MODIFIED NK-92 haNK003 CELLS FOR CLINIC

Abstract

To provide a pharmaceutical for treating cancer.SOLUTION: The present invention provides a pharmaceutical comprising a population of modified NK-92 cells, wherein the modified NK-92 cells comprise CD16 and IL-2 and comprise a specific sequence on chromosome 17, and more than 90% of the cells in the cell population express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the cell population express CD3.SELECTED DRAWING: Figure 1

Description

[Background technology]

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

[0002] NK-92 is a cytolytic cancer cell line that was found in the blood of subjects suffering from non-Hodgkin's lymphoma and was subsequently immortalized ex vivo. NK-92 cells are derived from NK cells, but while retaining the majority of activating receptors, they lack 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. Characterization of the NK-92 cell line is disclosed in International Publication WO 1998 / 49268 (Patent Document 1) and US Patent Publication No. 2002-0068044 (Patent Document 2). NK-92 cells are also being evaluated as a potential therapeutic agent in the treatment of certain cancers.

[0003] Although NK-92 cells retain almost all of the activation receptors and cytolytic pathways associated with NK cells, they do not express CD16 on their cell surface.CD16 is an Fc receptor that recognizes and binds to the Fc portion of antibodies to activate NK cells for ADCC effector mechanism.Unmodified NK-92 cells lack CD16 receptors and therefore cannot lyse target cells via ADCC mechanism. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 1998 / 49268 [Patent Document 2] U.S. Patent Application Publication No. 2002-0068044 Summary of the Invention

[0005] overview Provided herein are populations of modified NK-92 cells, compositions and kits comprising the cells, and methods of making and using the populations of cells. [The present invention 1001] A population of modified NK-92 cells with antibody-dependent cell-mediated cytotoxicity (ADCC) comprising a heterologous nucleic acid molecule comprising both CD16 (SEQ ID NO:3) and IL-2 (SEQ ID NO:5), greater than 90% of the cells in the population of cells express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the population of cells express CD3; Modified NK-92 cell populations. [The present invention 1002] The cell of the present invention 1001, wherein the nucleic acid molecule is a DNA molecule. [The present invention 1003] 1001. The cell of claim 1001, wherein the nucleic acid molecule comprises, from 5' to 3', a sequence encoding CD16, an IRES sequence, and a sequence encoding IL-2. [The present invention 1004] The cell of any one of claims 1001 to 1003, wherein the cell comprises SEQ ID NO:1 on chromosome 17. [The present invention 1005] The cell of any one of 1001 to 1004, having an average cell doubling time of 55 to 70 hours. [The present invention 1006] The cell of any of 1001 to 1005, wherein the population of cells maintains an average doubling time of 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, or 25 days. [The present invention 1007] The cell of any of claims 1001 to 1006, wherein the population of cells can be passaged every 1, 2, 3, or 4 days. [The present invention 1008] The cell of any of claims 1001 to 1007, wherein the cell secretes IL-2 at a concentration of 10 to 60 pg / hour per million cells. [The present invention 1009] The cell of any one of 1001 to 1007, wherein the cell is an irradiated cell. [The present invention 1010] The cell of any of claims 1001 to 1009, wherein the cell exhibits reduced downregulation of CD16 expression compared to a control. [The present invention 1011] The cell of any of claims 1001 to 1009, wherein the cell maintains a higher level of CD16 after ADCC compared to a control. [The present invention 1012] A kit comprising a population of any one of the cells of the present inventions 1001 to 1011. [The present invention 1013] The kit of claim 1012 further comprising an antibody. [The present invention 1014] A pharmaceutical composition comprising a population of any of the cells of the present inventions 1001 to 1011 and a pharma- ceutically acceptable excipient. [The present invention 1015] A method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition of the present invention. [Brief description of the drawings]

[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. Viability (%), cell density (cells / mL), and cumulative PDL were monitored during the expansion period. [Figure 2-1] Representative histograms of surface marker expression in aNK and haNK003 cells are shown. [Figure 2-2] Representative histograms of surface marker expression in aNK and haNK003 cells are shown. [Figure 3A] 1 is a graph showing the natural cytotoxicity of haNK003 cells against K562 cells. [Figure 3B] 1 is a graph showing the natural cytotoxicity of haNK003 cells against Raji cells. [Figure 3C] 1 is a graph showing the natural cytotoxicity of haNK003 cells against SKOV3 cells. [Figure 3D] 1 is a graph showing the natural cytotoxicity of haNK003 cells against SKBR3 cells. [Figure 4] Figure 4A is a graph showing the ADCC of haNK003 cells against Raji cells, Figure 4B is a graph showing the ADCC of haNK003 cells against SKOV3 cells, and Figure 4C is a graph showing the ADCC of haNK003 cells against SKBR3 cells. [Diagram 5] Graph showing the natural cytotoxic activity of irradiated vs. non-irradiated haNK003 cells against K562 cells. haNK003 cells were mock irradiated (solid line) or irradiated with 10 Gy. Cytotoxic activity of irradiated haNK003 against K562 cells was assayed at 6 hours (dashed line) or 24 hours (dotted line) after irradiation. [Figure 6] FIG. 1 is a graph showing the natural cytotoxic activity of irradiated vs. non-irradiated haNK003 cells against DOHH2 cells. haNK003 cells were mock 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) after irradiation. Note that data points for the 20:1 E:T ratio were not obtained for irradiated cells due to insufficient numbers of haNK003 cells due to cell death. [Figure 7]Graph showing ADCC activity of irradiated vs. non-irradiated haNK003 cells against DOHH2 cells. haNK003 cells were mock irradiated (closed symbols) or irradiated with 10 Gy (open symbols). ADCC activity of irradiated and non-irradiated haNK003 against DOHH2 cells was assayed at 6 hours (dashed line) or 24 hours (dotted line) after irradiation in combination with Rituxan (squares) or Herceptin (triangles), which does not react with DOHH2 cells. Note that data points for the 20:1 E:T ratio were not obtained for irradiated cells at the 24 hour time point due to insufficient numbers of haNK003 cells due to cell death. [Figure 8A] FIG. 1 is a graph showing IL-2 released (pg / mL) per 1 x 106 cells for irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by sandwich ELISA experiment number 1. [Figure 8B] FIG. 1 is a graph showing IL-2 released (pg / mL) per 1 x 106 cells for irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by sandwich ELISA experiment number 2. [Figure 8C] FIG. 1 is a graph showing IL-2 released (pg / mL) per 1 x 106 cells for irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by multiplex ELISA experiment number 1. [Figure 8D] FIG. 1 is a graph showing IL-2 released (pg / mL) per 1 x 106 cells for irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by multiplex ELISA experiment number 2. [Figure 9A]FIG. 1 is a graph showing the total intracellular IL-2 content (pg) per 1 x 106 cells in irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by sandwich ELISA experiment number 1. [Figure 9B] FIG. 1 is a graph showing the total intracellular IL-2 content (pg) per 1 x 106 cells in irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by sandwich ELISA experiment number 2. [Figure 9C] FIG. 1 is a graph showing the total intracellular IL-2 content (pg) per 1 x 106 cells in irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by multiplex ELISA experiment number 1. [Figure 9D] Graph showing total intracellular IL-2 content (pg) per 1 x 106 cells in irradiated vs. non-irradiated cells at 6, 12, 24, and 48 hours (h) as determined by multiplex ELISA experiment number 2. [Figure 10A] FIG. 1 is a graph showing the amount of soluble IL-2 (pg) per 1×10 6 cells in experiment no. 1 as determined by multiplex ELISA and sandwich ELISA. [Figure 10B] FIG. 1 is a graph showing the amount of soluble IL-2 (pg) per 1×10 6 cells in experiment no. 2 as determined by multiplex ELISA and sandwich ELISA. [Figure 11] Graph showing the effect of intravenously administered haNK003 on animal weight in NOD / SCID mice. NOD / SCID mice (3 males and 3 females per group) were treated by intravenous injection of non-irradiated or irradiated haNK003 cells at a dose of PBS or 1 x 107 cells, respectively, in a single dose. Animal weight was monitored twice weekly for 5 weeks. Values ​​are mean ± SEM, n = 6. [Figure 12]Graph showing the effect of haNK003 cells on animal weight in NOD / SCID mice. NOD / SCID mice (3 males and 3 females per group) were treated with PBS or 1 x 107 non-irradiated or irradiated haNK003 cells once a week for 4 weeks, and animal weight was monitored twice a week for 5 weeks. Values ​​are mean ± SEM, n = 6. [Figure 13] FIG. 1 is a graph showing a comparison of natural cytotoxicity of aNK cells versus haNK003 cells against K562 cells. [Figure 14] FIG. 1 is a graph showing a comparison of natural cytotoxicity of NK cells versus haNK003 cells against Daudi cells. [Figure 15] FIG. 1 is a graph showing a comparison of natural cytotoxicity of aNK cells versus haNK003 cells against DOHH2 cells. [Figure 16] 1 is a graph showing a comparison of natural cytotoxicity of aNK cells versus haNK003 cells against SKOV-3 cells. [Figure 17] FIG. 1 is a graph showing a comparison of natural cytotoxicity of aNK cells versus haNK003 cells against HL-60 cells. [Figure 18] FIG. 1 is a graph showing a comparison of natural cytotoxicity of aNK cells versus haNK003 cells against SR-91 cells. [Figure 19] Graph showing antitumor activity of haNK003 cells in MDA-MB-453 subcutaneous xenograft model in female NSG mice. Female NSG mice bearing MDA-MB-453 human breast cancer tumors were treated twice weekly for 4 weeks by intravenous injection of PBS or irradiated haNK003 cells at a dose of 2.5 x 106 or 1 x 107 cells, respectively. Tumor volumes were monitored twice weekly. Values ​​are mean ± SEM; n = 8. [Figure 20]Graph showing the effect of haNK003 cells on animal weight in female NSG mice. Female NSG mice bearing MDA-MB-453 human breast cancer tumors were treated twice weekly for 4 weeks by intravenous injection of PBS or irradiated haNK003 cells at a dose of 2.5 x 106 or 1.0 x 107 cells, respectively. Mouse weights were monitored twice weekly. Values ​​are mean ± SEM; n = 4. [Figure 21] 1 is a table showing the distance between pairs of samples for gene expression of haNK cells as well as normal NK cells 950, 962, and 996 under 20% oxygen and 0% oxygen (hypoxia) conditions. [Figure 22] 1 is a table showing genes showing the greatest variability in expression between 20% and 0% oxygen conditions in 950, 962, 996, and haNK cells. [Figure 23] 1 is a table showing the change in expression of genes that show the greatest change between 20% and 0% oxygen conditions in 950, 962, 996, and haNK cells. [Figure 24] 1 is a table showing changes in expression of genes associated with hypoxia between 20% and 0% oxygen conditions in haNK cells, and NK cells 950, 962, and 996. [Diagram 25] Graph showing flow cytometry analysis of CD16 expression in haNK003 cells and donor NK cells before and after PMA treatment. Downregulation of CD16 expression is 94.36% ± 3 in donor NK cells and 30% ± 0.04 in haNK003 cells. aNK (NK-92 cells without CD16) were used as a control. [Figure 26]Graph showing flow cytometry analysis of CD16 expression levels in haNK003 cells and donor NK cells co-cultured with K562 cells (E:T = 1:1). After 4 hours, haNK003 cells showed stable CD16 expression compared to donor NK cells. After 4 hours of co-culture with K562, CD16 downregulation in donor NK cells was 60.25% ± 0.9, compared to 4.9% ± 2.57 for haNK003 cells. After overnight recovery, there was still a downregulation of CD16 expression of 57.54% ± 26.82 in donor NK cells, while in haNK003 cells, CD16 levels had returned to near normal with only a downregulation of 2.78% ± 3.5. aNK (NK-92 cells without CD16) was used as a control. [Figure 27] Figures 27A and 27B are graphs showing the expression levels of CD16 after ADCC in haNK003 cells. ADCC was performed by co-culturing haNK003 cells and DoHH at E:T ratios of 1:0 (effector alone) to 1:4 for 4 hours in the presence of 1 μg / ml rituximab. The expression levels of CD16 were measured by flow cytometry at 4 hours and after 24 hours. Figure 27A shows the flow cytometry analysis of the expression levels of CD16 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 after 24 hours of ADCC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Detailed Description Provided herein is a modified NK-92 haNK003 cell. The cell expresses the Fc receptor CD16 and endoplasmic reticulum-bound IL-2. Thus, the cell is not dependent on external IL-2 for growth. Furthermore, the modified NK-92 cell has enhanced cytotoxicity by inserting a high affinity mutant of the CD16 receptor, and therefore has the ability of CD16-targeted antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is mediated by recognition of the Fc fragment of the antibody (IgG) bound to the target via the CD16 Fc receptor. Thus, for oncological applications, ADCC by the modified cell is triggered by binding of the CD16 receptor to the Fc fragment of the IgG bound to the tumor cell, and thus the modified NK-92 cell is activated for target killing. As described herein, the modified NK-92 cells provided were generated by stable transfection of a bicistronic plasmid-based vector that contains a sequence for CD16 high affinity Fc gamma receptor (FcγRIIIa / CD16a) along with a sequence for IL-2 targeted to the endoplasmic reticulum. The cells contain a plasmid sequence inserted at a single location on the +strand of chromosome 17 at position 15,654,977. The modified NK-92 cells produce endogenous IL-2 and are phenotypically CD56+, CD3-, and CD16+. The modified NK-92 haNK003 cells provided herein are sometimes referred to simply as haNK003 cells.

