Genetically modified NK-92 cells and monoclonal antibodies for cancer therapy

Abstract

To provide a therapeutic agent for a subject having cancer or suspected of having cancer.SOLUTION: A therapeutic combination for treating cancer in a subject in need thereof, comprising a monoclonal antibody having a cytotoxic effect and a genetically modified NK-92 cell, wherein: Wherein the genetically modified NK-92 cells express a CD16 polypeptide having a valine at position 158 of the mature form of the CD16 polypeptide; and express interleukin 2 (IL-2) targeted to the endoplasmic reticulum (ER). A combination therapy comprising a transgene encoding a CD16 polypeptide and ER-targeted IL-2, wherein the combination therapy is genetically modified using a plasmid expression vector to generate stable NK-92 cells; and further wherein the expression vector comprises in the 5' to 3' direction: a polynucleotide encoding a CD16 polypeptide, an IRES, and a polynucleotide encoding ER-targeted IL-2.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 139,258, filed March 27, 2015, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Background of the Invention Anti-cancer treatment using monoclonal antibodies (mAbs), especially when combined with chemotherapy, has significantly improved the clinical outcomes of cancer patients. However, in many cases, the patient ultimately relapses. Natural killer cells can also be used as cytotoxic effector cells for cell-based immunotherapy.

[0003] NK-92 is a cytolytic cancer cell line that was discovered in the blood of a patient with non-Hodgkin's lymphoma and subsequently immortalized ex vivo. NK-92 cells are derived from NK cells, but while retaining most of the activating receptors, they lack the major inhibitory receptors present in normal NK cells. However, NK-92 cells do not attack normal cells or induce unacceptable immune rejection responses in humans. Characterization of the NK-92 cell line is disclosed in WO 1998 / 49268 (Patent Document 1) and U.S. Patent Application Publication No. 2002-0068044 (Patent Document 2). NK-92 cells are also being evaluated as a potential therapeutic agent for the treatment of certain cancers.

[0004] NK-92 cells retain nearly all of the activating receptors and cytolytic pathways associated with NK cells, but do not express CD16 on their cell surface. CD16 is an Fc receptor that recognizes and binds to the Fc portion of antibodies to activate NK cells for antibody-dependent cellular cytotoxicity (ADCC). Due to the absence of the CD16 receptor, NK-92 cells are unable to lyse target cells via the ADCC mechanism.

[0005] The present invention provides a solution to the above-mentioned problems by enhancing the cytotoxic effect of some molecular antibodies by simultaneous or subsequent administration of NK-92 cells expressing Fc receptors to a subject in need of anti-cancer treatment. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 1998 / 49268 [Patent Document 2] U.S. Patent Application Publication No. 2002-0068044 Summary of the Invention

[0007] Summary of Aspects of the Invention In one aspect, the present invention involves co-administering to a subject in need of anti-cancer treatment a monoclonal antibody that has a cytotoxic effect on target cancer cells and NK-92 cells genetically engineered to express Fc receptors, resulting in a synergistic effect between the anti-cancer effects of the NK cells and the therapeutic antibody.

[0008] Thus, in one embodiment, the present invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a monoclonal antibody having a cytotoxic effect on target cancer cells and NK-92 cells expressing an FcR. In some embodiments, the FcR is CD16. In one aspect of the invention, the NK-92 cells are genetically modified to express an Fc receptor encoding a polypeptide having at least 90% sequence identity to SEQ ID NO:1 (FCγRIII-A or CD16(F-158) having a phenylalanine at position 158), or a polypeptide having at least 90% identity to SEQ ID NO:2 (CD16(F158V) having a valine at position 158, a higher affinity form). In an exemplary embodiment, the CD16 polypeptide has a valine at position 158.

[0009] In further embodiments, the NK-92 cells are further modified to express a cytokine, such as IL-2. In some embodiments, the cytokine is targeted to the endoplasmic reticulum. In particular embodiments, the cytokine is interleukin-2 or a variant thereof that targets the endoplasmic reticulum. In some embodiments, the NK-92 cells are modified to express a polypeptide having the sequence of SEQ ID NO:7.

