Genetically engineered antigen-specific natural killer cells for in-situ protein synthesis

Abstract

[Solution] Genetically engineered natural killer (NK) cells comprise an exogenous polynucleotide sequence including a receptor element, an actuator element, and an effector element. The receptor element encodes a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain and an intracellular signaling domain differentially linked to a transmembrane domain, where the extracellular antigen-binding domain recognizes a surface antigen on the surface of a target cell. The actuator element encodes a transcription factor binding site that upregulates synthesis of an effector protein. The effector element encodes an effector protein operably linked to a signal peptide, and the genetically engineered NK cells are configured to activate and synthesize and secrete the effector protein in response to binding of the antigen-binding domain of the CAR to an antigen on a target cell.

Description

[Technical Field]

[0001] This disclosure was made with government support from grants 1DP2EB024245 and R21CA236640 from the National Institutes of Health (NIH) and contract number D19AP00024 from the Defense Advanced Research Projects Agency (DARPA) on Bara Day. The government reserves certain rights with respect to this disclosure.

[0002] Created on October 28, 2021, and submitted concurrently with this specification, the following is incorporated as a reference: the entire computer-readable nucleotide sequence list, identified as "S1647132111_SequenceListing_ST25," which is an 83kb ASCII text file. [Background technology]

[0003] Many standard therapeutic drugs are designed to treat the disease at the time of diagnosis. While many pathogens and disease cells undergo significant changes within the body, current drugs are not designed to co-evolve with the disease microenvironment. Such drugs may include those administered in doses normalized to the patient's body weight. However, patients of the same body size may exhibit different disease states. If a drug is administered in excess, it can reach the circulatory system, potentially causing pathological conditions in normal tissues. Optimal drug delivery can lead to drug resistance. While monitoring patients and adjusting dosages according to their health status is possible, daily monitoring is costly. Furthermore, monitoring strategies and treatments do not exist for many diseases. Therefore, static therapeutics often fail to control evolving and / or persistently dynamic pathogens and diseases. The gap between dynamic disease states and static therapeutics poses a significant social and economic burden. Synthetic peptides and host defense peptides derived from other species have been shown to exert therapeutic effects against a variety of diseases. However, such synthetic peptides and host defense peptides are rapidly broken down by the immune system and become toxic to normal (host) cells, so they cannot be administered at present. [Overview of the project]

[0004] This disclosure aims to overcome the above and other challenges relating to therapeutic agents for treating diseases, including genetically modified natural killer (NK) cell lines (such as NK-92MI) that can be activated in-situ and induce the synthesis of human or non-human therapeutic proteins (effectors) for targeted diseases.

[0005] Various embodiments of this disclosure relate to genetically engineered natural killer (NK) cells comprising an exogenous polynucleotide sequence including: a receptor element encoding a chimeric antigen receptor (CAR) differentially linked to a transmembrane domain and comprising an extracellular antigen-binding domain and an intracellular signaling domain that recognize a surface antigen on the surface of a target cell; an actuator element encoding a transcription factor-binding site that upregulates the synthesis of an effector protein in response to the binding of the antigen-binding domain of the CAR to an antigen on the target cell; and an effector element encoding an effector protein that is operably linked to a signal peptide. The genetically engineered NK cells are configured to be activated and synthesize and secrete the effector protein in response to the binding of the antigen-binding domain of the CAR to an antigen on the target cell.

[0006] In some embodiments, genetically engineered NK cells are configured to synthesize and secrete effector proteins as a function of the target cells present. In some embodiments, the amount of effector protein is proportional to the amount of target cells present in the environment.

[0007] In some embodiments, the signal peptide is upstream of the effector protein and is in a non-native state relative to the effector protein.

[0008] In some embodiments, an intracellular signaling domain, an actuator element, The signal peptide is a constant domain, while the extracellular antigen-binding domain and effector protein are variable domains.

[0009] In some embodiments, the actuator element is bound to an effector protein, and the NK cells are NK-92MI cells.

[0010] In some embodiments, the exogenous polynucleotide sequence includes an actuator element that binds to an effector element that binds to a receptor element.

[0011] In some embodiments, the effector protein is selected from detectable reporter proteins, therapeutic proteins, downstream signaling proteins, and combinations thereof.

[0012] In some embodiments, the intracellular signaling domain includes one or more intracellular signaling portions of CD28, 4-1BB, and CD3 zeta.

[0013] In some embodiments, the transcription factor binding site is selected from the group consisting of activated T cell nuclear factor (NFAT) response elements, serum response elements (SREs), and cyclic AMP response elements (CREs).

[0014] Various embodiments involve a population of genetically engineered NK cells, each genetically engineered NK cell in the population comprising an exogenous polynucleotide sequence including an actuator element that binds to an effector element that binds to a receptor element, the receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain and an intracellular signaling domain operably linked to a transmembrane domain that recognizes a surface antigen on the surface of a target cell, the actuator element encoding a transcription factor-binding site that upregulates the synthesis of an effector protein in response to the antigen-binding domain of the CAR binding to an antigen on the target cell, and the effector element encoding an effector protein operably linked to a signal peptide. The population of genetically engineered NK cells is configured to be activated in response to the antigen-binding domain of the CAR binding to an antigen on the target cell, and to synthesize and secrete a calibrated amount of effector protein based on the presence of the target cell.

[0015] In some embodiments, the exogenous polynucleotide sequence includes an actuator element bound to an effector element and located upstream of the effector element, and an effector element bound to a receptor element and located upstream of the receptor element, wherein the signal peptide is located upstream of the effector protein.

[0016] In some embodiments, the effector protein is a therapeutic protein that acts directly on target cells, and the therapeutic protein is selected from the group consisting of cytotoxic proteins, immunostimulatory proteins, and immunosuppressive proteins.

[0017] In some embodiments, the calibration amount of the effector protein is a function of the amount of target cells present in multiple cells or in a sample.

[0018] Various embodiments relate to a method comprising contacting multiple cells with genetically engineered NK cells, the genetically engineered NK cells comprising a receptor element encoding a CAR having an extracellular antigen-binding domain and an intracellular signaling domain operably linked to a transmembrane domain, an actuator element encoding a transcription factor-binding site, and an effector element encoding an effector protein operably linked to a signal peptide. The method further comprises contacting multiple cells with genetically engineered NK cells to induce binding of the receptor element to an antigen on the surface of a target cell in response to the presence of a target cell within the multiple cells, initiating the expression of the effector element by the actuator element in response to the antigen-binding domain of the CAR binding to the antigen on the target cell to synthesize an effector protein and a secreted peptide, and secreting the effector protein by the secreted peptide.

[0019] In some embodiments, the method further comprises detecting the expression of an effector protein, the detectable expression of the effector protein indicating the presence of target cells.

[0020] In some embodiments, the method further includes activating NK cells in response to the antigen-binding domain of a CAR binding to an antigen on a target cell, and synthesizing and secreting a calibrated amount of effector protein based on the presence of the target cell.

[0021] In some embodiments, the amount of effector protein is proportional to the amount of target cells present in multiple cells.

[0022] In some embodiments, the effector protein comprises a therapeutic protein that acts directly on target cells, and the method further comprises neutralizing the target cells with the therapeutic protein.

[0023] Various embodiments can be better understood by considering the following detailed description related to the accompanying drawings. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows an example of genetically modified NK cells relating to this disclosure.

[0025] [Figure 2] Figure 2 shows an example of sequences of genetically modified NK cells and events triggered in a disease environment as described herein.

[0026] [Figure 3] Figure 3 shows an example of a population of genetically modified NK cells in a disease environment as described herein.

[0027] [Figure 4] Figure 4 shows an example of a method relating to this disclosure for contacting multiple cells with genetically modified NK cells.

[0028] [Figure 5] Figures 5A to 5D illustrate examples of the functional characterization of gene-scanned NK cells having specificity for folate receptor α (FRα) and mesoserine (MSLN) antigens as per the present disclosure.

[0029] [Figure 6] Figures 6A to 6H show examples of the cytotoxic function of genetically engineered NK cells with MSLN specificity and FRα specificity towards target and non-target cells, as described herein.

[0030] [Figure 7] Figures 7A to 7D show examples of artificial cell signaling pathways of genetically modified NK cells as described herein.

[0031] [Figure 8] Figures 8A and 8B show genetically modified NK cells that are redirected to different cancer antigens, as described herein.

[0032] [Figure 9]Figures 9A to 9F show examples of the cytotoxic function of genetically modified NK cells as described herein. [Modes for carrying out the invention]

[0033] In the following detailed description, reference is made to the accompanying drawings, which constitute part of this specification and are illustrated as examples illustrating specific embodiments in which this disclosure may be carried out. It is understood that other embodiments are available and that various modifications can be made without departing from the scope of this disclosure. Accordingly, the following detailed description should not be taken as limiting, and the scope of this disclosure is defined by the claims attached. Unless otherwise specified, it is understood that some or all of the various embodiments described herein can be combined with one another.

[0034] In some embodiments, cells can be engineered to express genetic elements including (multiple) transmembrane receptors that autonomously regulate intracellular transcription mechanisms. Furthermore, the genetic elements of cells may be modular and / or cells may contain multiple genetic elements to obtain engineered cells that have the ability to function as vectors for various in vitro, ex vivo, and in vivo applications. Such cells can be modularized in that parts can be stored and parts can be modified for different applications. Genetically engineered cells can be used in therapeutic agents and therapeutic methods applicable to various cell line diseases such as cancer and emerging pathogens, which self-regulate therapeutic responses in response to stimulation by disease cells and evade or malfunction the immune system. Multiple types of such genetically engineered cells, such as genetically engineered T cells, provide robust and reproducible cell lines for treating complex diseases in vivo. Also, such genetically engineered effector cells provide reliable in vivo imaging technologies and reliable in vitro sensor technologies in a variety of applications.

[0035] Embodiments relating to this disclosure include primary NK cells and NK cell lines genetically engineered with chimeric antigen receptors (CARs) that specifically detect (e.g., bind to) antigens expressed on the surface of target cells. By binding to antigens, genetically engineered NK cells may be more functional than native NK cells. For example, tumor cells may acquire resistance to NK cells by reducing the expression of NK cell-activating ligands on the tumor cell surface. Furthermore, genetically engineered NK cells can be designed to be relatively more effective than CAR T cell therapy. Somewhat surprisingly, given the differences between T cells and NK cells and the difficulty of manipulating primary NK cells, genetically engineered NK cells can utilize modular structures that are at least somewhat similar to those of modified T cells. Compared to T cells, genetically engineered NK cells can extract NK cell-activating domains and improve artificial cell signaling pathways. Even more surprisingly, in various experimental embodiments, including studies of syngeneic NK cells, genetically engineered NK cells do not exhibit graft-versus-host disease (GvHD), indicating that NK cells can be used as living vectors for producing different target proteins. Compared to T cells, NK cells have a relatively short half-life of about 1-2 weeks in vivo. However, this short half-life has the advantage of minimizing the targeting of healthy host cells. As mentioned above, genetic manipulation and proliferation of primary NK cells are particularly difficult compared to primary T cells. In many disease states, the host (e.g., patient or other subject) has fewer T cells compared to NK cells, and NK cells can reduce adverse events such as cytokine release syndrome, neurotoxicity, and GvHD.