[0008] As described in more detail in the Examples below, NK-92 cells were transformed with pNEUKv1_FcRIL2 plasmid (SEQ ID NO:1). The pNEUKv1_FcRIL2 plasmid is a bicistronic construct expressing a modified CD16 containing a valine at amino acid position 176 (when referring to the full-length CD16 peptide) and IL-2 with an endoplasmic reticulum retention signal. Whole genome sequencing (WGS) of the cells was performed and one plasmid insertion site on chromosome 17 was identified. WGS confirmed that the integration of the bicistronic plasmid in the haNK003 cell line was in a region of the genome away from any genes with oncogenic potential. The 5'-closest gene TBC1D26 is 10,722 bp upstream and the 3'-closest gene ADORA2B is 186,828 bp downstream. Passaged every 3-4 days, approximately 0.3-0.5 x 10 cells were cultured. 6 The modified NK-92 cells proliferate stably when seeded at a density of 1000 cells / mL. The average doubling time was 65 (48-95) hours from day 3 to day 29. Analysis of flow cytometry data indicates that the modified NK-92 cells express CD54, CD56, NKG2D, NKp30, and CD16 surface marker proteins, and lack CD3. The modified NK-92 cells can proliferate without the addition of IL-2 in the culture medium. In addition, the modified NK-92 cells that express IL-2 were confirmed to release low levels of IL-2 into the culture medium. Non-irradiated haNK003 cells alone secrete 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 naturally cytotoxic against several cancer cell lines and, when combined with antibodies, are capable of enhanced specific lysis 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 have high cytolytic activity against a variety of cancers. The NK-92 cell line is a homogenous NK cell population with a broad range of antitumor cytotoxicity with predictable yields after expansion. Phase I clinical trials have confirmed its safety profile. NK-92 was discovered in the blood of subjects suffering from non-Hodgkin's lymphoma and was subsequently immortalized ex vivo. NK-92 cells are derived from NK cells, but while retaining the majority of activating receptors, they lack 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. Characterization of the NK-92 cell line is disclosed in WO 1998 / 49268 and U.S. Patent Application Publication No. 2002-0068044.

[0010] NK-92 cells are known and include, but are not limited to, those described in, for example, 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 by reference herein in their entireties, e.g., 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] Provided herein is a population of modified NK-92 haNK003 cells that have antibody-dependent cell-mediated cytotoxicity (ADCC) and that contain a nucleic acid molecule that contains both CD16 (SEQ ID NO:3) and IL-2 (SEQ ID NO:5), where greater than 90% of the cells in the population of cells express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the population of cells express CD3. Optionally, the nucleic acid molecule is an mRNA molecule. Optionally, the mRNA molecule contains, from 5' to 3', a sequence encoding CD16, an IRES sequence, and a sequence encoding IL-2. Optionally, the cells contain SEQ ID NO:1 on chromosome 17. Optionally, the average doubling time of the cells is 55-70 hours. Optionally, the population of cells maintains an average doubling time of 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 25 days, or more days. Optionally, the population of cells can be passaged for 1 day, 2 days, 3 days, 4 days, or more days. 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 is cleaved near the cell membrane, resulting in the release of the extracellular portion of the receptor and downregulation of expression following activation (see Jing, et al., PLOS one, 10(3):e0121788 DOI:10.1371 / journal.pone.0121788 (2015)). Under normal conditions, this mechanism serves to control NK cell cytotoxicity, but in a tumor environment, it may reduce the efficacy of ADCC and the killing of cancer cells. Advantageously, the provided haNK003 cells have enhanced ADCC activity against cancer cells. Without being bound by theory, this is believed to be due to the event of stable expression of CD16 in haNK003 cells after ADCC. As shown in the examples below, after activation with phorbol-12-myristate 13-acetate or stimulation with K562 cells, the expression of CD16 remained high compared to control cells. Moreover, even after ADCC, CD16 expression remains high in haNK003 cells.Therefore, the provided haNK003 cells have reduced downregulation of CD16 expression compared to control.Furthermore, haNK003 cells have increased levels of CD16 after ADCC compared to control.In other words, the cells maintain higher levels of CD16 after ADCC compared to control.Therefore, haNK003 cells have more stable expression of CD16 compared to control, for example, normal NK cells.

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

[0014] As described above, the 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 the Fc receptor (FcR) of a cell stimulates the phagocytic or cytotoxic activity of the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified by the type of antibody they recognize. For example, Fc-gamma receptors (FCγR) bind to antibodies of the IgG class. FCγRIII-A (also referred to as CD16) is a low affinity Fc receptor that binds 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, the modified NK-92 cells comprise a nucleic acid sequence having 70%, 80%, 90%, or 95% identity to SEQ ID NO:3. Optionally, the modified NK-92 cells comprise a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3. Optionally, the modified NK-92 cells comprise a polypeptide having 70%, 80%, 90%, or 95% identity to SEQ ID NO:4. Optionally, the modified NK-92 cells comprise a polypeptide having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4.