[0010] In other embodiments, the NK-92 cells are further modified to express a suicide gene. In one aspect, the suicide gene is iCas9.

[0011] The compositions of the present invention are useful in the treatment of cancer, including, but not limited to, cancers such as multiple myeloma, leukemia, lymphoma, metastatic breast cancer or gastric cancer.

[0012] The monoclonal antibody administered to the patient can be a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody. In some embodiments, the monoclonal antibody is alemtuzumab, rituximab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, adotranstuzumab, blinatunomab, daratumumab, or elotuzumab.

[0013] In some embodiments, the monoclonal antibody and FcR-expressing NK-92 cells are administered to the subject simultaneously. In other embodiments, the subject is administered the monoclonal antibody followed by the FcR-expressing NK-92 cells, e.g., within 24 hours or within 24 to 72 hours after administration of the monoclonal antibody.

[0014] In some aspects, the present invention relates to the use of NK-92 cells genetically modified to express an FcR, such as CD16, and a cytotoxic monoclonal antibody for the treatment of cancer. Accordingly, in some embodiments, the present invention provides the use of NK-92 cells genetically modified to express CD16 and a cytotoxic monoclonal antibody for patients with cancer. In some embodiments, the Fc receptor is CD16 with a valine at position 158 of the mature form of CD16. In some embodiments, the Fc receptor comprises a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity to, or the polynucleotide encodes, SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the FcR-expressing NK-92 cells are genetically modified to express a cytokine, such as interleukin-2 or a variant thereof. In some embodiments, the interleukin-2 is targeted to the endoplasmic reticulum. In some embodiments, the FcR-expressing NK-92 cells are engineered to express the interleukin-2 sequence set forth in SEQ ID NO:7. In some embodiments, the Fc receptor and at least one cytokine are encoded by different vectors. Alternatively, the Fc receptor and at least one cytokine are encoded by the same vector. In some embodiments, the Fc receptor comprises a CD16 polypeptide having a V at position 158, and the NK-92 cells are further engineered to express human interleukin-2, thereby targeting the interleukin-2 to the endoplasmic reticulum. The FcR-expressing NK-92 cells may also be further engineered to express a suicide gene, such as iCas9. In some embodiments, the cancer is leukemia, non-Hodgkin's lymphoma, metastatic breast cancer, or gastric cancer. The monoclonal antibody can be a naked monoclonal antibody, a conjugated monoclonal antibody, or a bispecific monoclonal antibody.In some embodiments, the monoclonal antibody is alemtuzumab, rituximab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adotrastuzumab, blinatumomab, avelumamab, daratumumab, or elotuzumab. In some embodiments, the monoclonal antibody and FcR-expressing NK-92 cells are administered to the subject simultaneously. In some embodiments, the subject is administered the monoclonal antibody followed by treatment with the genetically modified FcR-expressing NK-92 cells. In some embodiments, the monoclonal antibody is injected intravenously into the subject. In other embodiments, the genetically modified FcR-expressing NK-92 cells are infused into the bone marrow.