[0036] Despite recent approvals of cell therapies by various government agencies, including the U.S. Food and Drug Administration (FDA), the full impact of T cell-based drugs has been limited. This is likely due to the homemade nature of adoptive cell therapies, which can contribute to manufacturing costs, product variability, and the potential for adverse events. Furthermore, the dynamic state of cellular pathology and inter-patient variability present challenges in optimal administration and necessitate continuous monitoring of individual patient conditions. As mentioned above, drug delivery is often performed using doses normalized to body weight or surface area. However, disease conditions can differ even among patients of the same body size. Embodiments relating to this disclosure concern genetically engineered NK cells used as cell chassis or vectors that function as biofactories for different target proteins. Engineered NK cells may be used to synthesize calibrated amounts of target proteins and to induce autocrine and paracrine signaling through artificial cellular signaling. NK cells are useful as therapeutic agents because they can exert rapid therapeutic effects, such as antitumor effects and doubling times of approximately 24 hours.

[0037] In various embodiments, genetically engineered NK cells are modular and antigen-specific. Antigen specificity can be used to overcome tumor resistance and direct cytolytic function to different antigen-presenting target cells, such as human or other organismal host cells. Furthermore, the artificial cell signaling pathways of such genetically engineered NK cells can be introduced to enable them to function as vectors by producing calibrated amounts of protein-based therapeutics and inducing intended autocrine and paracrine signaling when the genetically engineered NK cells engage with a target antigen. Genetically engineered NK cells can improve patient outcomes by enabling intensive synthesis of biologics at target sites and / or reducing systemic toxicity, thereby extending the duration of treatment.

[0038] Various embodiments demonstrate successful implementations of artificial cell signaling pathways in NK cell lines. An example of an NK cell line is NK-92MI, a rapidly growing cell-lysing cell line with a proven track record in clinical efficacy. In several experimental embodiments, the NK-92MI cell line is transformed into a vector for engagement with antigen-presenting target cells, inducing the synthesis of calibrated amounts of engineered proteins (often referred to herein as “effector proteins”) in situ. Genetically engineered NK cells can provide modular, homogeneous biovectors. For example, modularity can be used to combine different receptor elements with different effector elements, thereby allowing NK cells to be reprogrammed to target diseases with known biomarkers, such as cancer, viral infections, and / or autoimmune diseases.

[0039] As used herein, “genetically engineered NK cells” include and / or NK cells that have been genetically engineered or modified to comprise (i) receptor elements, (ii) actuator elements, and (iii) effector elements, each of which is modular. As used herein, the terms “module” and “modularity” include and / or refer to the versatility relating to recombinant sequence domains and the resulting recombinant polypeptides when assembled in various combinations for introduction into engineered NK cells. As used herein, “receptor elements” include and / or refer to polynucleotide sequences encoding transmembrane receptors such as CARs that can specifically interact with target cells. Depending on the specific application, receptor elements can be reprogrammed by replacing the single-chain variable fragment (scFV) portion of a CAR with an extracellular antigen-binding domain specific to a different disease-associated antigen. Other usable receptor elements include, but are not limited to, CARs specific to antigens associated with autoimmune diseases, CARs specific to antigens associated with neurological disorders (e.g., PTSD, Parkinson's disease, Alzheimer's disease), ligand-gated GPCRs (e.g., GPR1 glucose receptor), light-gated ion channels (e.g., melanopsin, rhodopsin, photopsin), pressure-sensing ion channels (e.g., TRPV1, TRPV2), and ligand-gated ion channels.

[0040] As used herein, “actuator element” includes and / or refers to a polynucleotide sequence encoding a transcription factor binding site that initiates transcription and translation events downstream of a trigger signal (e.g., binding of a sensing element to a target antigen). Generally, the underlying molecular mechanism of actuator elements is intracellular calcium [Ca 2+ Based on the dynamics of ]i, this is the mechanism that almost all types of cells use to regulate their function. Response elements include, but are not limited to, NFAT ("nuclear factor of activated T cells") response elements (NFAT-RE), serum response elements (SRE), and cyclic AMP response elements (CRE).

[0041] As used herein, “effector element” includes and / or includes polynucleotide sequences encoding effector proteins, and in some examples, effector proteins operably linked to signal peptides. For example, polynucleotide sequences encoding effector proteins include: sequences derived from human genes, sequences derived from non-human species genes, recombinant sequences, sequences encoding detectable reporter molecules, sequences encoding detectable imaging molecules, sequences encoding therapeutic molecules, etc.

[0042] The genetically modified NK cells into which receptor elements, actuator elements, and effector elements are introduced may be any NK cell type, including human NK cells or non-human NK cells (e.g., mammalian, reptile, plant, etc.). In this embodiment, the genetically modified cell “source” of the module elements provides, among other things, a cell chassis or frame that provides a transcription and translation machine for the expression and presentation of the receptor elements, actuator elements, and effector elements. In some embodiments, the NK cells may originate from a source (e.g., a first human), be modified, and administered to an organism different from the source (e.g., a host which is a second human). In other embodiments, the NK cells may originate from a source (e.g., a first human), be modified, and administered to return to the source (e.g., the source is the host).

[0043] Referring to the drawings, Figure 1 shows an example of genetically engineered NK cells according to the present disclosure. Genetically engineered NK cells 100 may be modular in that they can be prepared to synthesize different effector proteins for different target cells with different elements.

[0044] Genetically modified NK cells 100 possess an exogenous polynucleotide sequence that is functionally related and includes a receptor element 102, an actuator element 106, and an effector element 110. Various different types of NK cells can be used, such as NK cells from the NK-92MI cell line (often also referred to as NK-92MI cells).

[0045] Receptor element 102 encodes CAR104. CARs are often referred to as “chimeric receptors,” “T-bodies,” or “chimeric immune receptors (CARs).” As used herein, CARs include and / or refer to artificially constructed hybrid proteins or polypeptides comprising a transmembrane domain 105 and an extracellular antigen-binding domain 103 of an antibody (e.g., scFv) operably linked to at least one intracellular signaling domain 107. For example, CAR104 comprises an extracellular antigen-binding domain 103 operably linked to a transmembrane domain 105 and an intracellular signaling domain 107. CAR104 may be designed to identify a target surface antigen, such as a host target cell. In response to antigen binding, CAR104 transmits intracellular Ca +2 Internal Ca for release +2 This allows for the mobilization of stored antigens. For example, the extracellular antigen-binding domain 103 of CAR104 can recognize surface antigens on the surface of target cells, such as host disease cells.

[0046] As used herein, the extracellular antigen-binding domain 103 comprises and / or refers to a polynucleotide sequence complementary to the surface antigen of the target cell. As described above, the extracellular antigen-binding domain 103 can bind to the surface antigen of the target cell.

[0047] The transmembrane domain 105 includes and / or references a polynucleotide sequence encoding a transmembrane segment of a transmembrane protein, such as a membrane protein that spans the cell membrane, like the membrane of an NK cell 100. The transmembrane domain 105 may be derived from a native polypeptide or may be artificially designed. A transmembrane domain 105 derived from a native polypeptide can be obtained from any membrane-binding protein or transmembrane protein. For example, the transmembrane domains of T cell receptor α or β chains, CD3ζ chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR can be used.

[0048] The intracellular signaling domain 107 comprises and / or refers to a polynucleotide sequence encoding any oligopeptide or polypeptide known to function as a domain that transmits signals causing activation or inhibition of biological processes in cells. Examples of intracellular signaling domains include the intracellular signaling portion of CD28, the intercellular signaling portion of 4-1BB, and the intracellular signaling portion of CD3-zeta. In some embodiments, the intracellular signaling domain 107 comprises the intracellular signaling portion of CD28, the intercellular signaling portion of 4-1BB, and the intracellular signaling portion of CD3-zeta. However, embodiments are not so limited and may include other types and combinations of intracellular signaling domains. For example, the intracellular signaling domain 107 may include encoding any molecule that can transmit a signal to a cell when an extracellular antigen-binding domain 103, located within the same molecule, binds to (interacts with) an antigen.

[0049] Generally, the antigen-binding domain 103 of CAR104 is specific to a particular antigen expressed on the surface of a target cell. As described above, the extracellular antigen-binding domain 103 capable of binding to an antigen comprises any oligopeptide or polypeptide capable of binding to an antigen, such as the antigen-binding domain of an antibody and the ligand-binding domain of a receptor. The extracellular antigen-binding domain 103 binds to and interacts with an antigen, for example, an antigen present on the cell surface, thereby conferring specificity to genetically engineered NK cells 100 expressing CAR104. In some embodiments, the receptor element 102 encodes CAR104 having an extracellular antigen-binding domain 103 having specificity to folate receptor α (FRα), an antigen that has been found to be overexpressed in various cancers, including ovarian, cervical, lung, breast, kidney, and brain cancers. Other chimeric antigen receptors suitable for use as the antigen-binding portion of the receptor element 102 include those having specificity to a subset of immune cells, specificity to one or more tumor antigens, and / or specificity to one or more viral antigens.

[0050] Actuator element 106 encodes a transcription factor binding site 108. The transcription factor binding site 108 includes and / or refers to a protein binding site that upregulates the synthesis of effector protein 112 in response to the extracellular antigen-binding domain 103 of CAR 104 binding to an antigen on a target cell. The transcription factor binding site 108 is released in response to antigen binding as described above [Ca 2+ ] can trigger binding to transcription factors. In some embodiments, the transcription factor binding site 108 is selected from activated T cell nuclear factor (NFAT) response elements, serum response elements (SRE), and cyclic AMP response elements (CRE). Thereafter, the actuator element 106 can bind to [Ca 2+ [Ca may include a sequence for binding a factor triggered by ] 2+ ] i In response to the increase, the amplification of effector protein 112 synthesis may be triggered.

[0051] In some embodiments, the actuator element 106 encodes an NFAT transcription factor binding site for a transcription factor protein. The NFAT transcription factor family consists of five members: NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. For reviews, see Sharma S et al. (2011) PNAS, 108(28); Hogan PG et al. (2010) Ann Rev Immunol, 28; Rao A, Hogan PG (2009) Immunol Rev, 231(1); Rao A (2009) Nat Immunol, 10(1), M.R. Mueller and A. Rao, Nature Reviews Immunology, 2010, 10, 645 - 656; M. Oh-Hora and A. Rao, Curr. Opin. Immunol., 2008, 20, 250 - 258. Crabtree & Olson EN (Apr 2002), Cell 109 Suppl (2): S67 - 79, which are hereby incorporated in their entirety for their teachings. NFATc1 - NFATc4 are regulated by calcium signals. Since the known calcium sensor protein calmodulin activates calcineurin, a serine / threonine phosphatase, calcium signals are essential for NFAT activation. The molecular mechanism underlying this strategy is based on the dynamics of intracellular Ca +2 ([Ca 2+ i ) (further shown in Figure 2). The dynamics of [Ca 2+ i are common to almost all cell types, and thus this approach is widely applicable. The increase in [Ca 2+ i upon cell stimulation via CAR is (Ca +2 ​​​Through the calmodulin-dependent serine phosphatase calcineurin, this leads to the dephosphorylation of the nuclear factor of activated NK cell 100 protein, which then transfers to the nucleus and interacts with the NFAT response element (NFAT-RE), upregulating the expression of effector protein 112. In parallel, NFAT-RE also performs its intrinsic function of inducing IL-2 in activated genetically engineered NK cell 100, which controls clonal expansion proportional to the disease pathology. Furthermore, the expression of the NFAT-RE-inducing reporter protein can be used to quantitatively assess the activation level of genetically engineered NK cell 100.

[0052] The effector element 110 encodes an effector protein 112, which, in some embodiments, is operably linked to a signal peptide 114. As further shown herein, in some embodiments, the signal peptide 114 is upstream of the effector protein 112. The signal peptide 114 may be non-native to the effector protein 112. For example, the effector protein 112 may not be secreted into the extracellular environment without the addition of the signal peptide 114. However, embodiments are not so limited, and in some embodiments, the effector protein 112 contains the signal peptide in its native state. For example, the effector protein 112 may contain the signal peptide 114 (in its native state).