[0016] NK-92 cell cytotoxicity is dependent on the presence of cytokines, such as interleukin-2 (IL-2). The cost of exogenously added IL-2 required to maintain and expand NK-92 cells in commercial scale cultures is significant. Administration of IL-2 to human subjects in amounts sufficient to sustain NK-92 cell activation can cause adverse side effects. Optionally, IL-2 is expressed with a signal sequence that targets IL-2 to the endoplasmic reticulum. Targeting IL-2 to the endoplasmic reticulum allows expression of IL-2 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. A representative nucleic acid encoding IL-2 is shown in SEQ ID NO:5, and a representative polypeptide of IL-2 is shown in SEQ ID NO:6.

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

[0018] Nucleic acid, as used herein, refers to deoxyribonucleotides or ribonucleotides, as well as their polymers and complements. The term includes deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form. The term encompasses synthetic, natural, and non-natural nucleic acids that contain known nucleotide analogs or modified backbone residues or linkages that have similar binding properties and are metabolized in a manner similar to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, conservatively modified variants of nucleic acid sequences (e.g., degenerate codon substitutions), and complementary sequences can be substituted for the specific nucleic acid sequences described herein. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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 terms nucleic acid are used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

[0019] A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA encoding a presequence or secretory leader is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked means that the linked DNA sequences are near each other and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, do not have to be contiguous. For example, a nucleic acid sequence that is operably linked to a second nucleic acid sequence is covalently linked, either directly or indirectly, to such second sequence, although any effective three-dimensional association is permitted. A single nucleic acid sequence may be operably linked to multiple other sequences. For example, a single promoter may affect the transcription of multiple RNA species. Linkage may be accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0020] The term identity or percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides (i.e., about 60% identity over a particular region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity, when compared and aligned for maximum correspondence over a comparison window or designated region) when measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or when measured by manual alignment and visual inspection (see, for example, the NCBI website, etc.). Such sequences are therefore said to be substantially identical. This definition may also refer to or apply to the complement of a test sequence. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0021] For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using sequence comparison algorithm, test and reference sequences are input into computer; subsequence coordinates are designated if necessary; and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. Sequence comparison algorithm then calculates the percent sequence identity of test sequences to reference sequences based on program parameters.

[0022] A comparison window, as used herein, includes reference to a segment of contiguous positions of any one number selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, where a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison 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 method of searching for similarity of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988); by computerized implementations 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, which are 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 percent sequence identity of nucleic acids or proteins using parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information, as known in the art. This algorithm first involves identifying high scoring sequence pairs (HSPs) by identifying short words of a selected length (W) in a query sequence that either match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score.Extension of the word hits in each direction is stopped when the cumulative alignment score falls 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 either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The expectation (E) represents the number of different alignments that have a score equal to or better than that expected to occur by chance in a database search. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. The BLASTP program for amino acid sequences uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), alignments (B) of 50, expectation (E) of 10, M = 5, N = -4, and a comparison of both strands.

[0024] The term polypeptide, as used herein, is intended to have its art-recognized meaning of a polymer of at least three amino acids, and to include peptides and proteins. However, the term is also used to refer to specific functional classes of polypeptides, such as desaturases, elongases, etc. For each such class, the present disclosure provides several examples of known sequences of such polypeptides. Those skilled in the art will understand, however, that the term polypeptide is intended to be sufficiently general to include not only polypeptides having the complete sequences referred to herein (or in the references or in the databases specifically referred to herein), but also polypeptides that represent functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Furthermore, those skilled in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares an overall sequence identity of at least about 30-40%, often more than about 50%, 60%, 70%, or 80% with another polypeptide of the same class, and further typically includes 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 usually include at least 3-4 amino acids, and often up to 20 or more amino acids, is encompassed by the appropriate term polypeptide as used herein. Those skilled in the art can determine similarity and / or identity of other regions by analyzing the sequences of various polypeptides described herein. As known by those skilled in the art, a variety of strategies are known and tools are available for performing comparison of amino acid or nucleotide sequences 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 (generally implemented by computer) for performing sequence alignment 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.). Readily available computer programs incorporating such algorithms include, for example, BLASTN, BLASTP, Gapped BLAST, PILEUP, CLUSTALW, etc. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs may be used. Alternatively, the practitioner may use non-default parameters depending on experimental and / or other requirements (see, for example, the website having the URL www.ncbi.nlm.nih.gov).

[0025] As used herein, the terms promoter, promoter element, and control sequence refer to a polynucleotide that controls the expression of a selected polynucleotide sequence that is operably linked to a promoter and results in the expression of the selected polynucleotide sequence in a cell.

[0026] The term transformation, as used herein, refers to a process by which a foreign or heterologous nucleic acid molecule (e.g., a vector or a recombinant nucleic acid molecule) is introduced into a recipient cell or microorganism. The foreign or heterologous nucleic acid molecule may or may not be integrated (i.e., covalently linked) into the chromosomal DNA that constitutes the genome of the host cell or microorganism. For example, the 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 integrated 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 with polyethylene glycol (PEG); electroporation; magnetoporation; biolistic delivery; retroviral infection; lipofection; and microinjection of DNA directly into cells.

[0027] The term transformed, when used with reference to a cell, refers to a cell that has undergone transformation as described herein such that the cell carries foreign or heterologous genetic material (e.g., a recombinant nucleic acid). The term transformed may also, or alternatively, 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 reference to a cell, nucleic acid, polypeptide, vector, etc., indicate that the cell, nucleic acid, polypeptide, vector, etc. has been modified by or is the product of a laboratory method and is non-naturally occurring. Thus, for example, a modified cell includes cells that have been produced or modified by laboratory techniques, such as transformation methods to introduce a nucleic acid into a cell. Modified cells can contain nucleic acid sequences that are not found in a native (non-recombinant) form of the cell or can contain altered nucleic acid sequences, such as nucleic acid sequences linked to a non-native promoter.

[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, a test cell, such as a cell transformed with a nucleic acid sequence encoding a gene for an Fc receptor, can be compared with a known normal (wild type) cell (e.g., a standard control cell). A standard control can also represent an average measurement or average value obtained from a population of cells that do not express an Fc receptor or have no or minimal levels of Fc receptor activity (e.g., a standard control microorganism). Those skilled in the art will understand that a standard control 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 an IgG. The antibody may be a non-human antibody (e.g., 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] The term "monoclonal antibody" as used herein refers to a pure, target-specific antibody produced from a single clone of cells that can grow in culture and multiply indefinitely. Monoclonal antibodies that can be used include naked antibodies that bind to and inhibit antigens on cancerous cells. Optionally, the naked monoclonal antibody is alemtuzumab, which binds to the CD52 antigen in lymphocytes. Monoclonal antibodies that can be used also include conjugated monoclonal antibodies, such as tagged, labeled, or added antibodies. In particular, the antibody can be tagged or added with a drug or toxin, or can be radioactively labeled. Examples of such antibodies include, but are not limited to, ibritumomab, which targets the CD20 antigen; brentuximab, which targets the CD30 antigen, and trastuzumab, which targets the HER2 protein. Other monoclonal antibodies that may be used are bispecific monoclonal antibodies such as blinatumomab, which targets CD19 on lymphoma cells and CD3 on T cells.

[0032] As used herein, the term "antibody fragment" refers to any portion of an antibody that recognizes an epitope. The antibody fragment may be glycosylated. As non-limiting examples, the antibody fragment may be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a rIgG fragment, a functional antibody fragment, a single chain recombinant version of the above, etc. F(ab')2, Fab, Fab', and Fv are antigen-binding fragments that can be made from the variable regions of IgG and IgM. They differ in size, valency, and Fc content. The fragments may be made by any method, including expression of components (e.g., heavy and light chain portions) by a cell or cell line or multiple cells or cell lines. Preferably, the antibody fragment includes a sufficient portion of the Fc region such that it can recognize the epitope and bind to an Fc receptor.

[0033] As used herein, the term "cancer" refers to all types of cancer, neoplasms, or malignant tumors found in mammals, including leukemia, carcinoma, and sarcoma. Exemplary cancers include brain cancer, 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, gastric cancer, uterine cancer, and medulloblastoma. Additional examples include Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, cancer of the genitourinary tract, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, pancreatic endocrine and exocrine neoplasms, and prostate cancer.

[0034] Also provided is a method of treating a subject with modified NK-92 cells as described herein. Optionally, the subject is treated with modified NK-92 cells and an antibody.

[0035] The modified NK-92 cells can be administered to a subject by absolute number of cells, e.g., the subject can be administered about, at least about, or up to about 1 x 10 cells per infusion. 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 NK-92 cells, or any range between any two of these numbers, including the endpoints. Optionally, 1 x 10 8 ~1 x 10 10The cells are administered to the subject. Optionally, the cells are administered once or multiple times per week for one or multiple weeks. Optionally, the cells are administered once or twice per week for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks.

[0036] Optionally, the subject receives about, at least about, or up to about 1 x 10 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 (etc.) of NK-92 cells, or any range between any two of these numbers inclusive. 2 Approximately 10 billion cells / injection / m 2 Up to 20 mg / kg is administered.