[0015] [The present invention 1001] A method for treating cancer in a subject in need of cancer treatment, comprising administering to the subject a monoclonal antibody having a cytotoxic effect and NK-92 cells genetically modified to express FcR. [The present invention 1002] 1001. The method of claim 1001, wherein the Fc receptor is a CD16 polypeptide having a valine at position 158 of the mature form of CD16. [The present invention 1003] 1001. The method of claim 1001, wherein the Fc receptor comprises a polynucleotide sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2, and comprises a valine at position 158. [The present invention 1004] 1001. The method of claim 1001, wherein the Fc receptor comprises the amino acid sequence of SEQ ID NO:2. [The present invention 1005] The method of any of claims 1001 to 1004, wherein the NK-92 cells expressing FcR are genetically modified to express a cytokine. [The present invention 1006] The method of claim 1005, wherein the cytokine is interleukin-2. [The present invention 1007] The method of any one of claims 10 to 8, wherein interleukin-2 is targeted to the endoplasmic reticulum. [The present invention 1008] 1005. The method of claim 10, wherein the Fc receptor and the at least one cytokine are encoded by different vectors. [The present invention 1009] 1005. The method of claim 1005, wherein the Fc receptor and the at least one cytokine are encoded by the same vector. [The present invention 1010] 1001. The method of claim 1001, wherein the Fc receptor comprises a CD16 polypeptide comprising the amino acid sequence of SEQ ID NO:2, and the NK-92 cells are further genetically modified to express human interleukin-2, wherein the interleukin-2 targets to the endoplasmic reticulum. [The present invention 1011] The method of claim 1010, wherein the NK-92 cells expressing the FcR are further modified to express a suicide gene. [The present invention 1012] The method of claim 10, wherein the suicide gene is iCas9. [The present invention 1013] The method of any of claims 1001 to 1009, wherein the NK-02 cells expressing the FcR are further modified to express a suicide gene. [The present invention 1014] The method of the present invention 1013, wherein the suicide gene is iCas9. [The present invention 1015] The method of any one of claims 1001 to 1013, wherein the cancer is multiple myeloma, leukemia, non-Hodgkin's lymphoma, metastatic breast cancer, or gastric cancer. [The present invention 1016] 1016. The method of any of claims 1001 to 1015, wherein the monoclonal antibody is a naked monoclonal antibody, a conjugated monoclonal antibody or a bispecific monoclonal antibody. [The present invention 1017] 1016. The method of claim 1016, wherein the monoclonal antibody is alemtuzumab, rituximab, trastuzumab, ibritumomab, brentuximab, gemtuzumab, adotranstuzumab, blinatunomab, avelumamab, daratumumab, or elotuzumab. [The present invention 1018] The method of any of claims 1001 to 1017, wherein the monoclonal antibody and the FcR-expressing NK-92 cells are administered to said subject simultaneously. [The present invention 1019] The method of any of claims 1001 to 1017, wherein said subject is administered a monoclonal antibody and subsequently treated with FcR-expressing NK-92 cells. [The present invention 1020] 1019. The method of claim 1018 or 1019, wherein the monoclonal antibody is injected intravenously into said subject. [The present invention 1021] The method of any one of claims 1018, 1019, or 1020, wherein the genetically modified NK-92 cells are infused into the bone marrow. The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Other objects, advantages and novel features will become readily apparent to those skilled in the art from the following detailed description of the invention. [Brief explanation of the drawings]

[0016] The objects, features and advantages of the present invention will be more readily understood by reference to the following disclosure when considered in conjunction with the accompanying drawings.

[0017] [Figure 1] 1 shows a schematic diagram of a plasmid expressing a modified form of IL-2 with ERRS (endoplasmic reticulum retention signal) and CD16. [Figure 2]Exemplary data are provided showing CD16 expression in NK-92 cells modified to express CD16 using the plasmid vector shown in FIG. 1 at about 2 weeks ( FIG. 2 a) and about 4 weeks ( FIG. 2 b). [Figure 3] Exemplary data are provided showing CD16 expression in modified NK-92 cells that were frozen for storage and then thawed for culture. [Figure 4] 1 provides exemplary data showing the ADCC activity of CD16-expressing NK-92 cells used in combination with a monoclonal antibody. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description of the Invention In one aspect, the present disclosure relates to the use of NK-92 cells and monoclonal antibodies modified to express FcR for the treatment of cancer in subjects.Malignant cells can develop mechanisms of action to evade the immunological defense provided by innate immune cells such as dendritic cells and natural killer cells, and adaptive immune cells such as T cells and B cells.Therefore, there is an urgent need to reduce the incidence of tumor recurrence in subjects with or suspected of having cancer.

