HK1237796B - Polypeptides, cells, and methods involving engineered cd16 - Google Patents

Description

Polypeptides, cells and methods involving engineered CD16

Cross-reference to related applications

This application claims priority to U.S. Provisional Patent Application Serial No. 61/971,996, filed on March 28, 2014, which is incorporated herein by reference.

SUMMARY OF THE INVENTION

The present disclosure generally describes modified forms of CD16, genetically modified cells expressing modified CD16, and methods involving genetically modified cells. The modified forms of CD16 can exhibit increased anti-tumor and/or anti-viral activity due, at least in part, to reduced sensitivity to metalloproteinase-mediated shedding upon NK cell stimulation.

Thus, in one aspect, the present disclosure describes cells that are genetically modified to express a CD 16 polypeptide having a membrane proximal region and amino acid modifications in the membrane proximal region.

In another aspect, the disclosure describes a cell comprising a polynucleotide encoding a CD 16 polypeptide having a membrane proximal region and an amino acid modification in the membrane proximal region.

In any aspect, the amino acid medication reflects the addition of one or more amino acids, the deletion of one or more amino acids, or the substitution of one or more amino acids compared to the wild-type amino acid sequence of the CD16 membrane proximal region. In some of these embodiments, the substitution of one or more amino acids includes substitution of the serine residue at position 197 of SEQ ID NO: 1.

In any aspect, the cell can be a natural killer (NK) cell, a neutrophil, a monocyte, or a T cell.

In either aspect, the modified CD16 polypeptide exhibits reduced sensitivity to ADAM17-mediated shedding compared to a wild-type CD16 polypeptide.

In either aspect, the modified CD16 polypeptide exhibits reduced sensitivity to cleavage upon NK cell stimulation compared to wild-type CD1 polypeptide.

In another aspect, the disclosure describes methods generally directed to administering to a patient in need of such treatment comprising: (a) administering to the patient a therapeutic NK effector, and (b) administering to the patient any of the embodiments of the genetically modified cells outlined above.

In some embodiments, the therapeutic NK effector comprises a therapeutic agent. In some of these embodiments, the therapeutic agent may comprise an antibody, or a therapeutic antibody fragment. In some of these embodiments, the antibody or antibody fragment specifically binds to a viral antigen. In other embodiments, the antibody or antibody fragment specifically binds to a tumor antigen.

In some embodiments, the therapeutic agent may include a bispecific killer adaptor (BiKE) or a trispecific killer cell adaptor (TriKE).

In yet another aspect, the disclosure describes methods for improving immunotherapy of a patient, wherein the immunotherapy involves administering to the patient therapeutic NK effectors. Generally the methods comprise further administering to the patient any embodiment of the genetically modified cells outlined above.

The above summary of the present invention is not intended to describe every disclosed embodiment or every implementation of the present invention. The following description more specifically illustrates illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which can be used in various combinations. In each case, the recited list serves only as a representative group and should not be construed as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Location of extracellular domain cleavage sites in human CD16. (A) Tryptic peptides of soluble CD16 immunoprecipitated from cell supernatants of PMA-activated human NK cells or neutrophils were subjected to mass spectrometry analysis. Four high-confidence peptides with non-tryptic C-termini were identified: one peptide from soluble CD16 released by NK cells (peptide #1, upper left) and three peptides from soluble CD16 released by neutrophils (peptide #2, lower left; peptide #3, upper right; and peptide #4, lower right). (B) Illustration of peptides #1-4 (underlined) and the putative cleavage sites (arrows) in CD16a (SEQ ID NO:1) and CD16b (SEQ ID NO:2). Amino acid 176 in the identified peptides distinguishes CD16a (F) from CD16b (V). Amino acids 1-16 indicate the predicted signal sequences for CD16a and CD16b. Amino acids 210-229 indicate the transmembrane region of CD16a. Amino acid numbering begins with methionine in the signal sequence. The amino acid sequences of CD16a and CD16b are from NCBI reference sequences NM_000569.6 and NM_000570.4, respectively.

Figure 2. Schematic diagram of CD16 extracellular domain shedding, cleavage region, and engineered serine-197 to proline mutation. CD16a and CD16b undergo extracellular domain shedding by ADAM17 in the membrane proximal region, as indicated. The CD16 cleavage region in the membrane proximal region is based on mass spectrometry analysis, which shows three different cleavage sites (arrows) in close proximity. Site-directed mutagenesis was performed to replace serine-197 in CD16 (amino acids 190-202 of SEQ ID NO: 1) with proline (CD16/S197P).

Figure 3. Effect of the engineered S197P mutation on CD16a and CD16b shedding. Transfected HEK293 (human embryonic kidney) cells expressed CD16b and CD16b/S197P (A) or CD16a and CD16a/S197P (B) at similar levels, as determined by flow cytometry (left panel). Different transfectants were treated with or without PMA (15 ng/ml at 37°C for 30 minutes), and soluble CD16 levels in culture supernatants were quantified by ELISA (right panel). For each experiment, each treatment condition was repeated three times, and data are representative of three independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as ***P < 0.001. (C) Transfected HEK293 cells expressed L-selectin (CD62L) or L-selectin and CD16b/S197P. Surface levels of L-selectin and CD16b/S197P on transfected and mock-transfected cells were measured using flow cytometry (histograms). Transfectants expressing L-selectin or L-selectin and CD16b/S197P were incubated at 37°C for 30 minutes in the presence or absence of PMA, and the mean fluorescence intensity (MFI) of L-selectin staining was determined (bar graph). For each experiment, each treatment condition was repeated three times, and data are representative of two independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as *P < 0.05. For all histograms, x-axis = Log 10 fluorescence, y-axis = cell number.

Figure 4. Effect of the engineered S197P mutation on CD16a shedding in NK cells. NK92 cells transduced with empty vector (vector only), CD16a, or CD16a/S197P were treated with unstimulated (Unstim.) or PMA (100 ng/ml) at 37°C for 30 minutes (A), IL-12 and IL-18 (100 ng/ml and 400 ng/ml, respectively) at 37°C for 24 hours (B), or Raji cells and rituximab were treated at 37°C for 60 minutes (C). Cell surface levels of CD16a were determined by flow cytometry. The dashed line indicates staining with an isotype-matched negative control antibody. (D) Parental NK92 cells and transduced cells expressing CD16a or CD16a/S197P were treated with Raji cells and rituximab in the presence or absence of the ADAM17 inhibitor BMS566394 (5 μM) for 60 minutes at 37°C. Soluble CD16a levels were measured by ELISA. Each treatment condition was repeated three times, and data are representative of three independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as ***P < 0.001. (E) NK92 cells expressing CD16a or CD16a/S197P were stained with anti-ADAM17 mAbs M220, 623, 633, or an isotype-matched negative control antibody, as indicated. (F) CD56 ⁺ CD45 ⁺ NK cells derived from mock-transduced iPSCs (left panel) or iPSCs expressing recombinant CD16a or CD16a/S197P (right panel) were incubated with or without K562 target cells for 4 hours at 37°C. For all histograms, x-axis = Log 10 fluorescence, y-axis = cell number, and data are representative of at least 3 independent experiments.

