Polypeptides, cells, and methods involving modified CD16

Abstract

To provide generally a modified form of CD16, genetically-modified cells that express the modified CD16, and methods that involve the genetically-modified cells.SOLUTION: A modified form of CD16 can exhibit increased anti-tumor and / or anti-viral activity due to, at least in part, reduced susceptibility to ADAM17-mediated shedding upon NK cell stimulation.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 61 / 971,996, filed on 28 March 2014, which is incorporated herein by reference. [Overview of the project]

[0002] This disclosure generally describes modified CD16, recombinant cells expressing modified CD16, and methods involving recombinant cells. Modified CD16 may, at least in part, exhibit enhanced antitumor and / or antiviral activity due to lower sensitivity to metalloproteinase-mediated shedding upon NK cell stimulation. Therefore, in one embodiment, this disclosure describes a cell genetically modified to express a CD16 polypeptide comprising a membrane proximal region and amino acid modifications within the membrane proximal region.

[0003] In another embodiment, this disclosure describes a cell comprising a membrane-proximal region and a polynucleotide encoding a CD16 polypeptide having amino acid modifications in the membrane-proximal region. In any embodiment, amino acid therapy reflects the addition, deletion, or substitution of one or more amino acids compared to the wild-type amino acid sequence of the proximal CD16 membrane region. In some of these embodiments, the substitution of one or more amino acids includes the substitution of a serine residue at position 197 of SEQ ID NO: 1.

[0004] In any embodiment, the cells may be natural killer (NK) cells, neutrophils, monocytes, or T cells. In either embodiment, the modified CD16 polypeptide exhibits lower susceptibility to ADAM17-mediated shedding compared to the wild-type CD16 polypeptide. In either embodiment, the modified CD16 polypeptide exhibits lower sensitivity to cleavage upon NK cell stimulation compared to the wild-type CD1 polypeptide. In another embodiment, this disclosure describes a method that typically involves administering a treatment to a patient in need of such treatment, which includes (a) administering a therapeutic NK effector to the patient, and (b) administering one of the genetically modified cells of the embodiments outlined above to the patient.

[0005] In some embodiments, the therapeutic NK effector is a therapeutic agent. In some of these embodiments, the therapeutic agent may be 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 be a bispecific killer engager (BiKE) or a tripspecific killer cell engager (TriKE).

[0006] In yet another embodiment, this disclosure describes a method for improving immunotherapy for a patient, the immunotherapy comprising administering a therapeutic NK effector to the patient. Typically, the method further comprises administering to the patient one of the genetically modified cells of the embodiments outlined above. The prior summary of the present invention is not intended to describe any of the disclosed embodiments or any implementation of the invention. The following description provides more specific examples of embodiments that are helpful for explanation. Guidance is provided in several places in the application by lists of embodiments, which can be used in various combinations. In any case, the lists provided are representative only and should not be interpreted as exclusive lists. [Brief explanation of the drawing]

[0007] [Figure 1A]Location of ectodomain cleavage sites of human CD16. (A) Trypsin peptides of soluble CD16 immunoprecipitated from the supernatant of PMA-activated human NK cells or neutrophils were analyzed by mass spectrometry. Four high-confidence peptides with non-trypsin C-terminuses were identified: one peptide derived from soluble CD16 released by NK cells (peptide number 1, upper left), and three peptides derived from soluble CD16 released by neutrophils (peptide number 2, lower left; peptide number 3, upper right; and peptide number 4, lower right). [Figure 1B] (B) Examples of peptide numbers 1-4 (underlined) and the predicted cleavage sites (wedge-shaped) of CD16a (sequence number 1) and CD16b (sequence number 2). In the identified peptides, CD16a(F) is distinguished from CD16b(V) at amino acid 176. Amino acids 1-16 show the predicted signal sequences of CD16a and CD16b. Amino acids 210-229 show the transmembrane region of CD16a. Amino acid numbering begins with methionine in the signal sequence. The amino acid sequences of CD16a and CD16b are based on the NCBI reference sequences NM_000569.6 and NM_000570.4, respectively. [Figure 2] A schematic diagram of ectodomain shedding, cleavage regions, and genetically engineered serine-197 to proline mutations in CD16. CD16a and CD16b undergo ectodomain shedding by ADAM17 in the proximal membrane region, as shown. The CD16 cleavage region in the proximal membrane region is based on mass spectrometry analysis that revealed three very close, distinct cleavage sites (wedge-shaped). Site-directed mutagenesis was performed to replace serine-197 (amino acids 190-202 of SEQ ID NO: 1) of CD16 with proline (CD16 / S197P). [Figure 3]Effect of genetically engineered S197P mutations on CD16a and CD16b shedding. Transplanted HEK293 (human embryonic kidney) cells expressed CD16b and CD16b / S197P (A) or CD16a and CD16a / S197P (B) separately at the same level as measured by flow cytometry (left panel). Different transplants were treated with or without PMA (15 ng / ml, 37°C for 30 minutes), and the solubility level of CD16 in the culture medium supernatant was quantified by ELISA (right panel). Each treatment condition was repeated three times for each experiment, and the data represent three independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as ***P<0.001. (C) Transplanted HEK293 cells expressed L-selectin (CD62L) or L-selectin and CD16b / S197P. Surface levels of L-selectin and CD16b / S197P on transfused and mock transfused cells were measured using flow cytometry (histogram plots). Transfused cells expressing L-selectin or L-selectin and CD16b / S197P were incubated at 37°C for 30 minutes in or without PMA, and the mean fluorescence intensity (MFI) of L-selectin staining was determined (bar graph). Each treatment condition was repeated three times for each experiment, and the data represent two independent experiments. Bar graphs show mean ± SD. Statistical significance is indicated as *P<0.05. For all histogram plots, the X axis = Log10 fluorescence and the Y axis = cell number. [Figure 4]Effect of genetically engineered S197P mutations on CD16a shedding in NK cells. NK92 cells transduced with an empty vector (vector only), CD16a, or CD16a / S197P were treated with PMA (100 ng / ml) or without (unstimulated) at 37°C for 30 minutes (A), with IL-12 and IL-18 (100 ng / ml and 400 ng / ml, respectively) at 37°C for 24 hours (B), or with Raji cells and rituximab at 37°C for 60 minutes (C). Cell surface levels of CD16a were measured by flow cytometry. Antibody staining of isotype-corresponding negative controls is shown by dotted lines. (D) Parental NK92 cells and transduced cells expressing CD16a or CD16a / S197P were treated with Raji cells and rituximab at 37°C for 60 minutes in or without the ADAM17 inhibitor BMS566394 (5 μM). Soluble CD16a levels were measured by ELISA. Each treatment condition was repeated three times for each experiment, and the data represent three independent experiments. Bar graphs show the mean ± SD. Statistical significance is indicated as ***P<0.001. (E) NK92 cells expressing CD16a or CD16a / S197P were stained with anti-ADAM17 antibodies mAbs M220, 623, 633, or isotype-corresponding negative control antibodies as shown. (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 at 37°C for 4 hours. For all histogram plots, the X axis = Log10 fluorescence and the Y axis = cell number, and the data represent at least three independent experiments. [Figure 5]Effect of genetically engineered S197P mutation on CD16a function. (A) NK92 cells expressing equivalent levels of CD16a or CD16a / S197P (left panel) were treated with monomeric human IgG (0-20 μg / ml). As controls, cells were similarly treated with monomeric human IgA (20 μg / ml), and NK92 parent cells were treated with IgG (20 μg / ml) (bar graph). Antibody binding was measured by flow cytometry as described in Materials and Methods. The bar graph shows the mean ± SD of at least three separate experiments. Statistical significance is indicated as *P < 0.05 for IgG (0 μg / ml), IgA, or NK92 parent cells + IgG. (B) Mock transduced NK92 cells or NK92 cells expressing CD16a or CD16a / S197P were incubated at 37°C until the indicated time point, either in the absence of (unstimulated) or in the presence of Raji cells treated with or without anti-CD20 rituximab. NK92 cell activation was evaluated by flow cytometry with upregulation of CD107a staining. For the histogram plot, the X axis = Log10 fluorescence and the Y axis = cell number. Data represent at least three independent experiments.

