one of claims 1 to 7, wherein the cell secretes IL-2 at a concentration of 10 to 60 pg / hour per 1 million cells.

9. The cell according to any one of claims 1 to 7, wherein the cell is an irradiated cell.

10. The cells according to any one of claims 1 to 9, wherein the cells exhibit reduced downregulation of CD16 expression compared to a control.

11. Cells according to any one of claims 1 to 9, wherein the cells maintain higher levels of CD16 after ADCC compared to a control.

12. A kit comprising a population of cells according to any one of claims 1 to 11.

13. The kit according to claim 12, further comprising an antibody.

14. A pharmaceutical composition comprising a population of cells according to any one of claims 1 to 11, and a pharmaceutically acceptable excipient.

15. A method for treating cancer in a subject, comprising the step of administering the pharmaceutical composition according to claim 14 to the subject.

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