Figure 5. Effect of the engineered S197P mutation on CD16a function. (A) NK92 cells expressing equivalent levels of CD16a or CD16a/S197P were treated with monomeric human IgG (0-20 μg/ml) (left panel). As controls, cells were also treated with monomeric human IgA (20 μg/ml) and NK92 parental cells were treated with IgG (20 μg/ml) (bars). Antibody binding was determined by flow cytometry as described in Materials and Methods. Bar graphs show the mean ± SD of at least three separate experiments. Statistical significance is indicated as *P < 0.05 relative to IgG (0 μg/ml), IgA, or NK92 parental cells + IgG. (B) Mock-transduced NK92 cells or NK92 cells expressing CD16a or CD16a/S197P were incubated with or without anti-CD20 rituximab at 37°C in the absence or presence of Raji cells (Unstim.) for the indicated time points. NK92 cell activation was assessed by upregulation of CD107a staining by flow cytometry. For histograms, x-axis = Log 10 fluorescence, y-axis = cell number. Data are representative of at least three independent experiments.

Detailed Description of Illustrative Embodiments

The present disclosure generally describes modified forms of CD16a, genetically modified cells expressing modified CD16a, and methods involving genetically modified cells. The modified forms of CD16a can exhibit increased anti-tumor and/or anti-viral activity due, at least in part, to reduced sensitivity to metalloproteinase-mediated shedding upon NK cell stimulation.

Compared with many solid tumor types, the survival rate of women with epithelial ovarian cancer has hardly changed in the past 30 years. In addition, the current standard treatment for recurrent ovarian cancer provides a low (<20%) response rate. Although HER2 is generally overexpressed in ovarian cancer samples, only limited response is provided in patients with advanced ovarian cancer using anti-HER2 antibody trastuzumab. This resistance to trastuzumab may be caused by antibody-dependent cellular toxicity mediated by dysfunctional NK cells. Therefore, there is an urgent need for innovative treatment strategies. We have described a new approach for providing a treatment strategy.

One concern with ovarian cancer is the surrounding environment, in which tumor cells develop, which can be highly proinflammatory and therefore likely promote the cleavage of CD16a on infiltrating NK cells, thereby reducing antibody-dependent cellular cytotoxicity. Several antibodies have emerged as effective targeted therapies for the treatment of human malignancies. Their efficacy is partly due to antibody interaction with FcγRIIIa/CD16a on natural killer (NK) cells and induction of cancer cell killing through antibody-dependent cellular cytotoxicity. The human IgG Fc receptor CD16 (FcγRIII) consists of two isoforms: CD16a (FcγRIIIa) and CD16b (FcγRIIIb). CD16a is expressed by natural killer (NK) cells, and CD16b is expressed by neutrophils. NK cell activation leads to a rapid downregulation of surface levels of both CD16 isoforms through a process called ectodomain shedding (involving the metalloprotease ADAM17 and a proteolytic event occurring at a single ectodomain close to the plasma membrane) (Figure 1A).

As noted above, ovarian cancer patients may be resistant to NK cell-mediated immunotherapy—that is, the tumor is insensitive to NK cell-mediated therapy. For example, ovarian cancer cells often express the epidermal growth factor receptor HER2, but targeting it with the therapeutic antibody trastuzumab provides only limited clinical response. This resistance may be caused, at least in part, by ectodomain shedding—that is, NK cell activation by cytokines, target cell interactions, and/or tumor infiltration can lead to CD16 cleavage and impair antibody-dependent cellular cytotoxicity. Therefore, blocking the ectodomain shedding process has clinical significance.

We have used mass spectrometry to determine the cleavage sites of CD16a and CD16b and cloned the cDNAs of CD16a and CD16b from human leukocytes. Each cDNA was mutated in a directed manner to induce a single amino acid change. The serine at position 197 was changed to a proline (Figure 1B). This mutation blocks the cleavage of CD16a and CD16b, preventing it from being downregulated during cell activation. The expression of anti-cleavage CD16a in NK cells amplified in vitro maintains the high surface level of the IgG Fc receptor, which enhances NK cell stimulation, therapeutic antibody efficacy, and cancer cell killing.

ADAM17 has many cell surface substrates, but does not have a protein hydrolysis consensus sequence that can be used to predict the CD16a cleavage site. Therefore, we used LC-MS-MS to determine the C-terminal cleavage site in the soluble CD16 released from activated human peripheral blood leukocytes. We observed three very close putative cleavage sites (Fig. 2, arrows) in the membrane proximal region of CD16 (the same region between CD16a and CD16b). Although ADAM17 protein hydrolysis does not require a consensus sequence, the secondary structure of the cleavage region is very important. In an attempt to block CD16a cleavage, we replaced serine-197 (CD16a ^197P ) with proline to introduce conformational changes.

We identified the site of CD16 cleavage by immunoprecipitating CD16 from culture supernatants of activated NK cells and, separately, from culture supernatants of neutrophils. Immunoprecipitated CD16 was treated with PNGaseF to remove N-glycans, digested with trypsin, and the resulting peptides analyzed by mass spectrometry. Four distinct peptide patterns containing a non-trypsin C-terminus were identified with high confidence (Figure 1A).

For CD16 enriched from culture supernatants of activated NK cells, we observed only one peptide profile, corresponding to amino acids glycine-174 to alanine-195 of SEQ ID NO: 1 (peptide #1, Figure 1A). The membrane-proximal regions of CD16a and CD16b share identical amino acid sequences except for residue 176. The phenylalanine at this position represents CD16a, which is present in peptide #1 (Figures 1A and B). This peptide exhibits a non-trypsin P1/P1' cleavage site at alanine-195/valine-196 (Figure 1B).