[0008] Detailed description of exemplary embodiments This disclosure generally describes modified CD16a, recombinant cells expressing modified CD16a, and methods involving recombinant cells. Modified CD16a may, at least in part, exhibit enhanced antitumor and / or antiviral activity due to lower sensitivity to metalloproteinase-mediated shedding upon NK cell stimulation. In contrast to many solid tumor types, the survival rate of women with epithelial ovarian cancer has changed only slightly over the past 30 years. Moreover, current standard treatments for recurrent ovarian cancer have a low response rate (<20%). Despite HER2 overexpression in ovarian cancer samples, treatment with the anti-HER2 antibody trastuzumab yields only a limited response in patients with advanced ovarian cancer. This resistance to trastuzumab may stem from dysfunctional NK cell-mediated antibody-dependent cytotoxicity. Therefore, innovative therapeutic strategies are urgently needed. We describe a novel approach to provide a therapeutic strategy.

[0009] One concern with ovarian cancer is that the environment in which tumor cells progress is highly pro-inflammatory, which in turn promotes CD16a cleavage by invasive NK cells, thereby tending to reduce antibody-dependent cytotoxicity. Several antibodies have emerged as effective targeted therapies for treating human malignancies. Their effectiveness is partly due to antibody interaction with FcγRIIIa / CD16a on natural killer (NK) cells and the induction of cancer cell killing through antibody-dependent 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 involves the ectodomain shedding-metalloproteinase ADAM17 and leads to rapid downregulation of the surface levels of both CD16 isoforms through a process called a proteolytic event occurring in a single extracellular region close to the plasma membrane (Figure 1A).

[0010] As previously mentioned, ovarian cancer patients are resistant to NK cell-mediated immunotherapy—that is, the tumor may not be sensitive to NK cell-mediated therapies. For example, ovarian cancer cells commonly express the epidermal growth factor receptor HER2, but their targeting with the therapeutic antibody trastuzumab has yielded only limited clinical responses. This resistance may, at least in part, result from ectodomain shedding—that is, cytokine-mediated NK cell activation, target cell interactions, and / or tumor invasion may lead to CD16a cleavage and defective antibody-dependent cytotoxicity. Therefore, inhibiting the process of ectodomain shedding is clinically significant.

[0011] We determined the cleavage sites of CD16a and CD16b using mass spectrometry and cloned the cDNAs of CD16a and CD16b from human leukocytes. Each cDNA was mutated in a directional manner to induce a single amino acid change. Serine at position 197 was changed to proline (Figure 1B). This mutation inhibits the cleavage of CD16a and CD16b, preventing their downregulation by cell activation. Ex vivo expression of cleavage-resistant CD16a increased NK cells that maintain a high surface level of this IgG Fc receptor, which in turn promoted NK cell stimulation, the efficacy of therapeutic antibodies, and cancer cell killing. Although ADAM17 has many cell surface matrix components, there is no consensus sequence for proteolysis that can be used to predict the site of CD16a cleavage. Therefore, we used LCMS-MS to determine the C-terminal cleavage site within soluble CD16 released from activated human peripheral blood leukocytes.

[0012] We observed three very closely spaced putative cleavage sites within the membrane-proximal region of CD16 (Figure 2, wedge-shaped), one of which was identical between CD16a and CD16b. While ADAM17 proteolysis does not require a consensus sequence, the secondary structure of the cleavage region is important. In an attempt to prevent CD16a cleavage, we substituted serine-197 with proline (CD16a 197P), we introduced a change in three-dimensional structure. We identified the sites of CD16 cleavage by immunoprecipitation of CD16 from the culture supernatant of activated NK cells and separately from the culture supernatant of neutrophils. The immunoprecipitated CD16 was treated with PNGaseF to remove N-glycans, digested with trypsin, and the resulting peptides were subjected to mass spectrometry. We identified four different peptide patterns with high reliability, including a non-trypsinic C-terminus (Figure 1A).