[0053] As used herein, the terms “secretor,” “secretory peptide,” and “signal peptide” are used interchangeably and include and / or refer to peptides that assist or guide the synthesized effector protein 112 to the extracellular environment (e.g., assist in the translocation of effector element 110). The signal peptide 114 may be operably ligated or fused to the effector protein 112 for release into the extracellular environment. In this way, the signal peptide 114 can direct the movement of the effector protein 112 out of the genetically engineered NK cell 100. The signal peptide 114 is particularly beneficial when contained in genetically engineered NK cells 100 expressing an effector protein 112 that cannot and / or can only minimally translocate in nature, allowing the effector protein 112 to remain within the genetically engineered NK cell 100 in the absence of the signal peptide 114 and / or to translocate at a subthreshold rate. Generally, signal peptides are located at the N-terminus of newly secreted proteins and are characterized by three domains: (1) a basic N-terminal domain, (2) a central hydrophobic core, and (3) a carboxyl-terminal cleavage region. Any suitable signal peptide can be used. For example, signal peptide 114 may be a signal peptide of interleukin-6 (IL-6) or interleukin-2 (IL-2).

[0054] In various embodiments, genetically engineered NK cells 100 are configured to be activated and synthesize and secrete effector protein 112 in response to the binding of the extracellular antigen-binding domain 103 of CAR104 to an antigen on a target cell (e.g., a target host cell). For example, genetically engineered NK cells 100 may synthesize and secrete effector protein 112 in an amount proportional to the number of target cells present in the environment (e.g., the extracellular environment), as a function of the amount of target cells present in the environment.

[0055] Effector protein 112 may comprise a variety of different types of proteins that can be used to provide therapy to a host, such as a patient. For example, effector protein 112 may comprise detectable reporter proteins, therapeutic proteins, downstream signaling proteins, and combinations thereof. As used herein, a detectable reporter protein comprises and / or refers to a protein that is detectable upon expression, such as a protein that provides an optical, electrical, or other type of detectable signal. A therapeutic protein comprises and / or refers to a protein that provides a therapeutic effect to a patient. A downstream signaling protein comprises and / or refers to a protein that drives downstream elements of signaling pathways, such as the regulation of cell growth, proliferation, differentiation, and apoptosis.

[0056] Non-limiting examples of effector proteins include bacterial cytotoxic polypeptides (e.g., parasporin, plantaricin A); insect-derived (e.g., Polyvia-MP1); viral antiviral polypeptides (e.g., α-helix peptide (AHP)); viral antiviral polypeptides (e.g., antiviral peptide (AVP)); fungal immunosuppressive peptides (e.g., cortelin A); vasodilators (e.g., relaxin, bradykinin) and endopeptidases (e.g., heparanase, relaxin, collagenase); and cell-permeable cationic peptides (e.g., LL-37, TAT peptide). Regarding cortelin A, autoreactive NK cells can be manipulated from NK cells obtained from hosts with autoimmune diseases (e.g., type 1 diabetes, polymyositis, and lupus) or from other sources. In particular, NK cells may be manipulated to locally express cortelin A upon stimulation with a target autoantigen. Systemic infusion of immunosuppressants is not suitable for hosts with these conditions due to the risk of other opportunistic infections. Regarding vasodilators and endopeptidases, such NK cells can be used to improve perfusion (see Chauhan VP & Jain RK (2013) Nat. Mater. 12(11):958-962) and to assist in the efficient delivery of anticancer drugs that cannot be administered systemically due to structural tissue damage and tumorigenesis. Regarding cell-permeable cationic peptides, these target peptides can be used to target intracellular bacteria. For example, site-specific overexpression of such peptides could be a potent treatment for tuberculosis.

[0057] As described above, in some embodiments, effector protein 112 is a therapeutic protein. In some embodiments, the therapeutic protein may act directly on target cells. In other embodiments, the therapeutic protein may act on cells adjacent to the target cell or on non-cellular components. Examples of therapeutic proteins include cytotoxic proteins, immunostimulatory proteins, and immunosuppressive proteins.

[0058] Different parts of gene elements 102, 106, and 110 of the genetically engineered NK cell 100 may be modules, while other parts may be conserved (e.g., not changing in different implementations). For example, in some embodiments, the intracellular signaling domain 107, actuator element 106, and signal peptide 114 are constant domains, while the extracellular antigen-binding domain 103 and effector protein 112 are variable domains. As an example, the extracellular antigen-binding domain 103 can be modified for different targets and / or the effector protein 112 can be modified to induce in-situ synthesis of different proteins, while the intracellular signaling domain 107, actuator element 106, and signal peptide 114 remain the same in different implementations. By conserving parts, manufacturing time can be reduced. However, the embodiments are not so limited, and any part of the genetically engineered NK cell 100 can be modified.

[0059] In some embodiments, the genetically engineered NK cell 100 may comprise multiple (e.g., two or more) of some or all of the gene elements 102, 106, and 110. For example, the genetically engineered NK cell 100 may comprise multiple receptor elements 102, multiple actuator elements 106, and / or multiple effector elements 110. In some embodiments, the redundancy takes the form of providing a host with multiple genetically engineered NK cells (e.g., multiple cells) modified as described herein to provide multiple therapeutic tasks for treating or preventing a disease and / or for other purposes.

[0060] In some embodiments, the actuator element 106 binds to the effector element 110. In some embodiments, the exogenous polynucleotide 101 includes the actuator element 106 which binds to the effector element 110 which binds to the receptor element 102. For example, the exogenous polynucleotide sequence 101 binds to the effector element 110, and to the actuator element 106 upstream of the effector element 110, and to the receptor element 102, Upstream of receptor element 102Effector element 11 0 It may contain, and signal peptide 114 is located upstream of effector protein 112.

[0061] Figure 2 shows an example of genetically engineered NK cells and a set of events triggered in a disease environment, as relating to this disclosure. Genetically engineered NK cells are used or may act as living vectors to synthesize effector protein 212 using an artificial cell signaling pathway and / or to trigger a set of events 220. As shown in 222, genetically engineered NK cells 200 synthesize engineered effector protein 212 in-situ upon interaction with antigen-presenting target cells.

[0062] As described above, the genetically engineered NK cell 200 comprises a receptor element 202 encoding an extracellular antigen-binding domain 203, a transmembrane domain 205, and an intracellular signaling domain 207; an actuator element 206 encoding a transcription factor-binding site (e.g., NFAT); and an effector element 210 encoding an effector protein 212 and optionally a signal peptide 214. The genetically engineered NK cell 200 may comprise a single plasmid (e.g., a single construct containing each of these) containing three cis-configured constant domains (e.g., parts of receptor element 202 such as actuator element 206, signal peptide 214, transmembrane domain 205, and intracellular signaling domain 207) and two variable domains (e.g., the antigen-binding domain 203, labeled "sensor," and the effector protein 212).

[0063] The constant domain may be configured to provide functionality to genetically engineered NK cells 200. The constant domain includes a transmembrane molecule (e.g., transmembrane domain 205) that recruits a calcium-dependent transcription machine (e.g., actuator element 206) to upregulate an effector transgene (e.g., effector protein 212) fused to a signal peptide 214 that forms part of an intracellular signaling pathway and assists in transporting the effector transgene to the extracellular space 223.

[0064] The variable domain is responsible for the applicability of genetically engineered NK cells 200 to a variety of different diseases, target cells, therapies, and / or other applications. For example, the variable domain can confer specificity to genetically engineered NK cells 200 for specific diseases. The variable domain is used to identify antigen biomarkers on target cells (e.g., labeled "disease cells") independently of the peptide-major histocompatibility complex and effector transgenes (e.g., effector protein 212) using variable gravimetric analysis (V H -V L The chain may include (for example, the antigen-binding domain 203, which is represented as a “sensor” of receptor element 202). The variable domains are modular. For example, the antigen-binding domain 203 can be replaced or modified to reprogram genetically engineered NK cells 200 to target biomarkers specific to different cell-based diseases. As another example, the effector protein 212 can be replaced or modified with different therapeutic transgenes, for example, to essentially create a ready-made living vector that neutralizes activated pathology and is further enhanced by the innate cytolytic activity of the NK cells.

[0065] In some embodiments, receptor element 202 encodes a CAR. Features of the CAR include the ability to re-induce T cell specificity and responsiveness to a non-MHC-restricted target by leveraging the antigen-binding properties of a monoclonal antibody. Somewhat surprisingly, the CAR also provides such capabilities to NK cells. MHC-unrestricted antigen recognition gives NK cells expressing the CAR the ability to recognize antigens independently of antigen treatment, thus evading the primary mechanism of tumor escape. Referring to Figure 2, transmembrane CAR expression allows genetically engineered NK cells 200 to sense and bind to target antigens expressed on the surface of target cells. Binding of the CAR to the target surface antigen on the target cell activates the genetically engineered NK cell 200, triggering an activation cascade that leads to the expression of effector proteins 212, such as engineered reporter, imaging, and / or therapeutic proteins. For example, the expression of effector protein 212 is autonomously expressed as part of the NK cell 200 activation cascade in response to the binding of the transmembrane receptor to the antigen presented on the target cell.

[0066] More specifically, genetically engineered NK cells 200 expressing CAR bind to specific antigens via CAR, and in response, a signal is transmitted to NK cells 200, resulting in their activation. The activation of CAR-expressing NK cells 200 varies depending on the type of target cell and the intracellular domain of CAR, and can be confirmed by indicators such as cytokine release, increased cell proliferation rate, and changes in cell surface molecules. For example, when cytotoxic cytokines (e.g., tumor necrosis factor, lymphotoxins, etc.) are released from activated NK cells 200, the target cells expressing the antigen are destroyed. Furthermore, cytokine release and changes in cell surface molecules stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0067] As shown in Figure 2, examples of sequences of events 220 triggered or associated with genetically engineered NK cells 200 include: (1) active migration of NK cells 200 to a disease environment; (2) engagement of CARs on the surface of NK cells 200 with target cell antigens; (3) activation of NK cells; (4) upregulation of effector protein 212 comprising signal peptide 214 via NFAT; (5) cleavage of signal peptide 214 and transport of effector protein 212 to extracellular space 223; and (6) regulation of cytokines that regulate disease-dependent cell expansion in response to antigen stimulation.

[0068] Figure 3 shows an example of a population of genetically engineered NK cells in a disease setting as relating to this disclosure. Population 331 may include several genetically engineered NK cells 300-1, 300-2, 300-3, 300-4, 300-5, 300-6, and 300-N (referred to herein generally as “genetically engineered NK cell 300” for ease of reference). Each of the genetically engineered NK cells 300 may have at least substantially the same characteristics and elements as the genetically engineered NK cell 100 in Figure 1, the details of which will not be repeated.

[0069] In the example shown in Figure 3, the environment is an extracellular space 330 containing (multiple) target cells 332, which can be called a disease environment. A population 331 of genetically engineered NK cells 300 may bind to the antigens of (multiple) target cells 332 via the antigen-binding domain of their CARs. In response to binding, the genetically engineered NK cells 300 may be activated and, in response, synthesize and secrete a calibrated amount of effector protein based on the presence of (multiple) target cells 332. For example, the calibrated amount of effector protein is a function of the amount of target cells 332 present in the extracellular space 330 or in multiple (host) cells such as in a sample. As described above, the calibrated amount of effector protein may be proportional to the amount of target cells 332. While the extracellular space 330 represents genetically engineered NK cells 300 and target cells 332, the extracellular space 330 and the multiple (host) cells may further include other non-cellular components, such as other normal cells and / or pathological cells.