[0037] Optionally, NK-92 cells can be administered to such individuals in relative numbers of cells, e.g., the individual can be administered about, at least about, or up to about 1 x 10 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 From about 1000 cells to about 10 billion cells per kilogram of an individual may be administered, such as (and so on) NK-92 cells, or any range between any two of these numbers inclusive.

[0038] Optionally, the total dose is measured in m2 of body surface area. 2 may be calculated according to 1 m 2 Approximately 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , or any range between any two of these numbers, including the endpoints. Optionally, about 1 billion to about 3 billion NK-92 cells are administered to the patient. Optionally, the amount of NK-92 cells infused per dose may be calculated in square meters of body surface area, and may be calculated based on the number of cells infused per square meter. 2 1 x 10 per 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 Includes pieces.

[0039] The NK-92 cells, and optionally other anti-cancer agents, can be administered once to a patient with cancer, or multiple times, such as 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, 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 or more weeks, or any range between any two of these numbers inclusive endpoints, during treatment.

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

[0041] According to the methods provided herein, an effective amount of one or more agents provided herein is administered to a subject. The terms effective amount and effective dose are used interchangeably. The term effective amount is defined as any amount necessary to cause a desired physiological response (e.g., reduced inflammation). The effective amount and schedule for administration of the agent may be empirically determined by one of skill in the art. The range of doses for administration is large enough to cause 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, anaphylactic reactions, and the like. In general, the dose will vary according to age, health, sex, type of disease, extent of disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dose may be adjusted by the individual physician in the event of any contraindications. The dose may vary and may be administered once a day, for one or several days, in one or more doses. Guidance can be found in the literature regarding appropriate doses for a given class of pharmaceutical products. For example, an effective amount will exhibit at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least a 100% increase or decrease in a given parameter. Efficacy can also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.The exact dosage and dosage form will depend on the purpose of the treatment, and can be ascertained by one of skill in the art using known techniques (see, e.g., 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] Pharmaceutically acceptable compositions may include 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, and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Pharmaceutically acceptable carriers refer to materials that are not biologically or otherwise harmful. That is, the materials are administered to a subject without causing adverse biological effects or interacting in an adverse 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 include buffers and preservatives during storage, and depending on the route of administration, may include appropriate buffers and carriers for delivery.

[0043] The compositions may include acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride calcium chloride, sodium lactate, etc. The concentrations of cells and / or other agents in these formulations can vary and are selected primarily based on fluid volume, viscosity, weight, etc., according to the particular method of administration selected and the needs of the subject.

[0044] Optionally, NK-92 cells are administered to the subject together with one or more other treatments for the cancer to be treated.Without being bound by theory, it is believed that co-treatment of the subject with NK-92 cells and another therapy for cancer allows NK-92 cells and the other therapy to provide the endogenous immune system with the opportunity to eliminate the cancer that has previously overwhelmed such endogenous action.Optionally, the two or more other treatments for the cancer to be treated include, for example, antibody therapy, radiation therapy, chemotherapy, stem cell transplantation therapy, or hormone therapy.

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

[0046] Optionally, the antibodies may be used to target cancerous cells or cells expressing markers associated with cancer. Many antibodies are approved solely for the treatment of cancer.

[0047] Table 2: Examples of therapeutic monoclonal antibodies approved by the FDA TIFF2025038128000002.tif42142TIFF2025038128000003.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] Thus, NK-92 cells expressing CD16 are administered to subjects together with an effective amount 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 is a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody.Optionally, a bispecific antibody can be used that binds to cancer cells and also binds to cell surface proteins present on the surface of NK-92 cells.

[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 specific to cancer. 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 multiple types of advanced human cancers. It is believed that antibodies against YKL-40 can be used to inhibit 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 commercial sources or produced by any method known in the art. For example, antibodies can be produced by obtaining B-cell, bone marrow, or other samples from one or more patients who previously suffered from cancer and have recovered, or who were in recovery 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 produced by isolating RNA from a sample or cell of interest, preparing cDNA from the isolated RNA, enriching the cDNA for heavy and / or light chain cDNA, and creating a library using a phage display vector. Libraries can be prepared and screened, for example, as described in Maruyama et al., which is incorporated herein by reference in its entirety. Antibodies can be produced 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 by reference in its entirety.

[0052] Combinations of agents or compositions can be administered either together (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous routes), or sequentially (e.g., one agent is administered first, followed by administration of the second agent). Thus, the term combination is used to refer to the administration of two or more agents or compositions together, simultaneously, or sequentially. The course of treatment is best determined individually, depending on the particular characteristics of the subject and the type of treatment selected. Treatments such as those disclosed herein can be administered to the subject once a day, twice a day, once every two weeks, once a month, or on any suitable basis that is therapeutically effective. Treatments can be administered alone or in combination with any other treatments disclosed herein or known in the art. The additional treatments can be administered simultaneously with the first treatment, at different times, or on completely different therapeutic schedules (e.g., the first treatment can be once a day, while the additional treatment is once a week).

[0053] Also disclosed is a kit comprising the modified NK-92 cells provided. Optionally, the kit further comprises one or more additional agents, such as an antibody. The components of the kit may be contained in one or different containers, such as one or more vials. The antibody may be in liquid or solid form (e.g., after lyophilization) to increase shelf life. When in liquid form, the components may include additives such as stabilizers and / or preservatives, such as proline, glycine, or sucrose, or other additives that increase shelf life.

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

[0055] Optionally, the instructions for use of the kit include instructions for using the components of the kit 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), the antibodies, and the NK-92 cells (e.g., thawing and / or culturing). The instructions may further include guidance regarding dosage and frequency of administration.

[0056] Disclosed are materials, compositions, and components that can be used for, can be used with, can be used to prepare, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these materials are disclosed, and that even if specific reference to each of the various individual and collective combinations and modifications of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed, and several modifications that can be made to several molecules that comprise the method are discussed, each and every combination and modification of the method, and possible modifications, are specifically contemplated unless specifically indicated otherwise. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in a method using the disclosed compositions. Thus, if there are a variety of additional steps that may be performed, it is understood that each of these additional steps may be performed at any particular method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered as specifically contemplated and disclosed.

[0057] Publications cited herein, and the material for which they are cited, are hereby expressly incorporated by reference in their entireties.

[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 characterization of haNK003 NK-92 [CD16.176V, ER IL-2] (haNK003) was generated by modification of NK-92 cells. NK-92 cells were originally isolated in 1992 from a 50-year-old male patient with aggressive non-Hodgkin's 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 generated by stable transfection by electroporation of NK-92 cells with a bicistronic plasmid-based vector containing sequences for CD16 and IL-2. The transfected plasmids are shown in FIG. 1 and were constructed by GeneArt AG. The CD16 sequence encodes a valine at amino acid position 176 (176V), which allows for increased antibody-dependent cell-mediated cytotoxicity (ADCC) potential. The IL-2 sequence is tagged with an endoplasmic reticulum retention signal, KDEL, to inhibit IL-2 protein secretion from the endoplasmic reticulum (ER). Inclusion of the IL-2 sequence allows for haNK™ to be IL-2 independent.

[0060] EUFETS GmbH (Regensburg, Germany) performed transfection by electroporation and selected multiple clones by one round of limiting dilution. One clone from EUFETS was sent to BioReliance for establishing a GMP master cell bank, haNK003. Whole genome sequencing of selected clones confirmed the plasmid insertion site to 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 specifications provided. 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 resistance cassette. The promoter used for expression of the transgene is EF-1alpha with an SV40 polyadenylation sequence. The resulting plasmid is 5,491 base pairs (bp) long and contains sequences of human origin for CD16 and IL-2. Neither CD16 nor IL-2 have any transforming properties. Plasmid DNA was purified from transformed bacteria and its concentration was determined by UV spectroscopy. The final construct was verified by sequencing. The sequence identity within the restriction sites used was 100%. The plasmid was made under TSE-free production conditions.

[0062] The complete nucleotide sequence of the pNEUKv1_FcRIL2 plasmid (SEQ ID NO:1) is shown below. TIFF2025038128000004.tif89128TIFF2025038128000005.tif216128

[0063] To generate the haNK003 cell line, a vial of NK-92 (aNK) Master Cell Bank (MCB) (aNK COA) and 250 mg of pNEUKv1_FcRIL2 plasmid was sent to EUFETS GmbH. EUFETS thawed the MCB vial and cultured the NK-92 cells to the appropriate number for transfection with the plasmid. The 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 cells that were transfected and produced the appropriate amount of IL-2 continued to grow. Multiple clones were isolated by limiting dilution and preliminarily screened for phenotype and Fc receptor expression. Six (6) clones showing good viability (>70%), acceptable doubling time, expected phenotype, and positive Fc receptor expression were sent to the German Red Cross GMP Testing Laboratory (GRC) for more detailed screening and final selection of a single clone. At the GRC, all clones were tested for phenotype (including Fc receptor expression), ADCC, cytokine profile, growth characteristics, and radiosensitivity. The selected cell line, haNK003, was used to generate a master cell bank.

[0064] The NantKwest Master Cell Bank (MCB haNK003) was produced from selected cell lines and tested by BioReliance. The MCB was tested for purity, potency, identity, sterility, and viral / adventitious agents. The MCB is stored frozen in aliquots of 1 x 107 cells / vial in a formulation of 10% DMSO, 40% X-Vivo 10, 50% human AB serum. The total number of vials produced from cryopreservation for the MCB was 218.