[0013] Regarding CD16 enriched from the culture supernatant of activated NK cells, we observed only one peptide pattern, which corresponds to the amino acids from glycine-174 to alanine-195 in SEQ ID NO: 1 (peptide number 1, Figure 1A). The membrane proximal regions of CD16a and CD16b have identical amino acid sequences except for residue 176. Phenylalanine at this position suggests CD16a, which was present in peptide number 1 (Figures 1A and B). This peptide revealed a non-trypsinic P1 / P1' cleavage site at alanine-195 / valine-196 (Figure 1B).

[0014] Regarding CD16 concentrated from the supernatant of activated neutrophil culture media, we detected three distinct peptide patterns with non-trypsinic C-terminuses (peptide numbers 2-4, Figures 1A and 1B). Peptide number 2 corresponds to the amino acids glycine-174 to alanine-195 in SEQ ID NO: 2, peptide number 3 corresponds to the amino acids glycine-174 to valine-196 in SEQ ID NO: 2, and peptide number 4 corresponds to the amino acids asparagine-180 to threonine-198 in SEQ ID NO: 2.

[0015] Peptide No. 2 and Peptide No. 3, which are indicative of CD16b, contain valine at position 176, and the P1 / P1' positions at alanine-195 / valine-196 and valine-196 / serine-197 were revealed (Figure 1B). Peptide No. 4 had the P1 / P1' position at threonine-198 / isoleucine-199 (Figure 1B). This peptide is derived from soluble CD16 from concentrated neutrophils, but does not contain the amino acid at position 176 for identifying the isoform (Figure 1B). Regardless, high-confidence peptides revealed a third cleavage site of CD16. Collectively, these findings demonstrate the existence of a cleavage region of CD16 rather than a single specific cleavage site.

[0016] We further investigated the cleavage region of CD16 by using site-directed mutagenesis to determine whether CD16a and CD16b cleavage can be inhibited in cell-based assays. ADAM17 tends to select an α-helical structure in the substrate region that interacts with its catalytic site. Moreover, proteomic studies on ADAM17 cleavage site specificity have revealed a very low preference for proline residues at the P1', P2', or P3' positions. Therefore, we replaced serine-197 in the cleavage region of CD16a and CD16b with proline (S197P, as shown in Figure 2).

[0017] CD16b and CD16b / S197P were separately expressed in the human kidney cell line HEK293, which does not endogenously express CD16. HEK293 transfectants expressed CD16b or CD16b / S197P at the same level on their surface (Figure 3A). High levels of CD16b were released from transfected HEK293, which was further enhanced by their treatment with PMA, as measured by ELISA (Figure 3A). However, the soluble level of CD16b / S197P produced by untreated or PMA-treated HEK293 cells was significantly lower than that of CD16b (Figure 3A).

[0018] We also examined the effect of the S197P mutation on CD16a cleavage using the same approach. Surface expression of CD16a requires binding to a γ-chain dimer. Therefore, we used HEK293 cells that stably express human γ-chain. Compared with HEK293 transfectants expressing equivalent surface levels of CD16a or CD16a / S197P (Figure 3B), we measured the soluble levels of each receptor in the culture supernatant of untreated and PMA-treated cells, respectively. Again, we observed significantly lower levels of soluble CD16a / S197P compared with CD16a (Figure 3B).

[0019] To evaluate whether the genetically engineered S197P mutation in CD160 can interfere with ADAM17 activity, we also transfected HEK293 cells expressing or lacking CD16b / S197P with L-selectin, a well-documented ADAM17 substrate that is normally expressed in leukocytes. Both transfectants expressed equivalent levels of L-selectin, which was similarly downregulated following their activation with PMA (Figure 3C), demonstrating that the S197P mutation affected CD16 shedding rather than ADAM17 activity.

[0020] To evaluate the effect of S197P mutations 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 endogenous CD16a expression, but recombinant CD16a can be stably expressed. We transduced NK92 cells to express CD16a and CD16a / S197P separately. Cells expressing these receptors at equivalent levels were activated with PMA, and cell surface CD16 levels were examined by flow cytometry. CD16a, rather than CD16a / S197P, underwent significant downregulation in cell surface expression (Figure 4A). IL-12 and IL-18 are physiological stimuli of NK cells that can induce CD16a shedding individually or in combination. We demonstrated that NK92 cells treated with IL-12 and IL-18 significantly downregulated CD16a cell surface expression, but not with CD16a / S197P (Figure 4B). Direct involvement of cell-bound IgG by CD16a may also cause shedding, and we investigated this here by incubating NK92 cells expressing CD16a or CD16a / S197P with the CD20-positive Burkitt lymphoma cell line Raji in the presence or absence of the anti-CD20 mAb rituximab. Rituximab-treated Raji cells induced downregulation of CD16a, but not with CD16a / S197P (Figure 4C).

[0021] BMS566394 is an ADAM17 inhibitor that is orders of magnitude more selective to ADAM17 than other metalloproteinases. BMS566394 inhibited CD16a shedding with similar efficiency to the S197P mutation, but without additional blocking effect against activated NK92 cells expressing CD16a / S197P (Figure 4D). These findings provide further evidence that ADAM17 is the primary sheddase that cleaves CD16a within its cleavage region. However, the difference in ADAM17 expression levels between NK92 cells expressing CD16a or CD16a / S197P may have contributed to their different shedding. Therefore, we stained NK92 cells expressing CD16a or CD16a / S197P with multiple anti-ADAM17 mAbs and observed identical cell surface levels (Figure 4E).

[0022] To establish the effect of S197P mutations on CD16a shedding by primary NK cells, we generated genetically engineered NK cells using human iPSCs. We previously reported the induction 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). For gene insertion and stable expression in iPSC cells, CD16a and CD16a / S197P cDNAs were cloned into Sleeping Beauty transposon plasmids, which were then differentiated into mature NK cells.

[0023] NK cells obtained from mock transduced iPSC cells expressed low levels of endogenous CD16a, while transduced CD16a and CD16a / S197P were expressed at higher levels (Figure 4F). NK cell activation occurred through various receptors via interactions with K562 cells, including BY55 / CD160, resulting in ADAM17 activation and CD16a shedding. We found that stimulating iPSC-induced NK cells with K562 cells resulted in significant downregulation of CD16a cell surface expression, while CD16a / S197P expression remained stable (Figure 4F).