[0070] Figure 4 shows an example of a method relating to this disclosure for contacting multiple cells with genetically modified NK cells. Method 440 can be carried out using a population 331 of genetically modified NK cells 100 shown in Figure 1 and / or genetically modified NK cells 300 shown in Figure 3.

[0071] In 442, method 440 includes contacting a plurality of cells with genetically modified NK cells. The cells can be contacted by contacting a sample or by administering the genetically modified NK cells to a host such as a patient. The genetically modified NK cells may include at least some of substantially the same characteristics and components as those described above, as shown by the genetically modified NK cell 100 in Figure 1, details of which will not be repeated.

[0072] In method 444, multiple cells are brought into contact with genetically modified NK cells, and in response to the presence of target cells within the multiple cells, method 440 includes binding receptor elements to antigens on the surface of target cells. The multiple cells, including target cells, may include host cells (e.g., host cells and target host cells).

[0073] In 446, in response to the antigen-binding domain (of the CAR) binding to the antigen of the target cell, method 440 includes initiating the expression (e.g., transcription and translation) of an effector element by an actuator element to synthesize an effector protein and a signal peptide, and causing the effector protein to be secreted by the signal peptide. In some embodiments, method 440 may further include activating NK cells in response to the antigen-binding domain of the CAR binding to the antigen of the target cell, and in response to this, synthesizing and secreting a calibrated amount of effector protein based on the presence of the target cell. As described above, the calibrated amount of the effector protein may be a function (e.g., proportional) of the amount of target cells present in multiple cells in the environment.

[0074] In some embodiments, method 440 further includes detecting the expression of an effector protein. Detectable expression of an effector protein may indicate the presence of target cells. In some embodiments, as described above, the effector protein includes a therapeutic protein. The therapeutic protein may act directly on the target cell, such as by killing it. For example, method 440 may include neutralizing the target cell with the therapeutic protein. In another embodiment, the therapeutic protein may additionally and / or alternatively act on cells or non-cellular components adjacent to the target cell, such as by providing a chemotactic gradient that other immune cells can follow for an inflammatory response and for infiltration into a cold tumor.

[0075] Various embodiments relate to pharmaceutical compositions comprising genetically modified NK cells, such as the genetically modified NK cells 100 shown in Figure 1 and / or a population 331 of genetically modified NK cells 300 shown in Figure 3, and a pharmaceutically acceptable carrier or excipient.

[0076] For example, an NK cell composition, such as a pharmaceutical composition, may comprise a plurality of genetically modified NK cells as described herein and an acceptable carrier, diluent, or excipient (e.g., a pharmaceutically acceptable carrier, diluent, excipient, or a combination thereof). Means for preparing such a composition are described in the Art (see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980)). Preferably, the composition is prepared to facilitate the administration of NK cells to a living organism. In some embodiments, the pharmaceutical composition comprises a plurality of genetically modified NK cells as described herein and, for example, an equilibrium salt solution, preferably Hanks' equilibrium salt solution, or ordinary physiological saline.

[0077] Some embodiments relate to methods for forming genetically modified NK cells, such as genetically engineering or modifying NK cells to include components and characteristics as described by the genetically modified NK cell 100 in Figure 1.

[0078] The genetically modified NK cells and cell compositions provided herein possess properties advantageous for use in a variety of in vitro, ex vivo, and in vivo applications. For example, in vitro applications of the NK cells and cell compositions provided herein include, but are not limited to, detecting target cells based on antigens expressed on the surface of target cells. Target cells may be cancer cells (e.g., tumor cells), cells infected with pathogens such as viruses or bacteria, cell types associated with autoimmune diseases (e.g., type 1 diabetes, lupus), or cell types associated with neurodegenerative diseases such as Alzheimer's disease, ALS, or Huntington's disease. Furthermore, target (host) cells may be cell types associated with other pathological conditions in which infected (host) cells exhibit abnormal expression of cell surface antigens compared to uninfected (host) cells. Methods for using genetically modified NK cells or cell compositions for in vitro target cell detection are described below.

[0079] Ex vivo applications of genetically engineered NK cells and cell compositions provided herein include, but are not limited to, early disease detection and companion diagnostic or therapeutic applications of disease-target cells identified based on antigens expressed on the surface of disease-target cells. For example, NK cells can be used in ex vivo applications in companion diagnostics of cancer immunotherapy. As an example, NK cells engineered with NFAT_RE6X→Nluc-2A-GFP may be engineered to express different types of CARs. The expression of Nluc when a CAR is involved with a target antigen, compared to nonspecific Nluc expression, provides information on the relative quantitative robustness of each CAR in terms of its efficiency in causing intended on-target effects versus unintended off-target effects. Methods for using genetically engineered NK cells or cell compositions in ex vivo therapeutic applications are further described below. Other ex vivo applications of genetically modified NK cells and cell compositions include, but are not limited to, applications as companion diagnostics for cell therapies to treat infectious diseases, autoimmune diseases, neurodegenerative diseases, and other cell-based pathologies associated with the abnormal expression of cell surface antigens against unaffected (host) cells.

[0080] The in vivo applications of the genetically modified NK cells and cell compositions provided herein include, but are not limited to, methods for in vivo imaging of disease sites, targeted therapy at disease sites (e.g., targeted therapy for ovarian cancer) or sites of pathogen infection (e.g., targeted therapy for cells infected with dengue virus, Zika virus, West Nile virus, yellow fever, HIV, or hepatitis viruses (e.g., HepB, HepC)).

[0081] Various embodiments involve a panel of different types of genetically modified NK cells, such as multiple NK cells manipulated with different effector proteins and / or extracellular antigen-binding domains (among other differences), which are used to simultaneously target different cells and / or secrete different effector proteins.

[0082] In some embodiments, a method for detecting target cells includes (a) contacting genetically engineered NK cells with a cell population, (b) detecting the expression of an effector protein, and (c) detecting the expression of a reporter protein, wherein the detectable expression of the effector protein indicates the presence of the target cells of interest. In some embodiments, the NK cells include an NFAT response element and a reporter protein, and in the presence of target cells in the contacted cell population, the genetically engineered NK cells bind to a surface molecular antigen on the target cells, activating the NFAT response element, and the detectable expression of the reporter protein indicates the presence of the target cells.

[0083] In some embodiments, the detected target cells are cancer cells, and the antigen-binding domain of the CAR binds to cancer cell-specific surface antigens on the target cells. In other embodiments, the detected target cells are virus-infected host cells, such as Zika virus-infected cells. In some such embodiments, the surface molecular antigen expressed on the virus-infected cells may be Zika virus-specific envelope glycoprotein (Egp). For example, the antigen-recognition portion of the CAR may be modified or replaced to quantitatively evaluate different viral pathogens, such as dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV). In some embodiments, the method utilizes the translation machine of the infected host cell to process the viral RNA into virus-specific antigens detectable by the genetically engineered NK cells described herein.

[0084] Some embodiments relate to methods for treating or preventing diseases using genetically engineered NK cells expressing CAR as therapeutic agents. For example, this specification relates to a method comprising administering genetically engineered NK cells expressing CAR as an active therapeutic agent. The diseases to which CAR-expressing NK cells are administered are not particularly limited, as long as the disease is susceptible to NK cells. Examples of diseases include cancer (e.g., hematological malignancies (leukemia), solid tumors), inflammatory / autoimmune diseases (e.g., asthma, eczema), hepatitis, and infections caused by viruses, bacteria, or fungi such as Zika virus, influenza, and HIV, for example, tuberculosis, methicillin-resistant Staphylococcus aureus infection (MRSA), vancomycin-resistant enterococcal infection (VRE), and deep-seated mycoses. In some embodiments, genetically engineered NK cells expressing CAR are administered to treat or prevent such diseases by binding to antigens, such as tumor antigens, viral antigens, or bacterial antigens, that are expressed on the surface of target cells that are targeted to be reduced or eliminated for the treatment of the above diseases. The terms “treat” and “prevent” as used herein, and any words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, the degree of treatment or prevention that a person skilled in the art would recognize as having potential benefits or therapeutic effects varies. In this regard, the methods described herein can provide treatment or prevention of cancer in mammals at any level and in any amount. Furthermore, the treatment or prevention provided by exemplary methods may include the treatment or prevention of one or more conditions or symptoms being treated or prevented, such as cancer. Also herein, “prevention” may include delaying the onset of a disease, its symptoms or condition.

[0085] In some embodiments, genetically engineered NK cells are administered to a host (e.g., a subject) requiring them, as part of a composition comprising suitable genetically engineered NK cells as described herein, and as a carrier, diluent, or excipient. Any suitable method for providing a host with modified CAR-expressing cells may be used in the method herein. In some embodiments, the method for providing NK cells to a host may be adapted from clinical protocols of cell therapy and adoptive immunotherapy for injecting donor-derived immune cells into a human host. In some embodiments, adapted clinical protocols suitable for the method provided herein include obtaining NK cells from a host, genetically engineering (e.g., modifying) the NK cells to express the CAR and NFAT-RE regulatory protein transgenes described herein, and injecting the genetically engineered NK cells back into the host. The host as used herein includes and / or refers to any organism such as humans, animals (e.g., mammals, reptiles, birds), insects, plants, etc., which may be subjects of study or testing and / or patients.

[0086] The administration of genetically modified NK cells provided herein may be administered by any suitable route, including, but is not limited to, intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, intraarterial, or afferent lymphatic vessel administration, or parenteral administration, such as by injection or infusion. In some embodiments in which genetically modified NK cells or populations of such NK cells are administered, the NK cells may be allogeneic or autologous cells of a host, such as a mammal. Preferably, the NK cells are autologous to the host.

[0087] In some embodiments, a host provided with genetically modified NK cells is monitored or evaluated for increased tumor clearance (e.g., improved, more robust). Therefore, various embodiments cover methods used in cancer treatment. In some embodiments, a host provided with genetically modified NK cells is monitored or evaluated for the clearance of cells expressing a specific antigen.

[0088] Some embodiments relate to methods for cell-based treatment or prevention of a target pathogen. For such methods, genetically engineered NK cells contain a polynucleotide sequence encoding a therapeutic protein instead of, or in addition to, a polynucleotide sequence encoding a detectable reporter protein, and are fused with a signal peptide (sec) at the 3' end of the polynucleotide sequence to assist extracellular transport. When a cascade effector NK cell activation event and activation of NFAT response elements are triggered, the expression of the therapeutic protein is induced. The method may include local production of the therapeutic protein at the site of target cells (e.g., tumor cells, infected cells) and extracellular secretion of the therapeutic protein in the disease microenvironment.

[0089] Several embodiments relate to methods for using genetically engineered NK cells as a sensing technology in various applications. For example, the spread of emerging flavivirus pathogens such as Zika virus (ZIKV) and dengue virus (DENV) through blood transfusion has been recognized as a serious risk. To protect donated blood, screening of donors, including blood tests, is recommended. Only 20% of those infected with ZIKV develop clinical symptoms, and there are no reliable commercially available ZIKV diagnostic kits that can be used outside of clinical laboratories. Therefore, identifying infection is difficult, especially considering the similarity to symptoms of other diseases and the cross-reactivity of antibodies with other arboviruses (e.g., dengue, chikungunya). Accordingly, this specification relates to a method comprising contacting genetically engineered NK cells containing an antigen-binding domain for detecting and binding to an antigen specific to the virus of interest with a sample containing or suspected of containing cells infected with the virus of interest, and an NFAT-RE regulatory reporter transgene that signals the presence of cells infected with the virus of interest.