For CD16 enriched from the supernatant of activated neutrophils, we detected three different peptide spectra with non-trypsin C-termini (peptides #2-4, Figures 1A and 1B). Peptide #2 corresponds to amino acids glycine-174 to alanine-195 of SEQ ID NO: 2, peptide #3 corresponds to amino acids glycine-174 to valine-196 of SEQ ID NO: 2, and peptide #4 corresponds to amino acids asparagine-180 to threonine-198 of SEQ ID NO: 2. Peptide #2 and peptide #3 contain valine at position 176, representing CD16b, and show P1/P1' sites at alanine-195/valine-196 and valine-196/serine-197 (Figure 1B). Peptide #4 has a P1/P1' site at threonine-198/isoleucine-199 (Figure 1B). Although the peptide was derived from soluble CD16 from enriched neutrophils, it did not contain the amino acid at position 176 to identify the isoform (Figure 1B). Regardless, the high-confidence peptide showed a third cleavage site in CD16. Together, these findings demonstrate the presence of a cleavage region in CD16 rather than a single specific cleavage site.

We further examined the cleavage region of CD16 by using site-directed mutagenesis to determine whether CD16a and CD16b cleavage can be destroyed in cell-based assays. ADAM17 tends to preferably form an α-helical conformation that interacts with its catalytic site in the substrate region. In addition, proteomic studies of ADAM17 cleavage site specificity show a very low preference for the proline residues at P1 ', P2 ' or P3 ' positions. Therefore, we replaced the serine-197 (S197P, as indicated in Figure 2) in the CD16a and CD16b cleavage regions with proline.

CD16b and CD16b/S197P were expressed in the human kidney cell line HEK293, which does not express endogenous CD16. HEK293 transfectants expressed CD16b or CD16b/S197P (Fig. 3A) at similar levels on their surfaces. High levels of CD16b were released from transfected HEK293 cells, and these levels were further increased when treated with PMA, as measured by ELISA (Fig. 3A). However, the soluble levels of CD16b/S197P produced by untreated or PMA-treated HEK293 cells were significantly lower than those of CD16b (Fig. 3A).

We also used the same method to examine the effect of the S197P mutation on CD16a cleavage. The surface expression of CD16a requires association with the γ chain dimer. We therefore used HEK293 cells stably expressing human γ chain. Comparing HEK293 transfectants expressing equal surface levels of CD16a or CD16a/S197P (Figure 3B), we determined the soluble levels of each receptor in the culture supernatant of untreated and PMA-treated cells. Again, significantly lower levels of soluble CD16a/S197P were observed when compared to CD16a (Figure 3B).

To assess whether the engineered S197P mutation in CD16 could disrupt ADAM17 activity, we also transfected HEK293 cells expressing or lacking CD16b/S197P with L-selectin, a well-described ADAM17 substrate normally expressed by leukocytes. Both transfectants expressed equivalent levels of L-selectin, which were similarly downregulated after activation with PMA (Figure 3C), demonstrating that the S197P mutation affects CD16 shedding but not ADAM17 activity.

To evaluate the effect of the S197P mutation on CD16a shedding in NK cells, we used the human NK cell line NK92 (Gong et al., 1994, Leukemia 8: 652-658). These cells lack the expression of endogenous CD16a, but can stably express recombinant CD16a. We transduced NK92 cells to express CD16a and CD16a/S197P respectively. Cells expressing equal levels of these receptors were activated with PMA and cell surface CD16 levels were examined by flow cytometry. CD16a, but not CD16a/S197P, experienced significant downregulation of cell surface expression (Figure 4A). IL-12 and IL-18 are physiological stimulants of NK cells, which can induce CD16a shedding alone or in combination. NK92 cells treated with IL-12 and IL-18 showed considerable downregulation of CD16a, but not CD16a/S197P, in their cell surface expression (Figure 4B). Direct engagement of CD16a by IgG-bound cells can also induce its shedding, which we examined here by incubating NK92 cells expressing CD16a or CD16a/S197P with the CD20-positive Burkitt's lymphoma cell line Raji in the presence or absence of the anti-CD20 mAb rituximab. Treatment of Raji cells with rituximab induced downregulation of CD16a, but not CD16a/S197P (Figure 4C).

BMS566394 is a highly selective ADAM17 inhibitor, which has a higher order of magnitude of effectiveness for ADAM17 than for other metalloproteinases. BMS566394 blocks CD16a shedding with an efficiency similar to that of the S197P mutation, but does not have an additional blocking effect (Fig. 4 D) on the NK92 cells expressing CD16a/S197P of activation. These findings provide further evidence that ADAM17 is the main shedding enzyme for cutting CD16a in its cleavage region. However, it is possible that ADAM17 expression levels are unequal in the NK92 cells expressing CD16a or CD16a/S197P, resulting in their different shedding. We therefore stain the NK92 cells expressing CD16a or CD16a/S197P with a variety of anti-ADAM17 mAbs and observe identical cell surface levels (Fig. 4 E).

In order to determine the effect of the S197P mutation on the shedding of CD16a of primary NK cells, we used human iPSCs to generate engineered NK cells. We have previously reported the derivation of functional NK cells from iPSCs and their similarity to peripheral blood NK cells (Knorr et al., 2013 Stem Cells Transl Med . 2:274-283; Ni et al., 2014, Stem Cells 32:1021-1031). CD16a and CD16a/S197P cDNA were cloned into the Sleeping Beauty transposon plasmid for gene insertion and stable expression in iPSC cells, which were subsequently differentiated into mature NK cells. NK cells derived from simulated transduced iPSC cells expressed low levels of endogenous CD16a, while transduced CD16a and CD16a/S197P were expressed at higher levels (Fig. 4F). NK cell activation occurs through multiple receptors (including BY55/CD160) upon interaction with K562 cells, leading to ADAM17 activation and CD16a shedding. We stimulated iPSC-derived NK cells with K562 cells and found that CD16a underwent significant cell surface downregulation, while CD16a/S197P expression remained stable (Figure 4F).

Endogenous and recombinant CD16a have enough affinity to bind monomeric IgG. In order to check the effect of S197P mutation on CD16a function, we compared the IgG binding capacity of CD16a and CD16a/S197P. NK92 cells expressing CD16a or CD16a/S197P at equal levels bind IgG in a similar dose-dependent manner (Fig. 5A). The control consists of IgA bound to NK92 cells expressing CD16a or CD16a/S197P and IgG bound to NK92 parent cells. Both occur at basic background levels (Fig. 5A). These findings demonstrate specific and equal IgG binding by CD16a and CD16a/S197P.

CD16a is an effective activating receptor on NK cells. We have examined whether the engineered S197P mutation affects the ability of CD16a to induce cell activation when the tumor cells treated with the antibody participate. NK92 cell activation is assessed by measuring the increase in CD107a, which occurs very rapidly during degranulation and is a sensitive marker for NK cell activation. Simulated transduction NK92 cells incubated with or without rituximab-treated Raji cells show low levels and similarly increased CD107a (Fig. 5B). When incubated with Raji cells alone, NK92 cells expressing CD16a or CD16a/S197P at equal levels also increase CD107a on a small amount, while the incubation of the Raji cells treated with rituximab results in a considerable increase in CD107a (Fig. 5B). In short, the above findings show that the engineered S197P mutation in CD16a does not damage its function.