[0024] Endogenous and recombinant CD16a possess sufficient affinity to bind monomeric IgG. To investigate the effect of S197P mutations on CD16a function, we compared the IgG-binding ability of CD16a and CD16a / S197P. NK92 cells expressing CD16a or CD16a / S197P at comparable levels bound IgG in a similar dose-dependent manner (Figure 5A). The control consisted of IgA binding to NK92 cells expressing CD16a or CD16a / S197P, and IgG binding to NK92 parental cells. Both occurred at substantially background levels (Figure 5A). These findings demonstrate specific and comparable IgG binding by CD16a and CD16a / S197P.

[0025] Since CD16a is a potent activating receptor in NK cells, we investigated whether the genetically engineered S197P mutation affects the ability of CD16a to induce cell activation in response to antibody-treated tumor cells. NK92 cell activation was observed very rapidly by degranulation and assessed by measuring the upregulation of CD107a, a highly sensitive indicator of NK cell activation. Mock transduction NK92 cells incubated with Raji cells treated with or without rituximab demonstrated low levels of CD107a and similar upregulation (Figure 5B). NK92 cells expressing equivalent levels of CD16a or CD16a / S197P incubated with Raji cells alone also showed marginal upregulation of CD107a, but their incubation with rituximab-treated Raji cells also resulted in significant upregulation of CD107a (Figure 5B). In summary, the findings indicate that the S197P mutation in the CD16a gene did not impair its function.

[0026] Therefore, we demonstrate that genetically engineered S197P mutations in CD16a and CD16b effectively inhibited their shedding in cell-based assays with native ADAM17. The S197P mutation in CD16a also inhibited receptor shedding in the human NK cell line NK92, without impairing receptor function. NK92 cells expressing CD16a or CD16a / S197P at comparable levels bound monomeric IgG with similar efficiency across a wide range of antibody concentrations. In addition, NK92 cells expressing CD16a or CD16a / S197P upregulated the activation marker CD107a in a similar manner with the involvement of rituximab bound to Raji cells.

[0027] Multipotent stem cells enable genetic engineering to create genetically modified NK cells. This disclosure describes the creation of genetically modified NK cells from transduced iPSCs expressing wild-type CD16a or CD16a / S197P. When using NK92 cells, CD16a underwent shedding in iPSC-induced NK cells, demonstrating normal ADAM activity upon cell activation, while CD16a / S197P did not undergo shedding.

[0028] The cytotoxic functions of CD16a and NK cells are significantly downregulated in cancer patients. cDNAs encoding CD16a / S197P can be used to generate stable human induced pluripotent stem cells (iPSCs) 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 obtained from hESCs / iPSCs.

[0029] To create NK cell immunotherapies for use in human patients against various forms of cancer or infection, CD16a / S197P-expressing NK cells can mediate enhanced antibody-dependent cytotoxicity (ADCC) activity or other CD16a-mediated activity (e.g., IFNγ and TNFα production). For example, CD16a / S197P-expressing NK cells may be combined with therapeutic antibodies (e.g., trastuzumab or rituximab), bispecific killer engagers (e.g., BiKE, CD16×CD33, CD16×CD19, or CD16×EP-CAM bispecific killer cell engagers), or triplicate killer cell engagers (TriKE). Other therapeutic cell populations (e.g., neutrophils, monocytes, T cells, etc.) can also produce enhanced CD16-mediated activity.

[0030] The expression of CD16a / S197P in human iPSCs or human ESCs can create a population of NK cells with enhanced ADCC activity against neoplastic conditions, such as HER2 ovarian cancer. In some cases, the neoplastic condition may be treated with a therapeutic antibody, such as trastuzumab. Mature NK cells may be derived from human embryonic stem cells and iPSCs.

[0031] Wild-type CD16a and / or CD16a / S197P can be cloned to produce stable iPSC or ECS strains expressing individual CD16a receptors. Any suitable cloning method can be used. Typical cloning methods include, for example, virus-based methods, transposon vectors (e.g., Sleeping Beauty), or nucleofection.

[0032] For example, iPSCs can be modified using the Sleeping Beauty transposon vector. The vector contains a selection system, such as a GFP / zeosin-resistant fusion protein, which enables a binary selection system (zeosin resistance and flow cytometry cell sorting). The 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 the transgenic receptor in iPSCs can lead to high expression levels in induced NK cells. CD16 expression in undifferentiated iPSCs can disrupt NK cell differentiation. In such cases, CD16 expression may be delayed, for example, using a CD56 or native CD16a promoter, so that it is advantageous for CD16 expression to occur simultaneously with normal NK cell differentiation.

[0033] Those skilled in the art can compare NK cells expressing CD16a / S197P at equivalent levels to wild-type CD16a. CD16 construct expression levels can be equalized by FACS sorting based on GFP expression, which appears in a manner proportional to the CD16 construct. Equalized CD16a levels can be confirmed by FACS. NK cytotoxicity against HER2-expressing ovarian cancer cells can be evaluated by a standard chromium release assay, for example, in the presence or absence of a therapeutic antibody such as trastuzumab. Antibody-dependent cytotoxicity can be evaluated using unchromium-labeled ovarian cancer cells. Those skilled in the art can evaluate cytokine production (e.g., IFNγ, TNF) and CD16a solubility levels of NK cells by ELISA, and cell surface levels of CD16a and other activating markers (e.g., CD107a, CD62L) can be evaluated by FACS.

[0034] The human tumor xenograft model described in Example 3 can be used to evaluate the anticancer activity of NK cells expressing CD16a that is not cleavable in vivo. Unlike human CD16, mouse CD16 does not undergo ectodomain shedding in response to cellular stimulation; therefore, measuring the effect of CD16a shedding on NK cell-mediated ADCC cannot be modeled in normal mice. Table 1 provides representative combinations of experimental groups and treatments.