[0090] Additional uses of genetically modified NK cells described herein include:

[0091] To target anti-cancer chemotherapy prodrugs to tumor sites, genetically engineered NK cells can be loaded with enzymatically activatable prodrugs, and since the drug-activating enzyme is synthesized only at the tumor site, the prodrug can be locally converted to its active form. In some embodiments, the prodrug may not be loaded into the NK cells but may be administered multiple times following the injection of genetically engineered NK cells. Alternatively, the prodrug may be bound to imaging nanoparticles or other means of image-guided means for active drug delivery. By binding the prodrug to imaging nanoparticles or manipulating NK cells to express an imaging transgene, the engineered NK cells can visually identify and / or image tumor margins to guide the appropriate staging of the patient in preparation for surgery and to assist in cell reductive surgery.

[0092] Some embodiments relate to methods for locally delivering chemotherapeutic agents to a disease site (e.g., a tumor mass, a site of autoimmune disease), comprising contacting genetically engineered NK cells with a host cell population, wherein the genetically engineered NK cells comprise (i) an exogenous polynucleotide sequence encoding a CAR including an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and (ii) an NFAT response element operably linked to a polynucleotide sequence encoding an enzyme, wherein in the presence of target host cells in the contacted cell population, the genetically engineered NK cells bind to a surface molecular antigen on the target host cells, activating the NFAT response element to initiate enzyme expression, the enzyme acting on a prodrug pre-designed to be activated by this enzyme, and being released to the disease site using its hydrophobic membrane permeability.

[0093] For surgical interventions to treat cancer, genetically engineered effector NK cells can be used for visualization and / or imaging of tumor margins through the expression of detectable reporter proteins such as fluorescent proteins (e.g., GFP, GFP variants) or bioluminescent enzymes (e.g., luciferase). For example, such genetically engineered NK cells can be used to mark tumor margins to assist in surgical resection and to identify residual positive tumor margins.

[0094] In some embodiments, genetically engineered NK cells are used to non-invasively detect and image tumors based on the expression of imaging enzymes (e.g., thymidine kinase, which can capture radioactive probes or other detectable probes; tyrosinase, which can be detected by photoacoustic imaging or magnetic resonance imaging) expressed when tumor-specific CAR NK cells engage with antigens on tumor cells.

[0095] In some embodiments, genetically engineered NK cells may be used to circumvent safety concerns associated with vaccines against flaviviruses. For example, antigenic diversity among the four different dengue virus serotypes is the cause of antibody-mediated immunity deficiency and the possibility of multiple consecutive infections. Antibodies are effective in primary infections but their neutralization is reduced in secondary infections, activating the complement system against large infected cell masses in the acute phase and exacerbating hemorrhagic fever. It has also been found that prior dengue infection worsens Zika infection. As described above, NK cells can be engineered to express human, non-human, or synthetic antiviral proteins upon detection of viral E glycoprotein (Egp) expressed on the surface of virus-infected cells; therefore, the use of NK cells can circumvent these safety concerns related to flaviviruses.

[0096] In some embodiments, genetically engineered NK cells include a CAR that detects cancer-specific antigens on target cancer cells (e.g., HPV E6 or E7 antigen in the case of cervical cancer) and an NFAT-RE that drives the expression of such reporter proteins. Such embodiments can be used for the early detection of cancer.

[0097] In some embodiments, genetically engineered NK cells include a CAR that detects antigens on pathogen-infected cells (e.g., ZIKV or DENV E glycoprotein on Zika virus or dengue virus-infected cells) and an NFAT response element that induces the expression of a reporter polypeptide. Such embodiments can be used in transfusion medicine to detect the presence of emerging pathogens (e.g., Zika virus, dengue virus, West Nile virus, yellow fever virus).

[0098] For cell-based therapies that deliver the intended therapeutic effect without unintended side effects, different CARs can be used in genetically engineered NK cells equipped with an NFAT-RE controlled reporter that detects and measures the signal-to-noise ratio and selects the appropriate CAR.

[0099] Mammalian cells may be engineered NK cells comprising a glucose-sensing GPCR (GPR1) that mobilizes internal Ca2+ storage and an NFAT response element controlled to express engineered insulin. Such engineered NK cells can be used to sense glucose and autonomously synthesize insulin. Such embodiments can be used in β-cell replacement therapy.

[0100] Other non-limiting uses of genetically modified NK cells include: i) imaging the location of the disease microenvironment to assist in surgical excision or to monitor disease progression / regression; ii) cytotoxicity to kill disease cells; iii) proliferation to enhance the persistence of T cells; iv) immunostimulation to supplement other immune cells; v) chemokines to supplement other immune cells; vi) immunosuppression to create a local immunosuppressive microenvironment; and vii) regeneration to enhance tissue healing.

[0101] As used herein, target cells (often interchangeably referred to herein as “host target cells,” “target cells of interest,” “disease cells,” or “target disease cells”) include and / or cells of interest related to a living organism (e.g., a biological component of interest). Antigens of target cells include and / or structures of target cells (e.g., binding sites) to which the antigen-binding domain of a receptor element can bind (e.g., have affinity). NK cells can be obtained from various different types of cells, such as human and non-human cells, and may be referred to herein as “sources.” As used herein, the terms “genetic modification” and “genetic engineering” are interchangeable and include and / or prokaryotic or eukaryotic cells containing exogenous polynucleotides, regardless of the method used for insertion. In some embodiments, NK cells are modified to contain non-spontaneously occurring nucleic acid molecules, which are created or modified by human hands (e.g., using recombinant deoxyribonucleic acid (DNA) technology) or derived from such molecules (e.g., by transcription, translation, etc.). NK cells containing polynucleotides that have been modified by exogenous, recombinant, synthetic, and / or other means are considered genetically modified NK cells.

[0102] As used herein, “nucleic acid” includes and / or refers to “polynucleotides,” “oligonucleotides,” and “nucleic acid molecules,” and generally means polymers of DNA or ribonucleic acid (RNA), which may be single-stranded or double-stranded and may include natural, unnatural, or modified nucleotides that are synthesized or obtained from natural sources (e.g., isolated and / or purified from natural sources), and may include natural, unnatural, or modified internucleotide bonds such as phosphoramidate bonds or phosphorothioate bonds instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as described herein, in some embodiments, it may be preferable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions. In some embodiments, the nucleic acid may encode additional amino acid sequences that do not affect the function of the CAR and polynucleotide and may or may not be translated when the nucleic acid is expressed by the host cell.

[0103] Nucleic acids may be obtained using any suitable method, including those described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, pp. 280-281 (1982) and / or U.S. Patent Application Publication No. US2002 / 0190663, each of which is fully incorporated herein for teaching purposes. Nucleic acids obtained from biological samples are typically fragmented to produce fragments suitable for analysis.

[0104] Nucleic acids and / or other sites may be isolated. As used herein, “isolated” includes and / or means separating all or part of the components usually associated with them, whether from naturally occurring sources or synthesized. The nucleic acids and / or other sites of the present invention can be purified. As used herein, “purified” includes and / or means separating from the majority of other compounds or entities. Compounds or sites can be partially or substantially purified. Purity may be expressed on a weight basis and can be determined using a variety of analytical techniques such as mass spectrometry and HPLC, but is not limited to these.

[0105] Numerous experimental embodiments were carried out to generate genetically modified NK cells and to characterize the functionality of NK cells. Examples of constructs used to generate genetically modified NK cells include the nucleotide sequences described in SEQ ID NO:1-7. SEQ ID NO:1-7 are each synthetic DNA.

[0106] Figures 5A to 5D illustrate examples of characterizing the function of genetically engineered NK cells with specificity for FRα and MSLN antigens according to various embodiments. FRα and MSLN are antigens that are overexpressed in several human cancers and expressed to a limited extent in normal tissues. In experimental embodiments, OVCAR3 human ovarian cancer cells endogenously overexpressing FRα and MSLN were used as target cells. A2780cis human ovarian cancer cells were used as a non-target negative control. A2780cis human ovarian cancer cells lack expression of FRα and MSLN. For the extracellular antigen-binding domain (often referred to as the "sensor domain"), VH-VL sequences of anti-FRα antibodies (MORAb-003) and anti-MSLN (MORAb-009) codon-optimized for human expression were used. The NanoLuc® (Nluc) (Promega Corporation) reporter enzyme was used to represent an effector domain (e.g., an effector protein) that can be substituted with human or non-human proteins or peptides to induce autocrine and paracrine effects.

[0107] Figure 5A shows the role of the constant domain in the antigen-directed functional response of genetically engineered NK cells. An optimally configured artificial cell-signaling pathway involves a copy of the actuator element's nuclear factor (e.g., NFAT), and intracellular costimulatory domains (e.g., transmembrane domain and intracellular signaling domain) and signal peptides. In experimental embodiments, the constant domain is combined with an FRα-specific binding domain, and NK cells bind to FRα, which is a non-target cell. neg MSLN neg Although it did not respond to A2780cis cells, it was shown to synthesize and release effector proteins when involved with target ligands on the surface of FRα+MSLN+OVCAR3 target cells. The signal-to-noise ratio (S / N), as defined in the legend, quantifies the specificity of genetically engineered NK cells. The function of the signal peptide released by the effector protein in the extracellular space was quantified by comparing Nluc activity between fully assembled genetically engineered NK cells and control NK cells lacking the signal peptide. The presence of the signal peptide (secretary, etc.) correlated strongly with the accumulation of Nluc in the supernatant (p<0.0001), and its absence was associated with Nluc construction in the cell pellet (p<0.0001). Similarly, the function of receptor elements (sensors or extracellular antigen-binding domains, which are part of receptor elements) that control artificial cell signaling pathways and induce target cell specificity was quantified by evaluating the increase in Nluc activity in two NK cell pellets: one with receptor elements and one without (p<0.0001).

[0108] Figure 5B shows the dynamics of effector protein synthesis from MSLN-specific NK cells. Nluc activity, which reports effector synthesis, increased rapidly within 2 hours (approximately S / N of 2 at 2 hours; approximately S / N of 3.3 at 6 hours, p<0.0001) when 12,500 MSLN-specific NK cells were stimulated with 2,500 OVCAR3 target cells. This activity stabilized within 24 hours and persisted for at least 96 hours.

[0109] Figure 5C shows the activity of effector proteins from MSLN-specific NK cells as a function of target cell mass (data collected with NK cells = 12,500). At low target / non-target cell counts, Nluc activity induced by target cells in stimulated genetically engineered NK cells was not detectable, but it increased exponentially with increasing target cell counts, and at cell counts exceeding 25,000, it differed statistically significantly from stimulation with non-target cells, e.g., E:T ≤ 5:1 (p < 0.05). This demonstrates that the dose of genetically engineered NK cells does not need to scale up in proportion to the disease burden and shows that genetically engineered NK cells can self-regulate effector protein synthesis in situation.

[0110] To demonstrate that NK cells can be re-induced to different target antigens, the sensor domain (e.g., antigen-binding domain) from an MSLN-specific scFV was replaced with an FRα-specific scFV sequence. As shown in Figure 5D, when stimulated with the same number of OVCAR3 target cells over a 25-hour period, Nluc activity in FRα-specific NK cells was significantly upregulated compared to MSLN-specific NK cells (12,500 effector cells and 2,500 target cells, e.g., E:T=5:1). This is likely due to higher FRα expression on OVCAR3 cells compared to MSLN expression, and is similar to or higher than the MSLN and FRα specificity observed in modified T cells. Alternatively, it may be due to higher integration of FRα-specific artificial cell signaling pathways in genetically engineered NK cells compared to integration of MSLN-specific artificial cell signaling pathways. Additional validations at 24, 48, and 72 hours using different E:T levels are further shown in Figures 8A and 8B.