[0065] Integration site DNA extracts from haNK003 were provided to the CLIA / CAP certified NantOmics Sequencing Lab (Culver City, CA) for whole genome sequencing. Whole genome libraries were prepared using the KAPA Hyper prep kit for cell line samples and sequenced on an Illumina HiSeq instrument to provide a minimum coverage of 25x covering haNK003. DNA sequencing data was aligned against the 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), which contains the plasma sequences reported by bwa-mem, has duplicates marked by samblaster, and has been indel realigned and base quality recalibrated by Genome Analysis Toolkit (GATK). Mutation analysis was performed using the NantOmics Contraster analysis pipeline to identify variants including single nucleotide changes, small insertions or deletions (indels), copy number changes, translocations, and integration sites. The integrated plasmids and resulting integration sites were visualized by the NantOmics Genome Browser, and further comparison and visualization was performed on the UCSC Genome Browser to identify any potential interactions with existing genomic elements.

[0066] haNK003 showed evidence of incongruent reads mapping to chromosome 17:15654977-15661403. The 5'-nearest gene TBC1D26 (chromosome 17:15,635,591-15,644,255) was 10,722 bp upstream, and the 3'-nearest gene ADORA2B (chromosome 17:15,848,231-15,871,210) was 186,828 bp downstream. Little is known about TBC1D26 other than that it is annotated 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 for the coding sequence named pNEUKv1_FcRIL. The UCSC Encode track and lincRNA show evidence 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, indicating that this transcript is likely still intact. Examination of 100 multiple alignments from vertebrate species indicates little base-level conservation across the integration site, with negative log p-values ​​ranging from -3.874 to 1.507, with a mean conservation of 0.01 and a standard deviation of 0.58.

[0067] haNK003 did not contain any evidence of gene, transcript or regulatory disruption at the human genome integration site. The integration in cell line haNK003 was at least 10 kbp away from any gene. The cell line is permissive in that there is no evidence of disruption to any known genomic features in the human genome of the target cell line.

[0068] Growth characteristics The growth characteristics of the clonal cell line haNK003 used to generate MCB haNK003 are shown in Figures 1A and 1B. Data were analyzed from the cell culture history when cell line haNK003 was expanded for cryopreservation of the Master Cell Bank. The average doubling time was 65 (48-95) hours from day 3 to day 29. Comparable cell densities were achieved throughout the passages, which were passaged every 3-4 days and consisted of approximately 0.3-0.5 x 10 cells. 6 We demonstrate that haNK003 cells grow stably when seeded at a density of 100 / 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 chosen to be representative of natural killer (NK) cells.

[0070] aNK cells express surface markers typical of NK cells at an early differentiation stage, which express multiple activating receptors including NKG2D and NKp30, but lack FcγRIIIa (CD16) and inhibitory KIR (killer immunoglobulin-like receptors). This special surface marker expression profile of aNK cells confers unique cytotoxic properties to the cells. It was therefore important to establish that the generation of the haNK003 cell line by stable transfection of plasmids encoding high affinity FcγRIIIa and intracellularly retained IL-2 (ERIL-2) did not alter the expression profile of key surface markers of the parental aNK cell line. The surface markers CD54, CD56, NKG2D, NKp30, CD3, and CD16 were analyzed, and marker expression was determined by staining of cells with specific antibodies conjugated to fluorochromes and detection of bound antibodies by flow cytometry.

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

[0072] Table 1. Expression of surface markers TIFF2025038128000006.tif32142% = percentage of cells positive for expression ± standard deviation

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

[0074] The generation of the haNK003 cell line did not alter the expression of key surface markers of the parental aNK cell line, but only conferred additional functionality in the form of CD16 expression.

[0075] Cell damage The natural cytotoxicity of the haNK003 cell line was evaluated against the cell lines K562, Raji, SKOV3, and SKBR3 at different effector to target ratios (E:T). The ADCC activity of haNK003 was also evaluated against the cell lines Raji, SKOV3, and SKBR3 at different E:T ratios. The susceptibility to natural cytotoxic killing by haNK varied for the different target cell lines, with the K562 cell line being the most sensitive and the solid tumor cell lines (SKOV3 and SKBR3) being less sensitive (Figures 3A, 3B, 3C, and 3D). Some differences in the ADCC activity of haNK003 against the 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 natural cytotoxicity in the presence of some cancer cells and have the capacity for enhanced specific lysis via ADCC using antibodies. The specifications and performance of haNK™ MCB (haNK003) are provided in Table 2.

[0077] Table 2. Specifications of haNK003 cells TIFF2025038128000007.tif90142TIFF2025038128000008.tif159142 a - Although EBV viral genome was detected, tests with NK-92 (aNK) confirmed that the cells do not cause EBV infection. Infectivity studies were performed by co-culturing aNK cells (irradiated and non-irradiated) with B lymphocytes to see if aNK cells release viral particles capable of infecting normal cells. The results showed no signs of proliferation or growth of B lymphocytes, indicating that aNK cells do not pose a risk regarding EBV infection.

[0078] Example 2. Effect of irradiation on proliferation potential and functionality in vitro haNK™ cells are irradiated to mitigate the risk of uncontrolled proliferation. The effect of irradiation on in vitro proliferation capacity and functionality was evaluated. These studies demonstrate that irradiation with 10 Gy inhibits the proliferation capacity of at least 99.9% of haNK cells, while still maintaining functional activity for at least 6 hours after irradiation.

[0079] haNK003 cells exhibit both natural (direct) cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). In both cases, antigens of target cells are recognized by activating receptors on NK cells. For natural cytotoxicity, these target cell antigens are stress antigens characteristic of virally infected or transformed cells. For ADCC, the antigen is a tumor-specific antigen recognized by an antibody, which then binds to the NK cell activating receptor, FcγRIIIa (CD16), via its constant (Fc) region.

[0080] Interaction of NK cells with ligands on target cells (either through direct or antibody-mediated interactions) leads to the formation of cell junctions and the subsequent release of perforin and granzymes, which in turn induce the process of apoptosis within the target cell, leading to cell membrane breakdown and target cell death.

[0081] A study was conducted to determine the level of natural cytotoxicity and ADCC activity in preformulated cells and the duration of that activity following gamma irradiation at 10 Gy. For this study, 1.5 x 107 cells were irradiated per flask. Although the number of cells irradiated was not equivalent to that used in the manufacture of clinical doses, the assay of functionality after irradiation provides insight into the potential for commercialization.

[0082] For the purpose of these experiments, haNK003 cells were irradiated with 10 Gy using an X-ray irradiator. Non-irradiated cells received the same treatment without irradiation. Target cells were selected to represent susceptibility to different mechanisms of killing by NK cells. For example, K562 cells are highly susceptible to killing by natural cytotoxicity, whereas DOHH are incompletely susceptible to killing via natural cytotoxicity but susceptible to ADCC using appropriate antibodies. Irradiated and non-irradiated cells were assayed in parallel for specific cytotoxicity using a flow cytometry-based assay developed in-house. Target cells were labeled with a green fluorescent dye with a long aliphatic tail (PKH67), which was stably incorporated into lipid regions of the cell membrane. Cell lysis was monitored by propidium iodide staining.

[0083] Analysis of natural cytotoxicity demonstrates that the impact of irradiation varies with target cells and with time after irradiation. For sensitive cells such as K562, natural cytotoxicity was maintained within 6 h of irradiation but was reduced by more than 40% after 24 h across all effector to target ratios (E:T) (Figure 5). For DOHH2, natural cytotoxicity was also retained at 6 h after irradiation but was reduced by more than 14% after 24 h 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 DOHH2 targets was also maintained by irradiated cells at 6 hours. However, the rituximab-mediated ADCC activity of irradiated cells at 24 hours was reduced by about 16% or more from the activity observed in cells at 6 hours and in non-irradiated cells at 24 hours (Figure 7). As expected, Herceptin (trastuzumab) in combination with haNK003 did not induce any ADCC killing of DOHH2 target cells (Figure 4), and antibodies alone (rituximab or trastuzumab) did not induce any killing of DOHH2 target cells (data not shown).

[0085] Appropriate levels of cytotoxicity and ADCC activity were maintained for at least 6 hours following irradiation of preformulated 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 at various time points, as well as the amount of IL-2 retained intracellularly in haNK003 cells. The amount of IL-2 was measured in the supernatant to determine the release of IL-2 from haNK003. The amount of IL-2 was measured in the cell pellet lysate to determine the total level of intracellular IL-2 in haNK003. Samples were analyzed before and after irradiation to determine the impact of irradiation on IL-2 release and intracellular IL-2 levels.

[0087] Two separate assays were performed (Experiment No. 1 and Experiment No. 2), in which haNK003 cells were cultured in T-75 flasks and irradiated with 0 Gy (non-irradiated) or 10 Gy (irradiated) using an X-ray irradiator. haNK003 cells (non-irradiated and irradiated) were then cultured in X-Vivo 10 supplemented with 5% heat-inactivated human AB serum for up to 48 hours. Samples were collected from the culture supernatant as well as the cell pellets at various time points for analysis. The cell pellets were lysed using a detergent-based solution to quantify total intracellular IL-2. IL-2 concentrations in the culture supernatant and in the cell lysate samples were measured independently using two different detection methods (sandwich ELISA or multiplex ELISA) at NantKwest or by AllCells, LLC.