[0035] [Table 1]

[0036] Tumor growth and / or regression can be observed weekly by conventional methods, including, for example, bioluminescence imaging, ultrasound, CT, MRI, other imaging techniques, and / or mouse weighing (Woll et al, 2009, Blood 113:6094-6101). Mice can also be collected (e.g. weekly) to quantify human NK cell viability. Expression and / or cell surface levels of various effector functional markers (e.g., IFNγ, CD16a) can be evaluated using conventional methods, such as by FACS. Mice can be followed for any suitable period, such as 60 days. At the time of sacrifice, the organs (e.g., spleen, liver, lungs, kidneys, and / or ovaries) can be examined for signs of metastatic cancer (e.g., by bioluminescence), as previously described (Woll et al, 2009, Blood 113:6094-6101).

[0037] Our analysis enables those skilled in the art to define and compare the antibody-dependent cytotoxic activity and in vivo efficacy of iPSC-induced NK cells expressing CD16a / S197P in comparison to wild-type CD16a. Accordingly, we describe in this specification modified CD16a, recombinant cells expressing modified CD16a, and methods involving recombinant cells (e.g., NK cells, neutrophils, monocytes, T cells, etc.). For example, NK cells expressing modified CD16a, CD16a / S197P, exhibit enhanced anti-ovarian cancer activity, at least in part, due to lower sensitivity to ADAM17-mediated shedding upon NK cell stimulation. This similarly enhances antibody-dependent cytotoxic activity with the involvement of antibody-tagged cancer cells, such as cancer cells tagged with therapeutic antibodies. Furthermore, antibody recognition by NK cells enhances contact stability with tumor cells and strengthens NK cell activity through other activating receptors such as NKG2D.

[0038] The terms "and / or" mean one or all of the enumerated elements, or any combination of two or more of the enumerated elements; the terms "including" and their variations do not have a restrictive meaning when they appear in the specification and claims; unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more; and details of numerical ranges by endpoints include all numbers encompassed within that range (for example, 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0039] In the preceding description, specific embodiments may be described separately for clarity. Unless otherwise explicitly stated, a specific embodiment includes a combination of compatible features described herein with respect to one or more embodiments. Any method disclosed herein that includes separate steps may be carried out in any feasible order. Furthermore, any combination of two or more steps may be carried out simultaneously as appropriate. The present invention is illustrated by the following embodiments. It should be understood that specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein. [Example 1]

[0040] Example 1 mass spectrometry Peripheral blood samples were collected from healthy individuals according to a protocol approved by the University of Minnesota Institutional Ethics Committee under protocol number 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 × 10⁶ in PBS) were collected. 7 CD16 (15 ng / ml; Mediatech, Inc., Manassas, VA) was activated with PMA (15 ng / ml or 50 ng / ml, respectively; Sigma-Aldrich, St. Louis, MO) at 37°C for 30 minutes. The cell supernatant was 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. The purified CD16 was deglycosylated with Remove-iT PNGase F (New England BioLabs, Inc., Ipswich, MA) with a tagged chitin-binding domain according to the manufacturer's instructions. In short, 10-20 μg of purified CD16 was denatured in the presence of 40 mM DTT at 55°C for 10 minutes, and then incubated with 3 μl of REMOVE-IT PNGase F (New England BioLabs, Inc., Ipswich, MA) at 37°C for 1 hour. Next, REMOVE-IT PNGase F was removed from the reaction mixture using chitin magnetic beads.

[0041] CD16 was subjected to SDS-PAGE, and the gel band corresponding to soluble CD16 was detected with krypton fluorescent protein dye (Thermo Fisher Scientific, Rockford, IL). This was confirmed by CD16 immunoblotting of adjacent lanes on the same gel, which were then excised and subjected to standard in-gel digestion with trypsin. The digested peptide extracted from the gel was dried down and reconstituted in water:acetonitrile:formic acid, 98:2:0.01 for liquid chromatography-mass spectrometry. <1 μg aliquots were analyzed by mass spectrometry (VELOS ORBITRAP, 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), employing the Paragon scoring algorithm (Shilov et al, 2007, Mol Cell Proteomics 6: 1638-1655) against the NCBI reference sequence Homo sapiens protein FASTA database with a contaminant database (thegpm.org / cRAP / index, 10⁹ proteins). Search parameters included: cysteine-iodoacetamide; trypsin; instrumentation: Orbi MS (1-3 ppm) Orbi MS / MS; biological modification ID focus (including asparagine deamidation); overall search results; and false discovery rate analysis (using a regressive database).

[0042] Creation of cDNA expression constructs CD16b gives rise to two allele variants called NA1 and NA2, which differ in four amino acids in the N-terminal region of its extracellular domain. Both allele variants of CD16b are cleaved by ADAM17 with similar efficiency. For this test, we examined only the NA1 variant. There are also two allele variants of CD16a, which have either a valine or phenylalanine residue at position 176. These two allele variants of CD16a were cleaved by ADAM17 with similar efficiency. For this test, we examined only the valine allele variant CD16a.

[0043] CD16a and CD16b were amplified from human leukocyte cDNA and cloned separately into pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) at the BamHI and EcoRI 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, converting the serine at position 197 of CD16a and CD16b to proline. All constructs were sequenced to confirm the presence of planned mutations and the absence of any spontaneous mutations.

[0044] Next, the CD16a cDNA was cloned into the two-cistronic retroviral expression vector pBMN-IRES-EGFP, provided by Dr. G. Nolan (Stanford University, Stanford, CA), at the BamHI and EcoRI restriction enzyme sites. The CD16a construct was also cloned into the two-cistronic 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).

[0045] In short, wild-type CD16a and CD16a / S197P were PCR amplified using primers: 5'-CCG GAA TTC CAG TGT GGC ATC ATG TGG CAG CTG CTC-3' (sense, SEQ ID NO: XX) and 5'-CCG GAA TTC TCA TTT GTC TTG AGG GTC CTT TCT-3' (antisense, SEQ ID NO: YY). The EcoRI region is underlined. The EcoRI-digested CD16a and CD16a / S197P PCR fragments were separately cloned into pKT2-IRES-GFP:zeo. The correct CD16a orientation and sequence were confirmed by PCR and sequencing analysis. We previously cloned 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 at restriction enzyme site Xba1. We cloned full-length human FcRγ cDNA with modification using the pcDNA3.1 vector as previously described (Dong et al., 2014, Arthritis Rheumatol. 66: 1291-1299).