[0111] Figures 6A to 6H illustrate examples of the cytolytic functions of MSLN-specific and FRα-specific genetically engineered NK cells against target and non-target cells as described herein. More specifically, FRα + MSKB + OVCAR3 target cells and FRαneg MSLN neg This study demonstrates the cell-lysing function of MSLN-specific and FRα-specific NK cells against A2780cis non-target cells. Target and non-target cells were engineered to express Luc2® (firefly luciferase) (Promega Corporation), a 60.6 kDA, ATP-dependent bioluminescence reporter related to in vitro and in vivo viability.

[0112] Figures 6A and 6B show the cytolytic activity of MSLN-specific NK cells (e.g., Figure 6A) and FRα-specific NK cells (e.g., Figure 6B) in co-culture with OVCAR-targeted cells for 6 hours, which is statistically significantly higher than that for non-targeted cells (Figure 6A shows p<0.05 for all E:T ≥0.94:1, and Figure 6B shows p<0.05 for all E:T ≥3.75:1). The parameter η(E:T) defines the target-specific cell lysis efficiency. 50 The E:T ratio was determined as the point at which the Luc2 activity of target or non-target cells, after co-culture with genetically modified NK cells for 6 hours, is 50% of the difference between the maximum and minimum normalized Luc2 activity of each cell. The η(E:T) of non-target A2780cis cells was also determined. 50 This was higher than that of target OVCAR3 cells (approximately 6.5 times higher in MSLN-specific NK cells and approximately 2.5 times higher in FRα-specific NK cells).

[0113] Figures 6C and 6D show the cytolytic activity of MSLN-specific NK cells (e.g., Figure 6C) and FRα-specific NK cells (e.g., Figure 6D) as a function of time at E:T = 0.94:1. Killing of OVCAR (target) cells was again statistically higher than killing of A2780cis (non-target) cells (Figures 6C and 6D, p<0.05 for all co-culture periods). 50The stimulation period was defined as the period during which the Luc2 activity of target or non-target cells, when co-cultured with NK cells at E:T = 0.94:1, reached 50% of the difference between the maximum and minimum normalized Luc2 activity of each cell (Luc2 activity at each time point was normalized, with 100% = no use of NK cells (negative control) and 0% = 0.5% Tween20 (positive control)). The mean difference became more pronounced at 24 hours and decreased again over longer periods, allowing for a cumulative increase in nonspecific cytolytic activity. This effect is further illustrated in Figures 9A to 9F.

[0114] Figures 6E and 6F, and 6G and 6H, show the cytolytic activity of MSLN-specific NK cells (e.g., Figures 6E and 6G) ​​and FRα-specific NK cells (e.g., Figures 6F and 6H) at different E:T values ​​when co-cultured with OVAR3 (target) cells and A2780cis (non-target) cells for 6 and 24 hours, respectively. Lower E:T values ​​allowed for longer co-culture times while still achieving sufficient cytolytic effect, and this effect was also observed in vivo. Therefore, the duration for which E:T and target-specific cytolytic effect are observed needs to be carefully balanced when designing in vitro assays. Additional validation in a wider range of E:T values ​​is shown below.

[0115] NK-92MI is a clinically relevant cell line, and the cells were irradiated with 10 Gray (Gy) before injection into humans. This halted further proliferation, making the cells non-cancerous. Up to 10 billion NK cells / m2 were safely injected into humans without serious side effects. However, DNA damage can disrupt the sequence of actuator and effector elements, potentially rendering genetically engineered NK cells non-functional.

[0116] Figures 7A to 7D show examples of artificial cell signaling pathways in genetically modified NK cells as described herein. Figures 7A to 7D demonstrate that the integrity of the artificial cell signaling pathways, and consequently the integrity of the genetically modified NK cells, is maintained after exposure to 15 Gy of radiation, thereby making them safe for clinical use.

[0117] Figure 7A shows the dynamics of effector protein synthesis from FRα-specific NK cells. Nluc activity, which reports effector protein synthesis, increased at 5 hours when stimulated by OVCAR3 (target) cells compared with A2780cis (non-target) cells (approximately 3 S / N ratio at 5 hours, p<0.02) (12,500 NK cells and 2,500 target cells, e.g., E:T=5:1) and increased exponentially up to at least 72 hours.

[0118] Figure 7B shows the effector protein activity from irradiated FRα-specific NK cells as a function of target cell mass. In 125,000 NK cells, often referred to as a biofactory, Nluc activity induced by target cells was statistically increased when stimulated by OVCAR3 (target) cells compared with A2780cis (non-target) cells (p=<0.50).

[0119] Figure 7C shows the effector protein activity from the same irradiated FRα-specific NK cells as a function of the increase in their number at a given target / non-target cell mass. At all cell numbers exceeding E:T = 0.625:1 (1562 NK cell biofactory for 2500 target / non-target cells), Nluc activity was statistically elevated when stimulated by 2500 OVCAR3 (target) cells compared to A2780cis (non-target) cells (p = < 0.01). Furthermore, a dose of 10 Gy was found to be acceptable in clinical trials in terms of its cytolytic effect.

[0120] Figure 7D shows the results of a cell lysis assay performed on FRα-specific NK cells irradiated with 15 Gy. When the irradiated NK cell biofactory was co-cultured with OVAR3 (target) cells for 6 hours, target-specific (OVCAR3) cell lysis was statistically significantly increased compared to that performed with A2780cis (non-target) cells (p<0.05 at E:T=100:1).

[0121] Synthesis and experimental information

[0122] (1) Materials and reagents Lentiviral particles were prepared in HEK293T / 17 (ATCC, Cat#CRL-11268) producing cells cultured in complete DMEM [DMEM growth medium captured with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Cat#F2442-500ML) and 1X penicillin-streptomycin solution (Corning, Cat #10-013-CV)]. Plasmid transfection was performed using Transporter5. TM The procedure was performed using reagents (Polyscience, Cat#26008-5). Plasmids encoding different gene payloads (transfer plasmids) were designed using SnapGene software (GSL Biotech LLC) and subcloned into lentiviral vector plasmids (System Biosciences, Cat#CD510B-1). Plasmids encoding second-generation packaging plasmids (psPAX2-Cat#12260, pMD2.G-Cat#12259) were obtained from Addgene. pAdvantage was obtained from Promega Corporation (Cat#E1711). Plasmid preparation services (chemical synthesis of DNA insertion sequences, subcloning into each vector backbone, and amplification) were obtained from Epoch Life Science, Inc. (Missouri, TX). The NK-92MI (ATCC, Cat#CRL-2408) cell line was maintained in complete NK-02MI medium [RPMI1640 (Corning, Cat#10-040-CV), 20% FBS (Sigma-Aldrich, Cat#F2442-500ML), 1X GlutaMAX solution (Gibco, Cat#35050-061), 1X penicillin-streptomycin solution (Corning, Cat#30-002-CI)]. OVCAR3 (ATCC, Cat#HTB-161) and A2780cis (Sigma-Aldrich, Cat#93112517) cell lines were maintained in complete RPMI [RPMI1640 (Corning, Cat#10-040-CV), 10% FBS (Sigma-Aldrich, Cat#F2442-500ML), and 1X penicillin-streptomycin solution (Corning, Cat-30-002-Cl)].+2 and Mg +2 Phosphate-buffered saline (PBS) (Corning, Cat#21-040-CV) without puromycin was used to minimize cell aggregation. In all cases, puromycin N-acetyltransferase was used as a selection marker. Puromycin dihydrochloride (Puromycin) (Thermo Fisher Scientific, Cat#A1113803) was used to select stable cell lines. The freezing medium consisted of 50% thermo-inactivated FBS (Sigma-Aldrich, Cat#F2442-500ML), 40% RPMI1640 (Corning, Cat#10-040-CV), and 10% DMSO (Sigma-Aldrich, Cat#D2650) to maintain the liquid nitrogen stock of the cell lines.

[0123] (2) Generation of lentivirus. Lentivirus particles were prepared by packaging the corresponding transfer plasmid using a second-generation lentivirus system, as described in Radhakrishnan H, Javitz HS, and Bhatnagar P, titled "Lentivirus Manufacturing Process for Primary T-Cell Biofactory Production," Advanced Biosystems, 1900288 (2020), the teaching of which is incorporated herein by reference as a whole.

[0124] (3) Construction of an NK cell biofactory. 92MI suspension cell lines were genetically engineered with lentiviral particles carrying appropriate (fully assembled (see Figure 2) or control) NK cell biofactories, as described in Radhakrishnan H, Javitz HS, and Bhatnagar P, Advanced Biosystems, 1900288 (2020). Briefly, cells were treated with lentivirus in the presence of 8 μg / mL polyblen (abm®, Cat#G062). After 48 hours, cells were selected using 0.5 μg / mL puromycin dihydrochloride. Unmodified parental cell lines were also selected as a positive control for puromycin-induced cell death. After selection, cells were expanded as required for different assays and frozen using freezing medium. In several experiments, differences were observed between the fully assembled biofactory and a control biofactory plasmid, as shown in Figure 2, as described in Repellin CE, Patel P, Beviglia L, Javitz H, Sambucetti L, and Bhatnagar P, titled "Modular Antigen-Specific T-cell Biofactories for Calibrated In Vivo Synthesis of Engineered Proteins," Advanced Biosystems, 2(12):1800210 (2018), the teachings of which are incorporated herein by reference.

[0125] (4) Creation of an irradiated NK cell biofactory. Using a 137Cs gamma ray irradiation device Mark I-68A (JL Shepherd and Associates), NK cell biofactories were irradiated with a dose rate of 222 mGy / min and a dose of 15 Gy. The control group underwent the same treatment except for irradiation.

[0126] (5) NK cells (E) [NK cell biofactory (MSLN-specific or FRα-specific)] and target cells (T) [Parental strain OVCAR3 or FRα + MSLN +Luc2-2A-E2Crimson + OVCAR3 (target) cells or parental strains A2780cis or FRα neg MSLN neg Luc2-2A-E2Crimson+A2780cis (non-target)] co-culture. Targeted OVCAR3 cells or non-targeted A2780cis cells were co-cultured in 100 μL of complete NK-92MI medium in a single well of a 96-well plate with MSLN-specific or FRα-specific NK cell biofactories at different NK cell-to-target cell ratios (E:T). After co-culture for a specific time, reporter activity was measured according to the manufacturer's protocol, for example, NanoLuc® (Nluc) activity of the NK cell biofactory using the Nano-Glo® assay (Promega Corporation, Cat#N1120) or Luc2 activity of A2780cis cells and OVCAR3 cells using the One-Glo® assay (Promega Corporation, Cat#E6110). Briefly, enzyme substrates (Nluc substrate or Luc2 substrate) were diluted with cell lysis buffers provided by the Nano-Glo® or One-Glo® assays and added to the co-culture in 96-well plates, respectively, to evaluate enzyme (Nluc or Luc2) activity. After a short incubation period (3 minutes for Nluc, 10 minutes for Luc2), use a microplate reader (PerkinElmer Corporation, EnVision). TM Bioluminescence was read using a Multilabel Plate Reader (Model: 2104-0010A).