Thus, we demonstrate that engineered S197P mutations in CD16a and CD16b effectively block shedding in cell-based assays involving native ADAM17. The S197P mutation in CD16a also blocks receptor shedding in the human NK cell line NK92, but does not impair receptor function. NK92 cells expressing equivalent levels of CD16a or CD16a/S197P bind monomeric IgG with similar efficiency across a range of antibody concentrations. Furthermore, when challenged with rituximab bound to Raji cells, NK92 cells expressing CD16a or CD16a/S197P upregulated the activation marker CD107a in a comparable manner.

Pluripotent stem cells allow genetic manipulation to generate engineered NK cells. This disclosure describes the generation of engineered NK cells from transduced iPSCs expressing wild-type CD16a or CD16a/S197P. Similar to NK92 cells, CD16a undergoes shedding in iPSC-derived NK cells, demonstrating normal ADAM17 activity upon cell activation, whereas CD16a/S197P is not shed.

CD16a and NK cell cytotoxic function can undergo considerable downregulation in cancer patients. cDNA encoding CD16a/S197P can be used to generate stable human induced pluripotent stem cells (iSPCs) and embryonic stem cells (ESCs). These stem cells can then be differentiated into primary NK cells expressing CD16a/S197P. Other cell populations expressing cleavage-resistant CD16a/S197P (e.g., monocytes) or CD16b/S197P (e.g., neutrophils) can also be derived from hESCs/iPSCs.

In order to produce NK cell immunotherapy for use in human patients against different forms of cancer or infection, CD16a/S197P-expressing NK cells can mediate increased antibody-dependent cellular cytotoxicity (ADCC) activity or other CD16a-mediated activity (e.g., IFNγ and TNFα production). For example, CD16a/S197P-expressing NK cells can be combined with therapeutic antibodies (e.g., trastuzumab or rituximab), bispecific killer adapters (BiKE, e.g., CD16×CD33, CD16×CD19 or CD16×EP-CAM bispecific killer cell adapters) or trispecific killer cell adapters (TriKE). Other therapeutic cell populations (e.g., neutrophils, monocytes, T cells, etc.) with increased CD16-mediated activity can also be produced.

Expression of CD16a/S197P in human iPSCs or human ESCs can generate NK cell populations with enhanced ADCC activity against tumors, such as HER2 ovarian cancer. In some cases, tumors can be treated with therapeutic antibodies, such as trastuzumab. Mature NK cells can be derived from human embryonic stem cells and iPSCs.

Wild-type CD16a and/or CD16a/S197P can be cloned to generate stable iPSC cell lines or stable ESC cell lines expressing a single CD16a receptor. Any suitable cloning method can be used. Exemplary cloning methods include, for example, viral-based methods, transposon vectors (e.g., Sleeping Beauty ), or nuclear transfection. In one example, iPSCs can be modified using a Sleeping Beauty transposon vector. The vector can contain a selection system, for example, a GFP/zeocin resistance fusion protein, which allows a dual selection system (zeocin resistance and flow cytometry sorting). iPSCs can be differentiated into mature NK cells as previously described (Ni et al., 2011, J. Virol . 85:43–50; Knorr et al., 2013, Stem Cells Transl Med 2:274–283; Woll et al., 2009, Blood 113:6094–6101). Expression of transgenic receptors in iPSCs can result in high levels of expression in derived NK cells. CD16 expression in undifferentiated iPSCs can impair NK cell differentiation. In this case, CD16 expression can be delayed using, for example, the CD56 or native CD16a promoter, so that CD16a expression is more consistent with normal NK cell differentiation.

Can compare the NK cell of expressing equal level wild-type CD16a and CD16a/S197P.The expression level of CD16 construct can be matched based on GFP expression by FACS sorting, and described GFP expression occurs in a proportional manner with CD16a construct.The CD16a level of matching can be verified by FACS.The cytotoxicity of NK cell anti-HER2-expressing ovarian cancer cell can be assessed by standard chromium release assay under therapeutic antibody, for example, trastuzumab exists or does not exist.Can assess the antibody-dependent cellular toxicity of non-chromium labeled ovarian cancer cell.Can assess the cytokine (for example, IFNγ, TNFα) production of NK cell and the soluble level of CD16a by ELISA, assess the cell surface level of CD16a and other activation markers (for example, CD107a, CD62L) by FACS.

The human tumor xenograft model described in Example 3 can be used to evaluate the anti-cancer activity of NK cells expressing non-cleavable CD16a in vivo. Unlike human CD16, mouse CD16 does not undergo extracellular domain shedding upon cell stimulation, so determining the effect of CD16a shedding on NK cell-mediated ADCC cannot be modeled in normal mice. Table 1 provides a representative set of experimental groups and treatments.

Table 1. Tumor xenograft models

Group n deal with# 1 5 No treatment 2 5 OVCAR3 cells only 3 5 OVCAR3 + NK cells/WT-CD16a 4 5 OVCAR3 + NK cells/WT-CD16a + trastuzumab 5 5 6 5 7 5 OVCAR3 + NK cells/vector only 8 5 OVCAR3 + NK cells/vector + trastuzumab

#Processing was performed at least twice and data were pooled.

Tumor growth and/or regression can be monitored weekly by conventional methods, including, for example, bioluminescent imaging, ultrasound, CT, MRI, another imaging technique and/or weighing mice (Woll et al., 2009, Blood 113:6094–6101). Mice can also be bled (e.g., weekly) to quantify human NK cell survival. The expression and/or cell surface levels of various effector function markers (e.g., IFNγ, CD16a) can be assessed using conventional techniques, for example, by FACS. Mice can be followed up at any appropriate time, for example, during 60 days. When sacrificed, evidence of metastasis (e.g., by bioluminescence) of viscera (e.g., spleen, liver, lungs, kidneys and/or ovaries) can be examined, as previously described (Woll et al., 2009, Blood 113:6094–6101).

Our analysis allows the definition and comparison of antibody-dependent cytotoxicity activity and in vivo efficacy of iPSC-derived NK cells expressing wild-type CD16a and CD16a/S197P. Therefore, we describe herein modified forms of CD16a, genetically modified cells expressing modified CD16a (e.g., NK cells, neutrophils, monocytes, T cells, etc.), and methods involving genetically modified cells. For example, NK cells expressing a modified form of CD16a, CD16a/S197P, showed increased anti-ovarian cancer activity, which was at least in part due to reduced sensitivity to ADAM17-mediated shedding upon NK cell stimulation. This, in turn, increased antibody-dependent cytotoxicity activity when engaging antibody-labeled cancer cells (e.g., cancer cells labeled with therapeutic antibodies). In addition, antibody recognition by NK cells increases contact stability with tumor cells and supports NK cell activity through other activating receptors (e.g., NKG2D).