[0046] Creation 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 laboratory's instructions. HEK293 cells were transiently transfused using pcDNA3.1 containing or not containing CD16b, CD16b / S197P, and / or L-selectin, using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. HEK293 cells stably expressing human FcRγ were transiently transfused using pcDNA3.1 containing or not containing CD16a or CD16a / S197P using the same approach. NK92 cells were stably transduced using pBMN-IRES-EGFP containing or not containing CD16a or CD16a / S197P, according to previously described retrovirus generation and infection procedures (Matala et al, 2001, J Immunol. 167: 1617-1623; Walcheck et al, 2003, JLeukoc Biol. 74:389-394; Wang et al, 2009, J Immunol. 182:2449-2457). Construct expression was evaluated by EGFP fluorescence and, when measured by flow cytometry, by CD16 staining. Human iPSCs (UCBiPS7, derived from umbilical cord blood CD34 cells) were maintained on mouse embryonic fibroblasts (Knorr et al., 2013, Stem Cells Trans I 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).In short, iPSCs were nucleofected using pKT2-IRES-GFP:zeo combined with transposase DNA in nucleofector solution V (Lonza Inc., Gaithersburg, MD) using program setting B16. The nucleofected cells were immediately suspended in iPSC growth medium containing zeosin (50 μg / ml) and then seeded onto mouse embryonic fibroblasts.

[0047] Induction 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). In short, 3000 single cells per well of a 96-well round-bottom plate were seeded in BPEL medium containing stem cell factor (SCF, 40 ng / ml), vascular endothelial growth factor (VEGF, 20 ng / ml), and bone morphogenesis protein 4 (BMP4, 20 ng / ml).BPEL medium consists of Iscove's Modified Dulbecco's Medium (IMDM, 86 ml, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA), F12 Nutrient Mixture containing 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 1 g / ml solution, Sigma-Aldrich, St. Louis, MO), linoleic acid (20 μl of 1 g / ml solution, Sigma), SYNTHECOL 500× solution (Sigma-Aldrich, St. Louis, MO), and α-monothioglycerol (3.9 μl / 100 ml, Sigma-Aldrich, St. Louis, It contained (MO), protein-free hybridoma mix 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).

[0048] On day 11 of hematopoietic cell differentiation, spin embryoids were transferred directly to 24-well plates in NK medium supplied with cytokines, either together with or without EL08-1D2 stromal cells (Le Garff-Tavernier et al, 2010, Aging Cell 9:527-535). After 4-5 weeks of culture, single-cell suspensions were stained with specific antibodies against APC-, PE-, FITC-, and PerCP-cy5.5-conjugated IgG or 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 evaluated by flow cytometry.

[0049] cell stimulation HEK293 and NK92 cells in RPMI1640 medium (Mediatech, Inc., Manassas, VA) were activated at 37°C for 30 minutes using 15 ng / ml and 100 ng / ml PMA, respectively. NK92 cells were further activated to the indicated time points using 100 ng / ml and 400 ng / ml IL-12 (PeproTech Inc., Rocky Hill, NJ) and IL-18 (R&D Systems, Inc., Minneapolis, MN), respectively. CD16a-mediated NK92 cell activation was mediated by incubation with CD20-positive Burkitt lymphoma cell line Raji (ATCC, cultured according to the laboratory's instructions) (1:1 ratio) treated with anti-CD20 mAb rituximab (1 μg / ml) (Genentech, Inc., South San Francisco, CA). Excess rituximab was removed by washing the Raji cells. In some experiments, NK92 cells were pre-incubated for 30 minutes with the selective ADAM17 inhibitor BMS566394 (5 μM) (Bristol-Myers Squibb Company, Princeton, NJ). iPSC-derived NK cells were stimulated using the human erythroleukemia cell line K562 (ATCC grown according to the laboratory's instructions), as previously described (Romee et al, 2013, Blood 121:3599-3608). In short, iPSC-induced NK cells were incubated with K562 target cells (in a 2:1 ratio) at 37°C for 4 hours.

[0050] Antibody binding assay Cells that bind monomeric human IgG and IgA (Sigma-Aldrich, St. Louis, MO) were realized using several modifications as previously described (Dong et al, 2014, Arthritis Rheumatol. 66: 1291-1299). 5 × 10⁶ cells in PBS. 6NK92 parental cells or transduced cells expressing CD16a or CD16a / S197P at 1 / ml were incubated with IgG or IgA at the concentrations indicated in the triplicate at 4°C for 1 hour. The cells were thoroughly washed 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. The cells were washed and immediately analyzed by flow cytometry.

[0051] Flow cytometry and ELISA For cell staining, nonspecific antibody binding sites were blocked as previously described (Wang et al, 2013, Biochim Biophys Acta. 1833:680-685; Romee et al, 2013, Blood 121:3599-3608), cells were stained with the indicated antibody, and then examined by flow cytometry. Flow cytometry analysis was performed using FACSCanto and LS RII instruments (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 by 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 by mAb LAMl-116 (Ancell Corp., Stillwater, MN). Isotype-matched negative control mAbs were used to assess the level of nonspecific staining. ELISA for CD16 was performed by a custom cytometry bead assay as previously described (Wang et al, 2013, Biochim Biophys Acta. 1833:680-685).

[0052] statistical analysis Statistical analysis was performed using Prism software (GraphPad, San Diego, CA) with ANOVA and Student's t-tests as needed. A p-value of <0.05 was considered statistically significant.