[0127] (6) Experiments and co-culture of signal peptides. Three different engineered NK cell biofactories (125,000 cells each) in Figure 2 [(i) fully assembled (FRα-specific); (ii) without signal peptide or secretion domain (FRα-specific); and (iii) without signal peptide and receptor element domain] were washed with serum-free RPMI and resuspended in 200 μL of fresh, complete NK-92MI medium. The parental strain OVCAR3(FRα + MSLN +Target cell lines) and A2780cis (FRα neg MSLN neg Non-target cell lines were seeded at 250,000 cells / well, processed similarly, and resuspended in 200 μL of fresh, complete NK-92MI medium. Three NK cell biofactories were mixed with OVCAR3-targeted cells or A2780cis non-targeted cells, and six individual co-cultures were performed in 400 μL 24-well plates, with the final volume reduced to 1 mL of complete NK-92MI medium. After 6 hours, 500 μL aliquots from each co-culture were centrifuged in 1.5 mL tubes at 200 RCF for 5 minutes.

[0128] a. Evaluation of the Nluc effect pedal during the upper part of the program. 100 μL of supernatant was collected without disturbing the cell pellet and transferred to a 96-well plate (four wells were prepared for each co-culture). Nluc activity was evaluated in the 96-well plate using the Nano-Glo® assay according to the manufacturer's protocol.

[0129] b. Evaluation of NK cells (Nluc) in cell pellets. Exactly 1 mL of complete NK-92MI medium was added to the remaining 100 μL in each 1.5 mL tube (containing the cell pellet). The cell pellet was washed three times by removing and adding exactly 1 mL of complete NK-92MI medium each time. Exactly 400 μL of complete NK-92MI medium was added to the remaining 100 μL in each 1.5 mL tube, and the cell pellet was resuspended in a total of 500 μL of complete NK-92MI medium. Exactly 100 μL of resuspended cells were taken from each 1.5 mL tube and transferred to a 96-well plate (four wells were prepared for each co-culture). Nluc activity was evaluated in the 96-well plate using the Nano-Glo® assay according to the manufacturer's protocol.

[0130] (7) Experimental design and statistical analysis All statistical analyses were performed using GraphPad Prism 8.1.1 (GraphPad Software, Inc.). The statistical methods for all group comparisons and fitting equations are reported below. The following provides additional details regarding the figures mentioned above.

[0131] Figures 5A to 5D (Manipulated functions of an NK cell biofactory). Statistical analysis of Figures 5A and 5D is based on a two-sample t-test [unpaired student two-sided t-test] with a common variance and a two-sided p-value of 0.05. No adjustments were made for multiple comparisons. Analysis of Figures 5B and 5C was performed with a false detection rate of less than 1% based on ANOVA and the subsequent two-step linear step-up procedure of Benjamini, Krieger, and Yekutieli. S / N was set to OVCAR3(FRα) of the parent strain. + MSLN + The average Nluc activity of the NK cell biofactory when stimulated with ) cells compared to the parent strain A2780cis(FRα neg MSLN neg The ratio was calculated by dividing by the average Nluc activity when the cells were stimulated. The error bars extend 1 standard deviation above and below the mean value and can be considered as the half-width of the 68% confidence interval relative to that mean value.

[0132] Figure 5A (Functional components of the NK cell biofactory). The vertical bar represents the target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The mean Nluc activity of three different engineered FRα-specific NK cell biofactories when stimulated by ) cells is shown. The first NK cell biofactory was fully assembled (Figure 2), the second NK cell biofactory was a control lacking the IFNα2 secretion domain (no signal peptide, Figure 2), and the third NK cell biofactory was a control lacking the IFNα2 signal peptide and the FRα-specific receptor element domain (no signal peptide and receptor element, Figure 2).

[0133] Figure 5B (NK cell biofactory activated within 2 hours). Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The Nluc activity of an MSLN-specific NK cell biofactory (without signal peptides, Figure 2) stimulated by ) cells is expressed by the formula Y = a + b * log 10 (X) is used for fitting, where X is the stimulation time in units of time.

[0134] Figure 5C (NK cell biofactory activation as a function of target cell mass). Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The Nluc activity of MSLN-specific NK cell biofactories (without signal peptides, Figure 2) stimulated by ) cells was measured using a 4-parameter logistic model Nluc=Nluc min +{Nluc max -Nluc min} / {1+10^[b*(log 10 [Target 50 Fitted using ]-X)}, where X is the log of the target cell count. 10 And Nluc max This is an estimated parameter that defines the upper limit asymptotic of Nluc activity, and Nluc min b is an estimated parameter that defines the lower limit asymptotic of Nluc activity, and b is a "Hill" parameter that defines the slope at the inflection point of the approximation curve, Target 50 (Nluc max -Nluc min This is an estimated parameter representing the X value corresponding to ) / 2.

[0135] Figure 5D (NK cell biofactories can be re-induced towards different cancer antigens). Tukey box plots target OVCAR3(FRα + MSLN+ ) or non-target A2780cis (FRα neg MSLN neg The Nluc activity of two NK cell biofactories stimulated by ) cells (FRα-specific receptor element using the VH-VL sequence from MORAb-003 or MSLN-specific receptor element using the VH-VL sequence from MORAb-009) is shown. Both NK cell biofactories were generated according to Figure 2, having either an FRα-specific or MSLN-specific receptor element but no signal peptide.

[0136] Figures 6A-6H (Innate cell lysis function of NK cell biofactories). Luc2 activity was normalized at each time point, where 100% = maximum Luc2 activity and 0% = 0.5% Tween 20 (complete cell killing). Statistical analyses of (A) and (B) were performed with a false detection rate of less than 1% based on ANOVA and the subsequent Benjamini, Krieger, and Yekutieli two-step linear step-up procedure. Analysis of (C) and (D) was based on a two-sample t-test [unpaired student two-sided t-test] with common variance and a two-sided p-value of 0.05. No adjustments were made for multiple comparisons. Error bars extend 1 SD above and below the mean and can also be considered as the half-width of the 68% confidence interval relative to the mean. All experiments were performed using FRα. + MSLN + Luc2-2A-E2Crimson + OVCAR3 (target) cells or FRα neg MSLN neg Luc2-2A-E2Crimson + The study included NK cell biofactories stimulated by A2780cis (non-targeted) cells, containing both FRα-specific receptor elements using VH-VL sequences from MORAb-003 and MSLN-specific receptor elements using VH-VL sequences from MORAb-009. Targeted or non-targeted Luc2 activity was evaluated as a surrogate biomarker in living cells.

[0137] Figures 6A and 6B (Cytolytic activity of two NK cell biofactories at 6 hours against the number of NK cell biofactories). NK cell biofactory (Figure 2 without a signal peptide) was Luc2 + target OVCAR3 (FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg ) When co-cultured with cells, the Nluc activity was fitted using the four-parameter logistic model Nluc = Nluc min +{Nluc max -Nluc min} / {1 + 10^[b*(log 10 [ηE:T 50 -X)]}, i) X is the log of the number of NK cells 10 and is often also referred to as effector cells or NK cell biofactories, Nluc max is an estimated parameter that defines the upper asymptote of the Nluc activity, Nluc min is an estimated parameter that defines the lower asymptote of the Nluc activity, and b is the "Hill" parameter that defines the slope at the inflection point of the approximate curve, ii) The parameter η(E:T) 50 for defining the target-specific cytolytic efficiency was determined as the E:T at which the Luc2 activity of the target cells or non-target cells when co-cultured with the NK cell biofactory is 50% of the difference between the maximum and minimum values of their respective normalized Luc2 activities. For example, η(E:T) 50 is the estimated E:T value corresponding to (Luc2 max -Luc2 min / 2).

[0138] Figures 6C and 6D (Cytolytic activity of two NK cell biofactories at an E:T of 0.94:1 against the duration of NK cell biofactory stimulation). NK cell biofactory (Figure 2 without a signal peptide) was Luc2 + target OVCAR3 (FRα + MSLN +) or non-target A2780cis (FRα neg MSLN neg )Normalized Nluc activity when co-cultured with cells is expressed using a 4-parameter logistic model Nluc = Nluc min +{Nluc max -Nluc min} / {1+10^[b*(log 10 [Time 50 Use ]-X)} to fit, i) X is the log of the duration of NK cell biofactory stimulation. 10 And Nluc max This is an estimated parameter that defines the upper limit asymptotic of Nluc activity, and Nluc min b is an estimated parameter that defines the lower limit asymptotic of Nluc activity, and b is a "Hill" parameter that defines the slope at the inflection point of the approximation curve. ii) Time 50 This is determined as the duration of stimulation at which the Luc2 activity of target or non-target cells, when co-cultured with an NK cell biofactory, becomes 50% of the difference between the maximum and minimum values ​​of their respective normalized Luc2 activity. For example, Time 50 (Luc2 max -Luc2 min This is the estimated duration corresponding to / 2).

[0139] Figures 6E and 6F (Cytolytic activity of two NK cell biofactories at different E:T intervals over a 6-hour duration of NK cell biofactory stimulation). Tukey's box plot shows that two NK cell biofactories (FRα-specific or MSLN-specific) are Luc2 + Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The normalized Luc2 activity after co-culture with NK cells for 6 hours is shown. Both NK cell biofactories were generated according to Figure 2, possessing FRα-specific or MSLN-specific receptor elements and lacking signal peptides. E:T ratios of 3.75:1 and 7.5:1 were used.

[0140] Figure 6G and 6H (Cytolytic activity of two NK cell biofactories at different E:T intervals over 24 hours of NK cell biofactory stimulation). Tukey's box plot shows that two NK cell biofactories (FRα-specific or MSLN-specific) are Luc2 + Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The normalized Luc2 activity after co-culture with NK cells for 24 hours is shown. Both NK cell biofactories were generated according to Figure 2, possessing FRα-specific or MSLN-specific receptor elements and lacking signal peptides. E:T ratios of 3.75:1 and 7.5:1 were used.

[0141] Figures 7A to 7D (Engineering and innate functions of irradiated NK cell biofactories). Statistical analysis was performed with a false detection rate of less than 1% based on multiple t-tests and the subsequent Benjamini, Krieger, and Yekutieli two-step linear step-up procedure. No adjustments were made for multiple comparisons. Error bars extend 1 standard deviation above and below the mean and can also be considered as the half-width of the 68% confidence interval relative to the mean. All experiments were performed using FRα. + MSLN + Luc2-2A-E2Crimson + OVCAR3 (target) cells or FRα neg MSLN neg Luc2-2A-E2Crimson + The biofactory contained irradiated (15 Gy) NK cell biofactories stimulated with A2780cis (non-targeted) cells. The signal-to-noise ratios of (A), (B), and (C) were compared to the parent strain's OVCAR3(FRα + MSLN + The average Nluc activity of the NK cell biofactory when stimulated with ) cells compared to the parent strain A2780cis(FRα neg MSLN negThis was calculated as a ratio obtained by dividing by the average Nluc activity when stimulated in cells.

[0142] Figure 7A (Activation of irradiated NK cell biofactories within 5 hours). Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The Nluc activity of irradiated NK cell biofactories (without signal peptides, Figure 2), stimulated by irradiated cells, is fitted using the equation Y = a + b*X, where X is the stimulation time in units of time. (The manipulated function of non-irradiated NK cell biofactories is modeled based on a logarithmic curve. However, it is understood that this function decreases for irradiated NK cell biofactories and can be explained using a linear regression model representing the initial part of the logarithmic curve).

[0143] Figure 7B (Activation of irradiated NK cell biofactories as a function of target cell mass). Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The Nluc activity (without signal peptide, Figure 2) of irradiated NK cell biofactories stimulated by ) cells was defined using a 4-parameter logistic model Nluc = Nluc min +{Nluc max -Nluc min} / {1+10^[b*(log 10 [Target 50 ]-X)} is used to fit, Nluc max This is an estimated parameter that defines the upper limit asymptotic of Nluc activity, and Nluc min b is an estimated parameter that defines the lower limit asymptotic of Nluc activity, b is a "Hill" parameter that defines the slope at the inflection point of the approximation curve, and Target 50 (Nluc max -Nluc min This is an estimated parameter that shows the X value corresponding to / 2).