The term "and/or" means one or all of the listed elements or a combination of two or more listed elements; the term "include" and variations thereof do not have a limiting meaning where these terms appear in the description and claims; unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably to mean one or more; the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the foregoing description, specific embodiments may be described separately for clarity. Unless otherwise clearly indicated that features of one specific embodiment are incompatible with features of another embodiment, certain embodiments may include a combination of compatible features described herein with respect to one or more embodiments.

For any method disclosed herein that includes discrete steps, the steps can be performed in any practicable order. Also, any combination of two or more steps can be performed simultaneously, where appropriate.

The present invention is illustrated by the following examples. It is to be understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

Example

Example 1

Mass spectrometry

Peripheral blood was collected from healthy individuals according to a protocol approved by the University of Minnesota Institutional Review Board (University of Minnesota Institutional Review Board) under protocol # 9708M00134. Human neutrophils and NK cells were isolated as previously described (Wang et al., 2013, Biochim Biophys Acta . 1833: 680-685; Long et al., 2010, J Leukoc Biol . 87: 1097-1101; Long et al., 2012, J Leukoc Biol . 92: 667-672). Enriched neutrophils or NK cells (1 × ¹⁰⁷ /ml in PBS; Mediatech, Inc. Manassas, VA) were activated with PMA (15 ng/ml or 50 ng/ml, respectively; Sigma-Aldrich, St. Louis, MO) at 37°C for 30 minutes. Cell supernatants were filtered (0.45 μm pore size) and CD16 was immunoprecipitated using mAb 3G8 (BioLegend, Inc., San Diego, CA) and the Pierce Direct Immunoprecipitation Kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's instructions. Purified CD16 was deglycosylated using Remove-iT PNGase F (New England BioLabs, Inc., Ipswich, MA), which targets the chitin-binding domain, according to the manufacturer's instructions. Briefly, 10–20 μg of purified CD16 was denatured at 55°C for 10 minutes in the presence of 40 mM DTT and then incubated with 3 μl of Remove-iT PNGase F (New England BioLabs, Inc., Ipswich, MA) at 37°C for 1 hour. Remove-iT PNGase F was then removed from the reaction using chitin magnetic beads.

CD16 was subjected to SDS-PAGE and the gel band corresponding to soluble CD16 was detected by krypton fluorescent protein staining (Thermo Fisher Scientific, Rockford, IL), and confirmed by CD16 immunoblotting analysis of adjacent lanes in the same gel, which were then excised and subjected to standard in-gel digestion with trypsin. The digested peptides extracted from the gel were dried and reconstituted in 98:2:0.01 water:acetonitrile:formic acid for liquid chromatography-mass spectrometry analysis, and aliquots of ≤1 μg were analyzed by mass spectrometry (VELOS OPBITRAP, Thermo Fisher Scientific, Rockford, IL) in data-dependent scanning mode as previously described (Lin-Moshier et al., 2013, J Biol Chem . 288:355-367). Database searches were performed using Protein Pilot 4.5 (AB Sciex, Framingham, MA) against the NCBI reference sequence human protein FASTA database with an additional contaminant database (thegpm.org/cRAP/index, 109 proteins) using the Paragon scoring algorithm (Shilov et al., 2007, Mol Cell Proteomics 6:1638-1655). Search parameters were: cysteine iodoacetamide; trypsin; instrument Orbi MS (1-3 ppm) Orbi MS/MS; biological modification ID focus, which included asparagine deamidation; exhaustive search effort; and false discovery rate analysis (using the reverse database).

Generation of cDNA expression constructs

CD16b exists as two allelic variants, designated NA1 and NA2, that differ by four amino acids in the N-terminal portion of its extracellular region. ADAM17 cleaves both CD16b allelic variants with similar efficiency. For this study, we examined only the NA1 variant. CD16a also exists in two allelic variants, harboring either a valine or a phenylalanine residue at position 176. ADAM17 cleaves both CD16a allelic variants with similar efficiency. For this study, we examined only the valine allelic variant, CD16a.

CD16a and CD16b were amplified from human leukocyte cDNA and cloned individually into pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) at the Bam HI and Eco RI restriction enzyme sites as previously described (Wang et al., 2013, Biochim Biophys Acta . 1833: 680-685; Dong et al., 2014, Arthritis Rheumatol . 66: 1291-1299). The constructs were then subjected to Quik-Change Site-directed Mutagenesis (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions to convert the serine at position 197 in CD16a and CD16b to a proline. All constructs were sequenced to confirm the presence of the expected mutations and the absence of any spontaneous mutations.

The CD16a cDNA was then cloned into the bicistronic retroviral expression vector pBMN-IRES-EGFP provided by Dr. G. Nolan (Stanford University, Stanford, CA) at the Bam HI and Eco RI restriction enzyme sites. The CD16a construct was also cloned into the bicistronic Sleeping Beauty transposon plasmid (pKT2-IRES-GFP:zeo) as previously described (Wilber et al., 2007, Stem Cells 25:2919-2927; Tian et al., 2009, Stem Cells 27:2675-2685). Briefly, wild-type CD16a and CD16a/S197P were PCR amplified using primers: 5'-CCG GAA TTC CAG TGT GGC ATC ATG TGGCAG CTG CTC-3' (forward, SEQ ID NO:XX) and 5'-CCG GAA TTC TCA TTT GTC TTG AGG GTCCTT TCT-3' (reverse, SEQ ID NO:YY). The EcoRI site is underlined. EcoRI -digested CD16a and CD16a/S197P PCR fragments were individually cloned into pKT2-IRES-GFP:zeo. Correct CD16a orientation and sequence were confirmed by PCR and sequencing analysis. We have previously cloned the full-length human L-selectin (CD62L) cDNA (Feehan et al., 1996, J Biol Chem . 271:7019-7024; Matala et al., 2001, J Immunol . 167:1617-1623) and transferred it into the pcDNA3.1 vector using the restriction enzyme site Xba I. The full-length human FcRγ cDNA was cloned as previously described (Dong et al., 2014, Arthritis Rheumatol . 66:1291-1299) and modified to use the pcDNA3.1 vector.