[0088] The values ​​of IL-2 concentration measured by the two different methods differ on average by a factor of 5 for the same sample. Both methods show a linear increase in IL-2 release over time for both irradiated and non-irradiated cells (Table 3 and Figures 8A, 8B, 8C, and 8D). Although the differences between the assays of Experiment #1 and Experiment #2 are different, the IL-2 concentration in the culture supernatant of irradiated cells tends to be higher than that of non-irradiated cells at all time points.

[0089] Table 3. Irradiated vs. non-irradiated haNK003 cells (1 x 10) at different time points. 6 IL-2 released per cell (pg / mL) (mean and standard deviation [StDev] of duplicate readings) TIFF2025038128000009.tif76142h-time

[0090] The values ​​of total intracellular IL-2 concentration measured by the two different methods differ on average by 5-fold for the same samples (Table 4 and Figures 9A, 9B, 9C, and 9D). Both methods show an increase in total intracellular IL-2 over time in non-irradiated cell lysates, while the amount of total intracellular IL-2 in irradiated cell lysates decreases over time. At 6 hours of culture, the levels of total intracellular IL-2 were comparable between irradiated and non-irradiated cells when measured by both methods. In irradiated cells, these levels decrease after 48 hours of culture.

[0091] Table 4. Irradiated haNK003 vs. non-irradiated haNK003 cells at different time points (1 x 10 6 Total intracellular IL-2 content (pg) per cell (mean and standard deviation [StDev] of duplicate readings) TIFF2025038128000010.tif118142h-time

[0092] To stimulate the release of IL-2 upon cell necrosis, cells were subjected to hypoosmotic shock. This level was compared to the total intracellular IL-2 concentration measured in cell lysates prepared using a detergent-based method. The values ​​of solubilized IL-2 concentration measured by the two methods (sandwich ELISA or multiplex ELISA) differed by approximately 10-fold for the same samples (Table 5, and Figures 10A and 10B). IL-2 concentrations in lysates from hypoosmotic shock ranged from 181.93 to 289.54 pg / 106 cells (sandwich ELISA) or 2619.15 to 3301.02 pg / 106 cells (multiplex ELISA), which corresponds to an average of 14% (sandwich ELISA) and 27% (multiplex ELISA) of the total intracellular IL-2 concentration measured in cell lysates prepared using detergent-based lysis.

[0093] Table 5. Amount of soluble IL-2 (pg) per 1 x 106 cells for haNK003 cells (mean and standard deviation [StDev] of duplicate readings). TIFF2025038128000011.tif81142

[0094] The multiplex ELISA IL-2 quantification values ​​were 5-10 fold higher than the data from the sandwich ELISA method. Although the absolute values ​​from these two methods have variability, the trends are consistent between both data sets and are summarized below. This data will be useful to allow NantKwest to continue to characterize IL-2 secretion and intracellular IL-2 levels from haNK003 cells as the product is further developed.

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

[0096] Irradiation of haNK003 cells with a dose of 10 Gy increased the amount of IL-2 released over a 48-h period, likely reflecting the presence of dead cells. Furthermore, irradiation did not result in a simultaneous release of IL-2, but rather a gradual release over time.

[0097] To stimulate the release of IL-2 during necrotic cell death, haNK003 cells were subjected to hypoosmotic shock. The amount of IL-2 released under these conditions represented either 14% or 27% of the total intracellular IL-2 determined in Triton X-100 lysates (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 non-irradiated cells over 6 hours when 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 of irradiated haNK003 cells in vivo suggest that IL-2 release by infused haNK003 is unlikely to cause adverse clinical effects.

[0099] The effect of irradiation on in vitro proliferation capacity and functionality, as tested in developmental studies with preliminary formulated cells, demonstrates that haNK003 cells are proliferation restricted in vitro (less than 0.1% of cells) and that levels of cytotoxicity and ADCC activity are maintained for at least 6 hours after irradiation.

[0100] IL-2 secretion and intracellular IL-2 levels of haNK003 cells before and after irradiation demonstrate that haNK003 cells secrete low levels of IL-2. haNK003 cells, irradiated or not, do not release amounts of IL-2 that would be expected to have adverse effects in humans.

[0101] Example 4 Permissiveness and tumorigenicity of a single dose of haNK003 cells administered intravenously Natural killer (NK) cells are effective cytotoxic effector cells for cancer therapy and potentially for viral infection. NantKwest has successfully established a unique NK cell-based platform for manufacturing GMP-grade activated NK (aNK) cells. aNK cells are being actively pursued in the clinic for cell therapy of patients with a variety of advanced hematological malignancies and solid tumors. Recently, a GMP-grade, plasmid-transfected variant of NK-92 expressing high affinity CD16 receptor was developed utilizing a novel transfection vector containing ER IL-2 gene that allows the resulting haNK003 cells to grow independent of IL-2. The expression of high affinity CD16 receptor allows haNK003 cells to exhibit high antibody-dependent cell-mediated cytotoxicity (ADCC) against target cell lines that were not killed by parental NK-92 cells in combination with rituximab and trastuzumab and daratumumab. One cell clone was selected to generate the master cell bank haNK003, which is 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 a single dose of haNK003 cells administered intravenously in NOD / SCID mice. Mice were obtained from Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US).

[0103] Eighteen NOD / SCID mice were selected and randomly assigned into three groups with six mice (three males and three females) per group based on the animals' weights. Mice were intravenously administered a single dose of PBS, non-irradiated haNK003 cells, or haNK003 cells irradiated at 10 Gy, as shown in Table 6. Animals were then monitored by daily observation and weighing the animals twice a week. After 5 weeks, the animals were euthanized, and major organs were collected and processed for further histopathological examination and immunohistochemical analysis using anti-CD56 antibody.

[0104] Table 6. Study design TIFF2025038128000012.tif56142

[0105] For cell culture, haNK003 cells were cultured in X-Vivo 10 medium (catalog no. BE02-055Q) supplemented with 5% heat-inactivated human AB serum (catalog no. IPLA-SERAB-HI, Innovative Research), 100 U penicillin / ml, and 100 μg / ml streptomycin (Corning, catalog no. 30-002-CI).

[0106] Upon irradiation, exponentially growing haNK003 cells were harvested and viable cell counts and viability were calculated. On the appropriate day, half of the haNK003 cells were cultured in a JL Shephard Mark 1 Model 68 137 The tumors were irradiated at a dose of 1000 cGy using a 1Cs irradiator (services provided by the Department of Radiation Oncology, the University of California, Irvine, CA 92697).

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

[0108] Eighteen NOD / SCID mice were selected and randomly assigned into three groups with six mice (three males and three females) per group 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 weight of the individual animal. The route of administration was intravenous injection via the tail vein and the dosing schedule was a single dose as indicated in the study protocol. On the appropriate day, each animal in groups B and C received 1 x 10 7 Each mouse received 100 μl of non-irradiated and irradiated haNK003 cells, respectively. The dose volume was 200 μl, the administration route was intravenous injection via the tail vein; the administration schedule was a single dose.

[0110] Animals were observed once daily for general appearance. Clinical observations were performed twice daily and recorded. Animals were routinely monitored after treatment for effects on normal behavior such as locomotion, 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 assessed using a repeated measures two-way ANOVA followed by the Bonferroni test. All data were analyzed using GraphPad Prism software version 5. p < 0.05 was considered statistically significant.

[0112] result 1 x 10 cells administered intravenously as a single agent 7 The doses of non-irradiated or irradiated haNK003 cells were well tolerated with a maximum mean body weight loss of 5.2% and 4.4%, respectively. As shown in Table 7 and Figure 11, the irradiated and non-irradiated haNK003 cells (1 x 10 7 There was no significant weight loss in NOD / SCID mice when either of the two drugs was administered as a single dose. As shown in Table 7, no treatment-related deaths occurred during the 5-week observation period in all treatment groups.

[0113] Regardless of whether the mice were male or female, all treatment groups were free of any visible tumor masses in all tissues and organs evaluated, including the brain, heart, liver, lungs, kidneys, spleen, and thymus. Results from histological analysis and IHC staining with anti-CD56 antibody confirmed that there were no lymphoid aggregates associated with haNK003 cells in tissues and organs, including the brain, bone marrow, heart, liver, lungs, kidneys, spleen, and thymus, suggesting that both non-irradiated and irradiated haNK003 cells have no tumor-forming ability in NOD / SCID mice.

[0114] Pathological examination of all specimens obtained in this study, including brain, bone marrow, heart, liver, lung, kidney, spleen, and thymus, showed that there was no significant toxicity associated with haNK003 treatment in either the non-irradiated or irradiated haNK003 cell treated groups compared to the PBS treated group.

[0115] Table 7. Effects of intravenously administered haNK003 on animal weight, mortality, and tumorigenicity in NOD / SCID mice. TIFF2025038128000013.tif38142Note: a MWL: maximum weight loss; b p values ​​relative to PBS treatment (two-way ANOVA for repeated measures followed by Bonferroni test); c (n / total): number of treatment-related animal deaths out of the total number of animals in each group.