[0053] Example 2 Equivalent levels of WT CD16a and CD16a 197P Comparison of NK cells expressing (CD16a / S197P)

[0054] The expression levels of the CD16 construct were equalized by FACS sorting based on GFP expression (as performed for NK92 cells described earlier, Figure 2), which occurs in a manner proportional to the CD16 construct. The equalized CD16a levels were confirmed by FACS for all assays. As a control, iPSC-induced NK cells modified with an empty Sleeping Beauty transposon vector (expressing only GFP) were evaluated. The iPSC-induced NK cells expressed low levels of endogenous CD16a (data not shown). NK cytotoxicity against HER2-expressing ovarian cancer cells was evaluated by a standard chromium release assay in the presence or absence of trastuzumab. Antibody-dependent cytotoxicity using unchromium-labeled ovarian cancer cells was also performed. Cytokine production (e.g., IFNγ, TNFα) and CD16a solubility levels of NK cells were evaluated by ELISA. Cell surface levels of CD16a and other activation markers (e.g., CD107a, CD62L) were evaluated by FACS.

[0055] Example 3 CD16a 197P A human tumor xenograft model to test whether iPSC-induced NK cells expressing (CD16a / S197P) have enhanced anti-ovarian cancer activity in vivo in the presence of trastuzumab.

[0056] NOD / SCID / γc designed to stably express firefly luciferase for bioluminescence imaging (Geller et al., 2013, Cytotherapy 15: 1297-1306) - / -A xenograft model using (NSG) mice and human ovarian cancer cell lines is used to test intraperitoneal (ip) delivery of NK cell activity against ovarian cancer cells. The HER2-overexpressing OVCAR3 ovarian cancer cell line is used as an in vivo target (Hellstrom et al, 2001, Cancer Res 61: 2420-2423). Female NSG mice that have received sublethal irradiation (225 cGY) are injected intraperitoneally with OVCAR3 (2×10 5 cells) engineered to express luciferase for bioluminescence imaging to quantify tumor growth or regression (Geller et al, 2013, Cytotherapy 15: 1297-1306). Tumors are allowed to grow for 7 days, after which mice receive a single intraperitoneal injection of 20×10 6 NK cells. Mice are then dosed with defined IL-2 (5 μg per mouse) every other day for 4 weeks as previously described (Woll et al, 2009, Blood 113: 6094-6101) to promote in vivo survival of NK cells. Trastuzumab is administered intraperitoneally weekly for 4 weeks at a dose of 50 μg, which is the dose 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 equivalent levels of WT CD16 or CD16a 197P (CDa6a / S197P) is compared. Controls include iPSC-derived NK cells expressing only GFP (vector only) and a cohort of mice that received only ovarian cancer cells. All mice receive the same IL-2 treatment.

[0057] Tumor growth / regression is observed weekly by bioluminescence imaging and mouse quantification, as previously described (Woll et al, 2009, Blood 113: 6094-6101). Mice are also collected weekly to quantify human NK cell viability. Expression / cell surface levels of various effector functional markers (e.g., IFNγ, CD16a) are evaluated by FACS. Mice are followed for approximately 60 days. At sacrifice, the internal organs (spleen, liver, lungs, kidneys, and ovaries) are examined for signs of metastatic cancer by bioluminescence, as previously described (Blood 113: Woll et al, 2009, 6094-6101).

[0058] Typical Embodiments Embodiment 1. Cells genetically modified to express a CD16 polypeptide containing a membrane proximal region and amino acid modifications within the membrane proximal region. Embodiment 2. A cell comprising a polynucleotide encoding a CD16 polypeptide that includes a membrane proximal region and amino acid modifications within the membrane proximal region. Embodiment 3. The cells according to Embodiment 1 or Embodiment 2, wherein the amino acid therapy reflects the addition, deletion, or substitution of one or more amino acids compared to the wild-type amino acid sequence of the proximal CD16 membrane region.

[0059] Embodiment 4. The cell according to Embodiment 3, wherein the substitution of one or more amino acids includes a substitution of a serine residue at position 197 of SEQ ID NO: 1. Embodiment 5. The cell according to any one of Embodiments 1 to 4, wherein the cell is a natural killer (NK) cell. Embodiment 6. The cell according to any one of Embodiments 1 to 5, wherein the cell is a neutrophil. Embodiment 7. The cell according to any one of Embodiments 1 to 6, wherein the cell is a monocyte. Embodiment 8. The cell according to any one of Embodiments 1 to 7, wherein the modified CD16 polypeptide exhibits lower sensitivity to ADAM17-mediated shedding compared to the wild-type CD16 polypeptide.

[0060] Embodiment 9. The cell according to any one of Embodiments 1 to 8, wherein the modified CD16 polypeptide exhibits lower sensitivity to cleavage upon NK cell stimulation compared to the wild-type CD16 polypeptide. Embodiment 10. For patients requiring such treatment, the following: Administer a therapeutic NK effector to the patient; and Administering the cells described in any one of claims 1 to 9 to the patient, A method that includes administering a treatment that includes [a specific treatment].

[0061] Embodiment 11. The method according to Embodiment 10, wherein the therapeutic NK effector contains a therapeutic agent. Embodiment 12. The method according to Embodiment 11, wherein the therapeutic agent specifically recognizes a tumor antigen. Embodiment 13. The method according to Embodiment 12, wherein the therapeutic agent comprises an antibody or antibody fragment that specifically recognizes a tumor antigen. Embodiment 14. The method according to Embodiment 13, wherein the tumor antigen comprises HER2. Embodiment 15. The method according to Embodiment 13 or Embodiment 14, wherein the antibody comprises trastuzumab or rituximab.

[0062] Embodiment 16. The method according to Embodiment 10, wherein the therapeutic NK effector includes a bispecific killer engager (BiKE). Embodiment 17. The method according to Embodiment 16, wherein the BiKE includes a CD16×CD33 BiKE, a CD16×CD19 BiKE, or a CD16×EP-CAM BiKE. Embodiment 18. The method according to Embodiment 10, wherein the therapeutic NK effector includes a triple-specific killer cell engager (TriKE).

[0063] Embodiment 19. The method according to any one of Embodiments 11 or 16-18, wherein the therapeutic agent specifically recognizes a viral target. Embodiment 20. A method for improving a treatment for a patient, comprising administering a therapeutic NK effector to the patient, the following: Administering the cells described in any one of claims 1 to 9 to the patient, A method that includes this.