Generation of cell lines expressing recombinant L-selectin, CD16a, and CD16b

HEK293 cells (human embryonic kidney cell line) and NK92 cells (human NK cell line) (ATCC, Manassas, VA) were cultured according to the repository's instructions. HEK293 cells were transiently transfected with pcDNA3.1 containing or without CD16b, CD16b/S197P, and/or L-selectin using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. HEK293 cells stably expressing human FcRγ were transiently transfected with pcDNA3.1 containing or without CD16a or CD16a/S197P using the same method. NK92 cells were stably transduced with pBMN-IRES-EGFP with or without CD16a or CD16a/S197P by a previously described retroviral production and infection procedure (Matala et al., 2001, J Immunol . 167:1617-1623; Walcheck et al., 2003, J Leukoc Biol . 74:389-394; Wang et al., 2009, J Immunol . 182:2449-2457). Construct expression was assessed by EGFP fluorescence and CD16 staining, as determined by flow cytometry. Human iPSCs (UCBiPS7, derived from umbilical cord blood CD34 cells) were maintained on mouse embryonic fibroblasts (Knorr et al., 2013, Stem Cells Transl Med . 2:274-283; Ni et al., 2014, Stem Cells 32:1021-1031). Stable expression of CD16a or CD16a/S197P was achieved using the Sleeping Beauty transposon system as previously described (Wilber et al., 2007, Stem Cells 25:2919-2927; Tian et al., 2009, Stem Cells 27:2675-2685). Briefly, iPSCs were nucleofected with a combination of pKT2-IRES-GFP:zeo and transposase DNA in nucleofector solution V (Lonza Inc., Gaithersburg, MD) using program B16. The nucleofected cells were immediately suspended in iPSC growth medium containing bleomycin (50 μg/ml) and plated onto mouse embryonic fibroblasts.

Derivation of NK cells from CD16a-hESC and CD16a-iPSC cells

Hematopoietic differentiation of hESCs and iPSCs was performed as previously described (Ng et al., 2005, Blood 106: 1601–1603; Ng et al., 2008, Nat Protoc 3:768–776; Le Garff-Tavernier et al., 2010, Aging Cell 9: 527–535). Briefly, 3000 single cells were seeded per well of a 96-well round-bottom plate in BPEL medium containing stem cell factor (SCF, 40 ng/ml), vascular endothelial growth factor (VEGF, 20 ng/ml), and bone morphogenetic protein 4 (BMP4, 20 ng/ml). BPEL medium contains Iscove's Modified Dulbecco's Medium (IMDM, 86 ml, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA), F12 Nutrient Mixture with Glutmax I (86 mL, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA), 10% deionized bovine serum albumin (BSA, 5 ml, Sigma-Aldrich, St. Louis, MO), 5% polyvinyl alcohol (10 ml, Sigma-Aldrich, St. Louis, MO), linolenic acid (20 μl of a 1 gm/ml solution, Sigma), Synthecol 500x solution (Sigma-Aldrich, St. Louis, MO), α-monothioglycerol (3.9 μl/100 ml, Sigma-Aldrich, St. Louis, MO), and 1% dapoxetine (5 μl/100 ml, Sigma-Aldrich, St. Louis, MO). MO), protein-free hybridoma cocktail II (Invitrogen, Thermo Fisher Scientific, Inc., Waltham. MA), ascorbic acid (5 mg/ml, Sigma), Glutamax I (Invitrogen, Thermo Fisher Scientific, Inc., Waltham. MA), insulin-transferrin-selenium 100x solution (Invitrogen, Thermo Fisher Scientific, Inc., Waltham. MA), and penicillin/streptomycin (Invitrogen, Thermo Fisher Scientific, Inc., Waltham. MA).

On day 11 of hematopoietic differentiation, spin embryoid bodies were directly transferred to 24-well plates with or without EL08-1D2 stromal cells in NK medium supplemented with cytokines (Le Garff-Tavernier et al., 2010, Aging Cell 9:527–535). After 4–5 weeks of culture, single-cell suspensions were stained with APC-, PE-, FITC-, and PerCP-cy5.5-conjugated IgG or specific antibodies against human blood surface antigens: CD45-PE, CD56-APC, CD56-PE, CD16-PerCP-cy5.5, NKG2D-PE, NKp44-PE, NKp46-PE, CD158b-FITC, CD158e1/2-FITC (BD Pharmingen, San Jose, CA), CD158a/h-PE, and CD158i-PE (Beckman Coulter, Inc., Pasadena, CA). Antibody staining was assessed by flow cytometry.

Cell stimulation

HEK293 and NK92 cells were activated with 15 ng/ml and 100 ng/ml PMA, respectively, in RPMI 1640 medium (Mediatech, Inc., Manassas, VA) for 30 minutes at 37°C. NK92 cells were activated with 100 ng/ml IL-12 (PeproTech Inc, Rocky Hill, NJ) and 400 ng/ml IL-18 (R&D Systems, Inc., Minneapolis, MN) for the indicated time points. NK92 cell activation by CD16a was mediated by incubation with the CD20-positive Burkitt's lymphoma cell line Raji (ATCC, grown according to the instructions of the repository) treated with the anti-CD20 mAb rituximab (1 μg/ml) (Genentech, Inc., South San Francisco, CA) (1:1 ratio), as previously described (Romee et al., 2013, Blood 121:3599-3608). Excess rituximab was removed by washing the Raji cells. In some experiments, NK92 cells were pre-incubated with the selective ADAM17 inhibitor BMS566394 (5 μM) (Bristol-Myers Squibb Company, Princeton, NJ) for 30 minutes. NK cells derived from iPSCs were stimulated with the human erythroleukemia cell line K562 (ATCC, grown according to the repository's instructions) as previously described (Romee et al., 2013, Blood 121:3599-3608). Briefly, iPSC-derived NK cells were incubated with K562 target cells (2:1 ratio) at 37°C for 4 hours.

Antibody binding assay

Binding of cells to monomeric human IgG and IgA (Sigma-Aldrich, St. Louis, MO) was performed as previously described (Dong et al., 2014, Arthritis Rheumatol . 66:1291-1299) with some modifications. 5 × 10 ⁶ /ml of NK92 parental cells or transduced cells expressing CD16a or CD16a/S197P were incubated in triplicate at the indicated concentrations for 1 hour at 4°C. Cells were washed extensively and incubated with APC-conjugated donkey anti-human Fc (heavy and light chain) antibodies (Jackson Immunoresearch, West Grove, PA) according to the manufacturer's instructions. Cells were washed and immediately analyzed by flow cytometry.