[0116] 1 x 10 cells administered intravenously as a single agent 7 Both doses of irradiated and non-irradiated haNK003 cells were well tolerated in both male and female NOD / SCID mice. There was no significant weight loss associated with either irradiated or non-irradiated haNK003 treatment. There were no treatment-related deaths in all treatment groups during the 5-week observation period. There were no significant pathological changes in major organs including brain, bone marrow, heart, liver, lungs, kidneys, spleen, and thymus. Most importantly, neither irradiated nor non-irradiated haNK003 cells had tumorigenic potential in both male and female NOD / SCID mice.

[0117] Example 5 Permissiveness and tumorigenicity of repeated doses of haNK003 cells administered intravenously material and method Eighteen (18) NOD.CB17-Prkdcscid / J (NOD / SCID) mice (9 males and 9 females) were used to investigate the tolerance and tumorigenicity of a single dose of haNK003 cells administered intravenously in NOD / SCID mice. Mice were obtained from Jackson Laboratory (610 Main Street Bar Harbor, ME 04609 US).

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

[0119] Table 8. Study design TIFF2025038128000014.tif49128

[0120] For cell culture, haNK003 cells were cultured in X-Vivo 10 medium (catalog no. BE02-055Q) supplemented with 5% heat-inactivated human AB serum (catalog no. IPLA-SERAB-HI, Innovative Research), 100 U penicillin / ml, and 100 μg / ml streptomycin (Corning, catalog no. 30-002-CI).

[0121] Upon irradiation, exponentially growing haNK003 cells were harvested and viable cell counts and viability were calculated. On the appropriate day, half of the haNK003 cells were cultured in a JL Shephard Mark 1 Model 68 137The tumors were irradiated at a dose of 1000 cGy using a 1Cs irradiator (services provided by the Department of Radiation Oncology, the University of California, Irvine, CA 92697).

[0122] For cell preparation for administration, non-irradiated or irradiated haNK003 cells were kept on ice during transportation to the animal facility (1124 W. Carson Street, Torrance, CA, 90502). Cells were washed twice with cold PBS and then resuspended in an appropriate amount of cold PBS and passed through a 40 μm cell strainer to generate 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 more than 85% were used for this study. These non-irradiated or irradiated haNK003 cells were then stored at room temperature for intravenous administration to the appropriate group of animals.

[0123] Eighteen NOD / SCID mice were selected and randomly assigned into three groups with six mice (three males and three females) per group based on the animals' body weight.

[0124] On the appropriate day, each animal in group A received 200 μl of PBS regardless of the individual animal's weight. As indicated in the study protocol (Appendix 1), the route of administration was intravenous injection via the tail vein, and the dosing schedule was once a week for a total of 4 weeks. Each animal in groups B and C received 1 x 107 non-irradiated and irradiated haNK003 cells in 200 μl of PBS, respectively. As indicated in the study protocol, the dose was 200 μl, the route of administration was intravenous injection via the tail vein, and the dosing schedule was once a week for a total of 4 weeks.

[0125] Animals were observed once daily for general appearance. Clinical observations were performed twice daily and recorded. Animals were routinely monitored after treatment for effects on normal behavior such as locomotion, 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 assessed using a repeated measures two-way ANOVA followed by the Bonferroni test. All data were analyzed using GraphPad Prism software version 5. p < 0.05 was considered statistically significant.

[0127] result As single agents, non-irradiated or irradiated haNK003 cells administered intravenously once a week for 4 weeks were well tolerated, with a maximum mean body weight loss of 3.4% and 4.9%, respectively. As shown in Table 9 and Figure 12, compared to the PBS-treated control group, non-irradiated and irradiated haNK003 cells (1 x 10 7 There was no significant weight loss in NOD / SCID mice administered either the 100 mg or 100 mg dose of 100 mg ...

[0128] Table 9. Effect of haNK003 cells on animal weight, mortality, and tumorigenicity in NOD / SCID mice. TIFF2025038128000015.tif34142Note: a MWL: maximum weight loss; b p values ​​relative to PBS treatment (two-way ANOVA for repeated measures followed by Bonferroni test); c (n / total): number of treatment-related animal deaths out of the total number of animals in each group.

[0129] Macro- and histopathological examination of all specimens obtained in this study, including brain, bone marrow, heart, liver, lung, kidney, spleen, and thymus, showed that there was no significant toxicity associated with haNK003 treatment in these organs in both non-irradiated and irradiated haNK003 cell treated groups compared to the PBS treated control group. Splenomegaly was not observed in any animals. No tumor masses were found grossly in all treatment groups. Results from IHC using anti-CD56 antibody confirmed that there were no lymphoid aggregates associated with haNK003 cells in tissues and organs including bone marrow, brain, liver, lung, heart, kidney, spleen, thymus, etc. during the 5-week follow-up, suggesting that there was no leukemia or tumor development associated with irradiated or non-irradiated haNK003 cells in NOD / SCID mice over these 5 weeks.

[0130] There was no edema, degeneration, or necrosis in all these tissues. Compared with the PBS control group, very focal slight and minimal steatosis in liver tissue was observed in the non-irradiated haNK003-treated group and the irradiated haNK003-treated group, respectively. It is also noteworthy that the liver parenchyma in the PBS-treated group, the non-irradiated haNK003-treated group, or the irradiated haNK003-treated group showed few minute foci of mixed inflammatory cells, including neutrophils and mononuclear cells. These small cluster cells were CD56 negative, suggesting that these mixed inflammatory cells were most likely due to the repeated treatment (tail vein injection) and not related to haNK003 treatment.

[0131] In summary, non-irradiated and irradiated HaNK003 cells were well tolerated, and there were no significant toxicity or tumorigenicity issues associated with haNK003 treatment in NOD / SCID mice at this dosing regimen.

[0132] Example 6 Comparison of natural 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 granzymes. This then induces the process of apoptosis in the target cell, which leads 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 and subsequent killing of target cells through the engagement of stress antigens with activating receptors on NK cells is referred to as natural (or direct) cytotoxicity.

[0133] Since natural cytotoxicity is a major functional feature of NK cells, analysis of this functionality in cell mutants and comparison with activated NK-92 cell (aNK) activity will help to ascertain the impact of specific genetic modifications of aNK cells.

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

[0135] For cell culture, aNK cells were cultured in X-Vivo 10 medium supplemented with 5% heat-inactivated human AB serum (from a CMV-negative test donor) and 500 IU / ml recombinant human IL-2. aNK cultures were passaged every 1-4 days to maintain cell density >10e5 cells / mL and <10e6 cells / mL. haNK003 cells were cultured in X-Vivo 10 medium supplemented with 5% heat-inactivated human AB serum (from a CMV-negative test donor) without IL-2. haNK003 cultures were passaged every 1-4 days to maintain cell density >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% heat-inactivated fetal bovine serum (FBS) and an antibiotic / antimycotic mix. Cells growing in suspension were passaged by simple dilution, while adherent cells (SKOV3) were passaged by trypsinizing the culture with TrypLE™. Passages were every 2-5 days (depending on the cell line's specific doubling time) or when the culture medium turned yellow (acidic), indicating consumption of medium.

[0136] For sample preparation, cell lines growing in suspension were resuspended by pipetting the cell culture up and down. Adherent target cell line (SKOV3) was enzymatically detached from the culture vessel using TrypLE™ and resuspended by pipetting the trypsinized cell pellet up and down. Cell viability was determined by manual counting (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 antibiotic / antimycotic. Effector and target cells were mixed in 96-well plates at different effector to target (E:T ratios 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 with 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 without and with PI were used to determine the B1 / B3 compensation parameter.

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

[0139] result The aNK 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 and effector cell cultures were passaged a maximum of 48 hours prior to the assay.

[0140] The results shown in Figures 13-18 confirm the susceptibility of the target cell lines to killing by aNK, 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 of greater than 10:1 to achieve approximately 40% specific lysis. In each case, the natural cytotoxic activity of haNK003 was comparable to that of aNK, and generally followed the same activity profile, within experimental error.

[0141] aNK cells and haNK003 cells exhibited comparable cytotoxic activity against the six cancer cell lines tested, demonstrating that the natural cytotoxic activity of aNK cells is essentially the same despite the genetic modifications used to generate haNK003 cells. haNK003 cells and aNK cells are equally functional with respect to natural cytotoxic activity.

[0142] Example 7 Evaluation of the antitumor activity of haNK003 in a subcutaneous mouse model of MDA-MB-453 human breast cancer In this study, the antitumor activity of haNK003 cells as a single agent was significantly increased in female NOD.Cg-Prkdc scid Il2rg tm1Wjl The results were evaluated in a subcutaneous MDA-MB-453 human breast cancer xenograft model in / 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] MDA-MB-453 HER2-positive human breast cancer subcutaneous xenograft models were established in female NSG mice. The average tumor size was approximately 100 mm 3 Treatment was initiated when the tumor cell count reached 100%, and the antitumor activity of haNK003 cells as a single agent was evaluated in this xenograft model. Other test articles were evaluated in parallel under protocol LABC-X01612, but only the performance of haNK003 compared to PBS is presented here. The haNK003 treatment groups and the dosing regimen design are described in Table 10.