[0064] All disclosures of all patents, patent applications, and publications described herein, as well as electronically available materials (e.g., nucleotide sequence deposits, e.g., GenBank and RefSeq, and amino acid sequence deposits, e.g., SwissProt, PIR, PRF, PDB, and translations from the annotated translation areas of GenBank and RefSeq), are incorporated by reference in their entirety. In the event of any inconsistency between the disclosures of this application and the disclosures of any documents incorporated by reference herein, the disclosures of this application shall prevail. The above-mentioned detailed descriptions and examples are provided solely for the purpose of clarifying understanding and should not be interpreted as unnecessarily limiting. The present invention is not limited to the exact and detailed descriptions shown and described herein, and the claimed invention includes modifications that would be obvious to those skilled in the art.

[0065] Unless otherwise specified, all numerical values ​​representing components, molecular weights, and other quantities used in the specification and claims should be understood to be modified in all cases by the term "approximately." Therefore, unless otherwise stated, the numerical parameters described in the specification and claims are approximate values ​​and may vary depending on the properties intended to be obtained by the present invention. While not limiting the doctrine of equivalents regarding the claims, at a minimum, when interpreting each numerical parameter, the number of significant digits of the reported value should be considered, and conventional rounding techniques should be used.

[0066] While the numerical ranges and parameters defining the broad scope of the invention are approximate values, the numerical values ​​described in each example have been reported as accurately as possible. However, each numerical value inevitably has an inherent range depending on the standard deviation observed in the measured values ​​of the corresponding tests.

[0067] Headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows them, unless otherwise explicitly stated.

Claims

1. The following array: MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVPTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK A polynucleotide encoding a modified CD16 polypeptide having the following characteristics.

2. In vitro human cells comprising the polynucleotide described in claim 1.

3. The in vitro human cells according to claim 2, wherein the human cells containing the polynucleotides have improved antibody-dependent cytotoxicity (ADCC).

4. The polynucleotide according to claim 1, wherein the modified CD16 polypeptide has reduced sensitivity to cleavage compared to an unmodified CD16 polypeptide comprising the amino acid sequence defined in SEQ ID NO:

1.

5. The in vitro human cell according to claim 2, wherein the human cell is a hematopoietic cell.

6. The aforementioned human cells are as follows: (i) Natural killer (NK) cells; (ii) T cells; (iii) neutrophils; (iv) Monocyte; or (v) Pluripotent stem cells or differentiated cells derived therefrom The in vitro human cell according to claim 2.

7. (i) Whether the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), (ii) The in vitro human cells according to claim 6, wherein the differentiated cells generated from the pluripotent stem cells are hematopoietic cells.

8. A polypeptide encoded by a polynucleotide according to claim 1 or 4.

9. The polypeptide according to claim 8, which exhibits reduced sensitivity to cleavage mediated by ADAM17 compared to an unmodified CD16 polypeptide containing the amino acid sequence defined in SEQ ID NO:

1.

10. (i) The cell is a mammalian cell, and (ii) an in vitro cell or population comprising the polynucleotide described in claim 1 or 4.

11. The aforementioned mammalian cells, (i) Hematopoietic cells; (ii) Natural killer (NK) cells; (iii) T cells; (iv) neutrophils; (v) monocyte; or (vi) Stem cells or differentiated cells derived therefrom The in vitro cells or population thereof according to claim 10.

12. The in vitro cells or population according to claim 11, wherein the cells express the encoded polypeptide and have reduced CD16 shedding compared to cells expressing an unmodified CD16 polypeptide comprising the amino acid sequence defined in SEQ ID NO:

1.

13. The in vitro cells or population thereof according to claim 12, wherein the cells are NK cells.

14. The in vitro cells or population thereof according to claim 13, wherein the NK cells are NK cells differentiated from stem cells.

15. The aforementioned cells, (i) Induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), (ii) The in vitro cells or population according to claim 10, wherein the genetically modified stem cells comprise a polynucleotide encoding the modified CD16 polypeptide.

16. The aforementioned induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) (i) It is a stable cell line, or (ii) Can differentiate into genetically modified hematopoietic cells, The in vitro cells or population thereof according to claim 15.

17. The in vitro cells or population thereof according to claim 10, further comprising a bispecific killer cell engager (BiKE) or a trispecific killer cell engager (TriKE).

18. The in vitro cells or population thereof according to claim 17, wherein the BiKE includes CD16xCD33 BiKE, CD16xCD19 BiKE, or CD16xEp-CAM BiKE.

19. A composition comprising the in vitro cells or population thereof as described in claim 10.

20. The composition according to claim 19, further comprising one or more therapeutic antibodies.

21. The use of in vitro cells or populations thereof according to claim 11 in the manufacture of a pharmaceutical product for a method of improving treatment to a patient, including the administration of a therapeutic NK effector to the patient, wherein the method comprises administering the cells or populations thereof to the patient.

22. Use of in vitro cells or population thereof according to claim 10 in the manufacture of a pharmaceutical product for neoplastic conditions in subjects requiring treatment.

23. An in vitro method for modifying human cells, the following: The method comprising introducing the polynucleotide described in claim 1 into human cells to obtain modified human cells.

24. The in vitro method according to claim 23, wherein the modified human cells are hematopoietic cells differentiated from pluripotent stem cells.

25. (i) Whether the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), (ii) Hematopoietic cells are differentiated cells generated from pluripotent stem cells. The in vitro method according to claim 24.

26. The aforementioned human cells are pluripotent stem cells, and the aforementioned method is By differentiating pluripotent stem cells into differentiated cells, The modification further includes differentiating the modified human cells to obtain cells having improved antibody-dependent cytotoxic activity. The in vitro method according to claim 23, wherein the differentiated cells express a polypeptide encoded by a polynucleotide.

27. The in vitro method according to claim 26, wherein the differentiated cells are hematopoietic cells.

28. The differentiated cells, (i) Natural killer (NK) cells; (ii) T cells; (iii) Neutrophil; or (iv) monocyte The in vitro method according to claim 26.

29. (i) Whether the pluripotent stem cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), (ii) The in vitro method according to claim 26, wherein the differentiated cells generated from pluripotent stem cells are hematopoietic cells.

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