Flow cytometry and ELISA

For cell staining, nonspecific antibody binding sites were blocked, and cells were stained with the indicated antibodies and examined by flow cytometry as previously described (Wang et al., 2013, Biochim Biophys Acta . 1833:680-685; Romee et al., 2013, Blood 121:3599-3608). Flow cytometric analysis was performed on a FACSCanto and LS RII instrument (BD Biosciences, San Jose, CA). Human CD16 was detected by mAbs 3G8 (BioLegend, Inc., San Diego, CA) and DJ130c (Santa Cruz Biotech, Santa Cruz, CA). CD107a was detected by mAb H4A3 (Biolegend, Inc., San Diego, CA). ADAM17 was detected using mAbs M220 (Doedens et al., 2000, J Biol Chem . 275:14598-14607), 111633, and 111623 (R&D Systems, Inc., Minneapolis, MN). Human L-selectin was detected using mAb LAM1-116 (Ancell Corp., Stillwater, MN). Isotype-matched negative control mAbs were used to assess nonspecific staining. CD16 ELISA was performed using a custom cytometric bead assay as previously described (Wang et al., 2013, Biochim Biophys Acta . 1833:680-685).

Statistical analysis

Statistical analysis was performed using Prism software (GraphPad, San Diego, CA) using ANOVA and Student's t-test when appropriate. P values < 0.05 were considered significant.

Example 2

Comparison of NK cells expressing equal levels of WT CD16a and CD16a ^197P (CD16a/S197P)

The expression level of the CD16 construct was sorted by FACS based on GFP expression matching (as described above for NK92 cells, Figure 2), and the GFP expression occurred in a manner proportional to the CD16 construct. The CD16a levels matched for all assays were verified by FACS. As a control, iPSC-derived NK cells (expressing only GFP) modified with an empty Sleeping Beauty transposon vector were evaluated. iPSC-derived NK cells express low levels of endogenous CD16a (data not shown). The cytotoxicity of NK cells to HER2-expressing ovarian cancer cells was assessed by standard chromium release assay in the presence or absence of trastuzumab. Antibody-dependent cellular toxicity using non-chromium labeled ovarian cancer cells was also performed. The cytokine (e.g., IFNγ, TNFα) production of NK cells and the soluble level of CD16a were assessed by ELISA. The cell surface levels of CD16a and other activation markers (e.g., CD107a, CD62L) were assessed by FACS.

Example 3

Human tumor xenograft model used to examine whether iPSC-derived NK cells expressing CD16a ^197P (CD16a/S197P) have increased anti-ovarian cancer activity in vivo in the presence of trastuzumab.

A xenograft model using NOD/SCID/γc ^−/− (NSG) mice and a human ovarian cancer cell line stably engineered to express firefly luciferase for bioluminescent imaging (Geller et al., 2013, Cytotherapy 15:1297–1306) was used to examine the anti-ovarian cancer activity of intraperitoneally (ip) delivered NK cells. The HER2-overexpressing OVCAR3 ovarian cancer cell line was used as an in vivo target (Hellstrom et al., 2001, Cancer Res 61:2420–2423). Sublethally irradiated (225 cGy) NSG female mice were intraperitoneally injected with OVCAR3 cells (2 × 10 ⁵ cells) expressing luciferase for bioluminescent imaging to quantify tumor growth or regression (Geller et al., 2013, Cytotherapy 15:1297–1306). Tumors were allowed to grow for 7 days before mice received a single intraperitoneal injection of 20 × 10 ⁶ NK cells. Mice were then given IL-2 (5 μg/mouse) every other day for 4 weeks, as previously described (Woll et al., 2009, Blood 113: 6094–6101) to promote the survival of NK cells in vivo. Trastuzumab was administered intraperitoneally at a dose of 50 μg once a week for 4 weeks (the dose was previously used in this model) (Warburton et al., 2004, Clinical cancer research 10: 2512–2524). The in vivo efficacy of iPSC-derived NK cells expressing equal levels of WT CD16 or CD16a ^197P (CDa6a/S197P) was compared. Controls included iPSC-derived NK cells expressing only GFP (vector only), and a group of mice of the same age that received only ovarian cancer cells. All mice received the same IL-2 treatment.

Tumor growth/regression was monitored weekly by bioluminescent imaging and weighing mice, as previously described (Woll et al., 2009, Blood 113: 6094–6101). Mice were also bled weekly to quantify human NK cell survival. Expression/cell surface levels of various effector function markers (e.g., IFNγ, CD16a) were assessed by FACS. Mice were followed up for ~60 days. At sacrifice, evidence of metastasis in the viscera (e.g., spleen, liver, lungs, kidneys, and/or ovaries) was examined by bioluminescence, as previously described (Woll et al., 2009, Blood 113: 6094–6101).

Exemplary embodiments

Embodiment 1. A cell genetically modified to express a CD16 polypeptide comprising a membrane proximal region and an amino acid modification in the membrane proximal region.

Embodiment 2. A cell comprising:

A polynucleotide encoding a CD16 polypeptide comprising a membrane proximal region and amino acid modifications in the membrane proximal region.

Embodiment 3. The cell of embodiment 1 or embodiment 2, wherein the amino acid medication reflects the addition of one or more amino acids, the deletion of one or more amino acids, or the substitution of one or more amino acids compared to the wild-type amino acid sequence of the CD16 membrane proximal region.

Embodiment 4. The cell of embodiment 3, wherein the one or more amino acid substitutions include substitution of the serine residue at position 197 of SEQ ID NO:1.

Embodiment 5. The cell of any preceding embodiment, wherein the cell is a natural killer (NK) cell.

Embodiment 6. The cell of any preceding embodiment, wherein the cell is a neutrophil.

Embodiment 7. The cell of any preceding embodiment, wherein the cell is a monocyte.

Embodiment 8. The cell of any preceding embodiment, wherein the modified CD16 polypeptide exhibits reduced sensitivity to ADAM17-mediated shedding compared to wild-type CD16 polypeptide.

Embodiment 9. The cell of any preceding embodiment, wherein the modified CD16 polypeptide exhibits reduced sensitivity to cleavage upon NK cell stimulation compared to wild-type CD16 polypeptide.

Embodiment 10. A method comprising administering to a patient in need of such treatment a treatment comprising:

administering therapeutic NK effectors to patients, and

Administering the cell of any one of claims 1 to 9 to a patient.

Embodiment 11. The method of Embodiment 10, wherein the therapeutic NK effector comprises a therapeutic agent.

Embodiment 12. The method of embodiment 11, wherein the therapeutic agent specifically recognizes a tumor antigen.

Embodiment 13. The method of embodiment 12, wherein the therapeutic agent comprises an antibody or antibody fragment that specifically recognizes a tumor antigen.

Embodiment 14. The method of embodiment 13, wherein the tumor antigen comprises HER2.

Embodiment 15. The method of embodiment 13 or embodiment 14, wherein the antibody comprises trastuzumab or rituximab.