[0145] Table 10. Study design TIFF2025038128000016.tif46142

[0146] For tumor cell culture, MDA-MB-453 human breast cancer cells (ATCC, Catalog No. HTB-131) were cultured in Leibovitz's L-15 medium formulated by ATCC (ATCC, Catalog No. 30-2008) supplemented with 10% heat-inactivated FBS (GeneTex, Catalog No. GTX73252), 100 U penicillin / ml, and 100 μg / ml streptomycin (Corning, Catalog No. 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 ml in 50% Matrigel (Corning, Cat. No. 354234) was injected subcutaneously in the left and right flank areas with a 25-gauge needle. Cell viability was determined using a Vi-CELL cell viability analyzer, and only cells with >95% viability were used for this in vivo study.

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

[0149] Upon irradiation, exponentially growing haNK003 cells were harvested and viable cell counts and viability were calculated. On the appropriate day, haNK003 cells were cultured in a JL Shephard Mark 1 Model 68 137The tumors were irradiated at a dose of 1000 cGy using a 1Cs irradiator (services provided by the Department of Radiation Oncology, the University of California, Irvine, CA 92697).

[0150] For cell preparation for administration, irradiated haNK003 cells were kept on ice during transportation to the animal facility (1124 W. Carson Street, Torrance, CA, 90502). Cells were washed twice with cold PBS and then resuspended in the appropriate volume of cold PBS and passed through a 40 μm cell strainer (Corning, Cat. No. 431750) to generate single cell preparations with a final cell density 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 each animal in the appropriate group.

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

[0152] The dose was 200 μl regardless of the weight of the individual animal. As indicated in the study protocol, the route of administration was intravenous injection via the tail vein, and the dosing schedule was twice a week for a total of 4 weeks. On the appropriate day, 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 route of administration was intravenous injection via the tail vein, and the dosing schedule was twice a week for a total of 4 weeks.

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

[0154] Tumor size was measured in three dimensions twice weekly using digital handheld calipers before the first dose (once tumors appeared) and twice weekly thereafter before euthanasia. The primary endpoint was inhibition or reduction of tumor growth. Tumor volumes were 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 volumes were then used to calculate T / C values: 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 for the treatment and control groups, respectively.

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

[0156] result Data are presented for 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 summarized in Table 11. As shown in Table 11, Figure 19, 2.5 x 10 cells 6 Pieces or 1.0 x 10 7haNK003 cells administered intravenously as a single agent at a dose of 10 x 10 twice a week for 4 weeks significantly inhibited tumor growth with T / C values ​​of 17.4% and 1.3%, respectively (p = 0.043 and p = 0.006 compared to the PBS group). As shown in Table 11 and Figure 20, 2.5 x 10 cells 6 Pieces or 1.0 x 10 7 The doses of HaNK003 were well tolerated, with maximum mean body weight losses 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 related to 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. TIFF2025038128000017.tif30130Note: a T / C (%) was calculated using the following formula: T / C (%) = ΔT / ΔC x 100, where ΔT and ΔC are the changes in mean tumor volume between the 26th and 1st day of measurement for the treated and control groups, respectively. b MWL: maximum weight loss of animals. c P value versus PBS treatment (two-way ANOVA for repeated measures followed by Bonferroni test).

[0158] cells 2.5 x 10 6 Pieces or 1.0 x 10 7 Irradiated haNK003 cells as a single agent at this dose significantly suppressed tumor growth in the MDA-MB-453 HER2-positive human breast cancer xenograft model in female NSG mice. Irradiated haNK003 cells were well tolerated at both doses. No deaths related to haNK003 treatment occurred.

[0159] Example 8 Expression of genes associated with hypoxia is not decreased in haNK cells The lytic activity of natural killer (NK) cells is suppressed in vitro under hypoxic conditions (1% O2) and is associated with downregulation of NKG2D, perforin, and granzymes. There is some variability in the sensitivity of NKs 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 (1000 IU / ml for 16 hours). Furthermore, NK cells retain the capacity for ADCC under conditions of 1% oxygen.

[0160] To determine whether hypoxic conditions alter gene expression in haNK cells, RNA expression was determined in NK cell populations of three normal donors and haNK cells exposed to 20% or 0% O2 for 5 hours. NK cell populations of three patient donors (950, 962, 996) were compared to haNK cells under two conditions, 0% oxygen and 20% oxygen. Clustering of samples between pairs reveals distinct clusters that distinguish haNK from donor NK cells (see FIG. 21). One patient sample appears to have little change in expression under hypoxic or pre-hypoxic conditions. Specifically, expression of 962 under 20% oxygen conditions appears very similar to 996 under 0% oxygen conditions and to 962 under 0% oxygen conditions. See FIG. 21. Genes showing the greatest variability across samples are shown in FIG. 22. Genes showing the greatest change between 20% and 0% oxygen conditions are shown in FIG. 23. Genes associated with hypoxia that show no change in expression between 20% and 0% oxygen conditions in haNK cells are shown in Figure 24. These same genes associated with hypoxia are shown to have decreased expression in the 950, 962, and 996 samples, although to a lesser extent in the 950 sample. See Figure 24.

[0161] Example 9 CD16 expression is more stable in hank003 cells It is desirable to have stable expression of CD16 to confer superior cytotoxicity by continuously killing target cells during antibody-dependent cellular cytotoxicity (ADCC). HaNK-003 (with ER-IL-2) expresses CD16 at higher levels compared to peripheral blood NK cells (donor NK cells) following activation with PMA (phorbol-12-myristate 13-acetate) or upon stimulation with K562 target cells. Furthermore, measurement of CD16 levels by flow cytometry after ADCC showed that CD16 levels in hank003 cells were not significantly affected during and after ADCC.

[0162] It is known that activation with PMA / ionomycin in NK cells induces the activation of CD16-specific proteases and the cleavage of CD16, resulting in downregulation of CD16 expression levels in NK cells. To determine the effect of PMA / ionomycin, both haNK003 cells and donor NK cells were contacted with 40 nM PMA and 669 nM ionomycin for 1 hour, and then CD16 expression levels were examined. PMA / ionomycin treatment resulted in a downregulation of CD16 expression of 94.36% ± 3.00 in donor NK cells, while in haNK003 cells the treatment resulted in only a downregulation of 30% ± 0.04, i.e., 3-fold less CD16 downregulation compared to donor NK cells (Figure 25).

[0163] It is also known that co-culture of NK cells with K562 cells stimulates CD16 cleaving proteases resulting in shedding of CD16 surface expression in NK cells. Thus, both donor NK cells and haNK003 cells were cultured with K562 cells and then CD16 expression was measured after 4 hours of co-culture. Standard co-culture conditions of haNK003 cells with K562 (effector:target = 1:1) result in complete cytotoxic killing of target cells within 4 hours. CD16 expression was measured again after an additional 24 hours to allow 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 after 4 hours of co-culture. However, in hank003 cells, CD16 expression was downregulated by only 4.9% ± 2.57. After 24 hours, downregulation of CD16 in donor NK cells was 57.54% ± 26.82, whereas in haNK003 cells it was only 2.78% ± 3.5, i.e. close to native CD16 levels (Figure 26).

[0164] The CD16 expression level in haNK003 cells was also measured after antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC was performed by incubating haNK003 cells with DOHH-2 (a 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, the expression of CD16 was downregulated by less than 10% in haNK003 cells (Figures 27A and 27B). The presence of high levels of CD16 even after ADCC indicated that CD16 expression in haNK003 cells was 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 <h2 style=";text-align:left;direction:ltr">ttattgtctc atgagcggat acatatttga a 5491<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 1872<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> artificial sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> synthetic construct<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaattcgcca ccatgtggca gctgctgctg cctacagctc tcctgctgct ggtgtccgcc 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggcatgagaa ccgaggatct gcctaaggcc gtggtgttcc tggaacccca gtggtacaga 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtgctggaaa aggacagcgt gaccctgaag tgccagggcg cctacagccc cgaggacaat 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcacccagt ggttcccacaa cgagagcctg atcagcagcc aggccagcag ctacttcatc 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gacgccgcca ccgtggacga cagcggcgag tatagatgcc agaccaacct gagcaccctg 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcgaccccg tgcagctgga agtgcacatc ggatggctgc tgctgcaggc ccccagatgg 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtgttcaaag aagaggaccc catccacctg agatgccact cttggaagaa caccgccctg 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cacaaagtga cctacctgca gaacggcaag ggcagaaagt acttccacca caacagcgac 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttctacatcc ccaaggccac cctgaaggac tccggctcct acttctgcag aggcctcgtg 540<h2 style=";text-align:left;direction:ltr"> 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 having antibody-dependent cell-mediated cytotoxicity (ADCC) comprising a heterologous nucleic acid molecule comprising both CD16 (SEQ ID NO:3) and IL-2 (SEQ ID NO:5), greater than 90% of the cells in the population of cells express CD56, CD16, CD54, and NKp30, and less than 5% of the cells in the population of cells express CD3; Modified NK-92 cell populations.

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

3. The cell of 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. The cell of any one of claims 1 to 3, wherein the cell comprises SEQ ID NO:1 on chromosome 17.

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

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

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

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

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

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

11. The cell of any one of claims 1 to 9, wherein the cell maintains a higher level 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. 13. The kit of claim 12, further comprising an antibody.

14. A pharmaceutical composition comprising the population of cells of any one of claims 1 to 11 and a pharma- ceutically acceptable excipient.

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

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