[0144] Figure 7C (Functions derived from target cells of an irradiated NK cell biofactory, proportional to the number of cells) Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg The Nluc activity (without signal peptide, Figure 2) of irradiated NK cell biofactories stimulated by ) cells was defined using a 4-parameter logistic model Nluc = Nluc min +{Nluc max -Nluc min} / {1+10^[b*(log 10 [Effector 50 It was fitted using ]-X)}.

[0145] Figure 7D (Innate cytolytic activity of irradiated NK cell biofactories 6 hours after irradiation, relative to the number of NK cell biofactories). Luc2 + Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg )Normalized Luc2 activity (without signal peptide, Figure 2), a surrogate marker of cell lysis in target cells derived from an irradiated NK cell biofactory when co-cultured with NK cells, was defined using a 4-parameter logistic model: Luc2 = Luc2 min +{Luc2 max -Luc2 min} / {1+10^[b*(log 10 [η(E:T) 50 Use ]-X)} to fit, i) X is the log of the duration of NK cell biofactory stimulation. 10 And Nluc max This is an estimated parameter that defines the upper limit asymptotic of Nluc activity, and Nluc min b is an estimated parameter that defines the lower limit asymptotic of Nluc activity, and b is a "Hill" parameter that defines the slope at the inflection point of the approximation curve. ii) Parameter η(E:T) for defining target-specific cell lysis efficiency 50 This is determined as E:T, where the Luc2 activity of target or non-target cells when co-cultured with an NK cell biofactory is 50% of the difference between the maximum and minimum values ​​of their respective normalized Luc2 activities, for example, η(E:T) 50 (Luc2 max -Luc2 min E: This is the estimated value of T corresponding to ( / 2).

[0146] As will be further explained below, for the statistical analysis of the figures, Figures 8A and 8B were based on a two-sample t-test [unpaired student two-sided t-test] with a common variance and a two-sided p-value of 0.05, without adjustment for multiple comparisons. The statistical analysis of Figures 9A to 9F was performed with a false detection rate of less than 1% based on ANOVA and the subsequent two-step linear step-up procedure of Benjamini, Krieger, and Yekutieli. The error bars extend 1 standard deviation above and below the mean and can also be considered as the half-width of the 68% confidence interval relative to the mean.

[0147] Figures 8A-8B show examples of genetically engineered NK cells that are re-induced towards different cancer antigens as per this disclosure. Tukey box plots show the target (FRα + MSLN + OVCAR3) or non-target (FRα neg MSLN neg Figure 8B shows the Nluc activity of (i) MSLN-specific (Figure 8A) and (ii) FRα-specific (Figure 8B) NK cell biofactories stimulated by A2780cis) cells. Both NK cell biofactories were generated according to Figure 2, which have FRα- or MSLN-specific receptor elements but no signal peptide. All data were collected with target / non-target cells = 2500. Using different E:T ratios, e.g., 20:1, 10:1, and 5:1, Nluc activity was observed at 24, 48, and 72 hours, as shown.

[0148] Figures 9A-9F illustrate the cytolytic function of exemplary genetically modified NK cells as described in this disclosure. All experiments were conducted using FRα + MSLN + Luc2-2A-E2Crimson + OVCAR3 (target) cells or FRα neg MSLN neg Luc2-2A-E2Crimson + NK cell biofactories stimulated by either A2780cis (non-target) cells contained both (i) MSLN-specific receptor elements (Figures 9A, 9C, 9E) and (ii) FRα-specific receptor elements (Figures 9B, 9D, 9F). Both NK cell biofactories were generated according to Figure 2, possessing either FRα- or MSLN-specific receptor elements but lacking signal peptides. Luc2 activity in target or non-target cells was evaluated as a surrogate biomarker for live cells. In all experiments, 0.5% Tween-20 was used as a positive control for cell elimination. Cell lysis activity as a function of E:T was fitted using a four-parameter logistic model at A) 24 hours (Figures 9A and 9B), B) 48 hours (Figures 9C and 9D), and C) 72 hours (Figures 9E and 9F). All data were collected with target / non-target cells = 2500. Luc2 activity was measured for all observations using n=3. Error bars extend 1SD above and below the mean, and can be considered as the half-width of the 68% confidence interval relative to the mean. Target OVCAR3(FRα + MSLN + ) or non-target A2780cis (FRα neg MSLN neg Normalized Luc2 activity in an NK cell biofactory (without signal peptides, Figure 2) stimulated by ) cells is expressed using a 4-parameter logistic model: Luc2 = Luc2 min +{Luc2 max -Luc2 min} / {1+10^[b*(log 10 [η(E:T) 50 It was fitted using ]-X)}.

[0149] Various embodiments are carried out in accordance with the underlying Provisional Application, cell number 63 / 106,838, filed on October 28, 2020, entitled “Modular Antigen-Specific NK-Cell Biofactory for In Situ Synthesis of Engineered Proteins,” which claims its interests and whose general and specific teachings are fully incorporated herein by reference. For example, embodiments herein and / or in the Provisional Application can be combined to varying degrees (including all of them). Experimental teachings and underlying references provided in the Provisional Application can also be referenced. The embodiments described in the Provisional Application are not intended in any way to limit the entire technical disclosure or any part of the requested disclosure unless otherwise specified.

[0150] While specific embodiments are illustrated and described herein, various alternative and / or equivalent embodiments can be substituted for the specific embodiments illustrated and described without departing from the scope of this disclosure. This application is intended to encompass any adaptations or modifications of the specific embodiments described herein.

Claims

1. Genetically modified natural killer (NK) cells containing an exogenous polynucleotide sequence, wherein the exogenous polynucleotide sequence is in a operable linkage, A receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain operably linked to a transmembrane domain that recognizes surface antigens on the surface of target cells, and an intracellular signaling domain, An actuator element encoding a transcription factor binding site that upregulates the synthesis of an effector protein in response to the antigen-binding domain of the CAR binding to the antigen on the target cell, An effector element encoding the effector protein that is operably linked to the signal peptide, Includes, The intracellular signaling domain includes the intracellular signaling portion of CD28, the intracellular signaling portion of 4-1BB, and the intracellular signaling portion of CD3 zeta. The CAR is configured to be activated in response to the antigen-binding domain of the target cell binding to the antigen, to synthesize the effector protein and the signal peptide, and to secrete the effector protein via the signal peptide. Genetically modified natural killer (NK) cells.

2. The genetically modified NK cells according to claim 1, wherein the genetically modified NK cells are configured to synthesize and secrete the effector protein as a function of the existing target cells.

3. The genetically engineered NK cell according to claim 2, wherein the amount of the effector protein is proportional to the amount of the target cells present in situ.

4. The genetically engineered NK cell according to claim 1, wherein the signal peptide is upstream of the effector protein and is in a non-natural state relative to the effector protein.

5. The genetically engineered NK cell according to claim 1, wherein the signal peptide is in its native state relative to the effector protein.

6. The intracellular signaling domain, the actuator element, and the signal peptide are constant domains. The extracellular antigen-binding domain and the effector protein are variable domains. Genetically modified NK cells according to claim 1.

7. The actuator element is bound to the effector protein, The aforementioned NK cells are NK-92MI cells. Genetically modified NK cells according to claim 1.

8. The genetically engineered NK cell according to claim 1, wherein the exogenous polynucleotide sequence includes an actuator element bound to an effector element, and the effector element bound to a receptor element.

9. The genetically engineered NK cell according to claim 1, wherein the effector protein is selected from a detectable reporter protein, a therapeutic protein, a downstream signaling protein, and a combination thereof.

10. The aforementioned transmembrane domain includes a transmembrane domain of CD8, The genetically engineered NK cell according to claim 1, wherein the transcription factor binding site comprises a plurality of activated T cell nuclear factor (NFAT) response elements.

11. The genetically engineered NK cell according to claim 1, wherein the transcription factor binding site is selected from the group consisting of activated T cell nuclear factor (NFAT) response element, serum response element (SRE), and cyclic AMP response element (CRE).

12. A population of genetically modified NK cells, Each of the aforementioned genetically engineered NK cells in the aforementioned population comprises an exogenous polynucleotide sequence in which actuator elements are bound to effector elements, and the effector elements are bound to receptor elements, The receptor element operably ligated to a transmembrane domain encodes a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that recognizes a surface antigen on the surface of a target cell and an intracellular signaling domain. The actuator element encodes a transcription factor binding site that upregulates the synthesis of effector proteins in response to the antigen-binding domain of the CAR binding to the antigen on the target cell. The effector element encodes the effector protein which is operably linked to the signal peptide. The intracellular signaling domain includes the intracellular signaling portion of CD28, the intracellular signaling portion of 4-1BB, and the intracellular signaling portion of CD3 zeta. The genetically modified population of NK cells is configured to be activated in response to the binding of the antigen-binding domain of the CAR to the antigen on the target cell, and to synthesize and secrete a calibrated amount of the effector protein based on the presence of the target cell. A group of genetically modified NK cells.

13. The exogenous polynucleotide sequence includes an actuator element bound to the effector element and located upstream of the effector element, and an effector element bound to the receptor element and located upstream of the receptor element, The signal peptide is located upstream of the effector protein. A population of genetically modified NK cells according to claim 12.

14. The effector protein is a therapeutic protein that acts directly on the target cells. The therapeutic protein is selected from the group consisting of cytotoxic proteins, immunostimulatory proteins, and immunosuppressive proteins. A population of genetically modified NK cells according to claim 12.

15. The genetically engineered NK cell population according to claim 12, wherein the calibration amount of the effector protein is a function of the amount of the target cells present in a plurality of cells or in a sample.

16. The method involves bringing multiple cells into contact with genetically modified NK cells in vitro, wherein the genetically modified NK cells are A receptor element encoding a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain and an intracellular signaling domain operably linked to a transmembrane domain, wherein the extracellular antigen-binding domain recognizes surface antigens on the surface of multiple cells, and the intracellular signaling domain includes the intracellular signaling portion of CD28, the intracellular signaling portion of 4-1BB, and the intracellular signaling portion of CD3 zeta, An actuator element that codes for a transcription factor binding site, It comprises a polynucleotide sequence having an effector element that encodes an effector protein operably linked to a signal peptide, The plurality of cells are brought into contact with the genetically modified NK cells, and in response to the presence of the target cells within the plurality of cells, the binding of the CAR to the antigen on the surface of the target cells is induced. In response to the antigen-binding domain of the CAR binding to the antigen of the target cell, the actuator element initiates the expression of the effector element to synthesize the effector protein and secreted peptide, and the secreted peptide secretes the effector protein. An in vitro method including [the specified method].

17. Further includes detecting the expression of the effector protein, The detectable expression of the effector protein indicates the presence of the target cells. The in vitro method according to claim 16.

18. The in vitro method of claim 16, further comprising activating the NK cells in response to the antigen-binding domain of the CAR binding to an antigen on a target cell, and synthesizing and secreting a calibrated amount of the effector protein based on the presence of the target cell.

19. The in vitro method according to claim 18, wherein the amount of the effector protein is proportional to the amount of the target cells present in the plurality of cells.

20. The effector protein includes a therapeutic protein that acts directly on the target cells. The further comprising killing the target cells with the therapeutic protein, The in vitro method according to claim 16.

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