Embodiment 16. The method of embodiment 10, wherein the therapeutic NK effector comprises a bispecific killing adaptor (BiKE).

Embodiment 17. The method of embodiment 16, wherein the BiKE comprises a CD16×CD33 BiKE, a CD16×CD19 BiKE, or a CD16×EP-CAM BiKE.

Embodiment 18. The method of embodiment 10, wherein the therapeutic NK effector comprises a trispecific killer cell engager (TriKE).

Embodiment 19. The method of any one of embodiments 11 or 16-18, wherein the therapeutic agent specifically recognizes a viral target.

Embodiment 20. A method for improving treatment of a patient comprising administering a therapeutic NK effector to the patient, the method comprising:

Administering the cell of any one of claims 1 to 9 to a patient.

The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions (e.g., in GenBank and RefSeq) and amino acid sequence submissions (e.g., in SwissProt, PIR, PRF, PDB) and translations of coding regions annotated in GenBank and RefSeq) are incorporated by reference in their entirety. If there is any inconsistency between the disclosure of this application and the disclosure of any document incorporated by reference herein, the disclosure of this application shall prevail. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be construed therefrom. The present invention is not limited to the exact details shown and described, as variations obvious to one skilled in the art will be encompassed by the invention as defined by the claims.

Unless otherwise indicated, all numbers used in the specification and claims expressing the amount of a component, molecular weight, etc. are to be understood as being modified in all instances by the term "about". Accordingly, unless otherwise indicated to the contrary, the numerical parameters listed in the specification and claims are approximate values that may vary depending on the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless so indicated.

Claims (31)

1. A cell comprising a polynucleotide encoding a functional CD16 polypeptide variant, said functional CD16 polypeptide variant having an amino acid sequence in which the serine residue at position 197 of SEQ ID NO:1 or SEQ ID NO:2 is replaced with proline. 2. The cell of claim 1, wherein the substitution of the serine residue at position 197 results in a conformational change that blocks CD16 cleavage. 3. The cell of claim 1, wherein the functional CD16 polypeptide variant further comprises a valine residue (176V) at position 176. 4. The cell of claim 1, wherein the cell is: (i) Natural killer (NK) cells; (ii) Neutrophils; (iii) Monocytes; or (iv) Pluripotent stem cells or differentiated cells derived from said pluripotent stem cells. 5. The cell of claim 4, wherein: (i) The pluripotent stem cells are induced pluripotent stem cells (iSPCs) or embryonic stem cells (ESCs); or (ii) Differentiated cells generated from the pluripotent stem cells are hematopoietic cells. 6. The cell of claim 1, wherein the functional CD16 variant exhibits: (i) Reduced sensitivity to extracellular domain shedding; (ii) Reduced sensitivity to ADAM17-mediated shedding compared to CD16 peptides without the same modifications; (iii) Reduced sensitivity to cleavage upon NK cell stimulation; or (iv) Increased CD16-mediated activity compared to CD16 peptides without the same modifications. 7. The cells of claim 4, wherein NK cells containing a functional CD16 peptide variant exhibit at least one of the following characteristics compared to NK cells expressing wild-type CD16 peptide: (a) Increased anti-tumor ability; (b) Increased antiviral capacity; (c) Improved antibody-dependent cell cytotoxicity; (d) Increased production of IFNγ or TNFα; (e) Increased CD16-mediated activity; (f) CD16 has a higher surface level; (g) Lower levels of soluble CD16; (h) Enhanced cell stimulation; and (i) Increased in vivo anticancer activity. 8. The cell of claim 1, wherein the cell further comprises a bispecific killer adaptor (BiKE) or a trispecific killer cell adaptor (TriKE). 9. The cell of claim 8, wherein the BiKE comprises CD16×CD33 BiKE, CD16×CD19 BiKE or CD16×EP-CAM BiKE. 10. Use of a cell population comprising the cells of claim 1 in the preparation of a medicament for a method, wherein the method comprises: The following therapies are given to patients who require this treatment: The patient is given a cell population comprising the cells of claim 1. 11. The use of claim 10, wherein the method further comprises administering a therapeutic NK effector to the patient. 12. The use of claim 11, wherein the therapeutic NK effector comprises a therapeutic agent. 13. The use of claim 12, wherein the therapeutic agent: (i) specifically recognizes a tumor antigen; or (ii) comprises an antibody or antibody fragment that specifically recognizes the tumor antigen; or (iii) specifically recognizes a viral target. 14. The use of claim 13, wherein: (i) the tumor antigen comprises HER2; or (ii) the antibody comprises trastuzumab or rituximab. 15. The use of claim 11, wherein the therapeutic NK effector comprises a bispecific killer adaptor (BiKE). 16. The use of claim 15, wherein the BiKE comprises CD16×CD33 BiKE, CD16×CD19 BiKE or CD16×EP-CAM BiKE. 17. The use of claim 11, wherein the therapeutic NK effector comprises a trispecific killer cell adaptor (TriKE). 18. The use of claim 12, wherein the therapeutic agent specifically recognizes a viral target. 19. Use of the cell of claim 1 in a medicament for preparing a method of improving a therapy for a patient, the therapy comprising administering a therapeutic NK effector to the patient, the method comprising: The patient is given the cells of claim 1. 20. A composition comprising the cells of any one of claims 1-9. 21. A therapeutic composition comprising a cell population, wherein the cell population comprises cells according to any one of claims 1-9. 22. The therapeutic composition of claim 21, wherein the composition comprises NK cell populations. 23. The therapeutic composition of claim 22, wherein the NK cells comprise a therapeutic effector. 24. The therapeutic composition of claim 23, wherein the therapeutic NK effector comprises a therapeutic agent. 25. The therapeutic composition of claim 24, wherein the therapeutic agent: (i) specifically recognizes a tumor antigen; or (ii) comprises an antibody or antibody fragment that specifically recognizes the tumor antigen; or (iii) specifically recognizes a viral target. 26. The therapeutic composition of claim 25, wherein: (i) the tumor antigen comprises HER2; or (ii) the antibody comprises trastuzumab or rituximab. 27. The therapeutic composition of claim 23, wherein the therapeutic NK effector comprises: (i) a bispecific killer adaptor (BiKE); or (ii) a trispecific killer cell adaptor (TriKE). 28. The therapeutic composition of claim 27, wherein the BiKE comprises CD16×CD33 BiKE, CD16×CD19 BiKE, or CD16×EP-CAM BiKE. 29. A cell population comprising the cells of any one of claims 1-9. 30. Use of the cells of any one of claims 1-9 in the preparation of a medicament for use in a subject with a tumor condition. 31. A pharmaceutical kit comprising the composition of any one of claims 20-28.