[0019] NK-92 cells exhibit the attractive feature of being easily proliferated and expanded in vitro. However, because they do not express the IgG Fc receptor FcγRIII, these cells cannot act via antibody-dependent cell-mediated cytotoxicity (ADCC). The present invention is based on the hypothesis that transformation of NK-92 cells to express the IgG Fc receptor FcγRIII will enhance NK cell-tumor cell interactions and allow NK cells to work in concert with monoclonal antibodies that kill target cells via ADCC. Thus, the separate cytotoxic effects of NK-92 cells and monoclonal antibodies can be enhanced when the monoclonal antibodies and NK-92 cells are administered simultaneously or in close temporal relationship to a subject with cancer or in need of cancer treatment.

[0020] Thus, the present invention provides for the use of NK-92 cells genetically modified to express a high affinity form of the transmembrane immunoglobulin gamma Fc region receptor III-A (FcγRII-A or CD16, in which a valine is present at position 158 of the mature form of the polypeptide).

[0021] In some embodiments, FcR-expressing NK-92 cells can be further modified to express IL-2. In such cells, the expression of IL-2 within the cells is typically directed to the endoplasmic reticulum. This characteristic prevents the undesirable effects of systemic administration of IL-2, such as toxicity affecting the cardiovascular, gastrointestinal, respiratory, and nervous systems. In some embodiments, when FcR-expressing NK-92 cells are further modified to express IL-2, a suicide gene can be further inserted into these cells to prevent unregulated endogenous expression of IL-2, which may potentially lead to the development of mutants with autonomous growth. In some embodiments, the suicide gene is iCas9.

[0022] FcR-expressing NK-92 cells produced according to the present invention are administered to a subject having or suspected of having cancer in combination with a monoclonal antibody that targets cancerous cells for the effective treatment of cancerous disease.

[0023] The administration of the FcR-expressing NK-92 cells can be simultaneous with or sequential to the administration of the monoclonal antibody, hi some embodiments, the FcR-expressing NK-92 cells are administered to the subject within 24 hours after the subject has been treated with the monoclonal antibody.

[0024] Terminology Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] In this specification and the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0027] All numerical designations, including ranges, e.g., pH, temperature, time, concentration, amount, molecular weight, etc., are approximations that are varied (+) or (-) by increments of 0.1 or 1.0, as appropriate. It should be understood, although not always explicitly stated, that all numerical designations can be preceded by the term "about." It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such reagents are known in the art.

[0028] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0029] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, refers to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and other than the substantial method steps recited. Embodiments defined by each of these transitional phrases are within the scope of the present invention.

[0030] "Immunotherapy," as used to describe the present invention, refers to the use of modified or unmodified NK-92 cells, either alone or in combination with antibodies, natural or modified NK cells, or T cells, that are capable of inducing cytotoxicity upon contact with target cells.

[0031] As used to describe the present invention, "natural killer (NK) cells" are cells of the immune system that kill target cells in the absence of specific antigenic stimulation and without MHC class restriction. Target cells can be tumor cells or virus-carrying cells. NK cells are characterized by the presence of the CD56 surface marker and the absence of the CD3 surface marker.

[0032] The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), as distinguished from the NK-92 cell line. Endogenous NK cells are generally a heterogeneous cell population enriched for NK cells. Endogenous NK cells can be for autologous or allogeneic treatment of a patient.

[0033] "NK-92 cells" refers to the immortal NK cell line NK-92, originally obtained from a patient with non-Hodgkin's lymphoma. In the present invention, unless otherwise indicated, the term "NK-92" is intended to refer to the original NK-92 cell line as well as NK-92 cell lines that have been modified (e.g., by the introduction of an exogenous gene). NK-92 cells and exemplary, non-limiting modifications thereof are described in U.S. Patent Nos. 7,618,817; 8,034,332; and 8,313,943, all of which are incorporated herein by reference in their entireties.

[0034] "Modified NK-92 cells" refers to NK-92 cells that further comprise a vector encoding a transgene, such as CD 16. In some embodiments, FcR-expressing modified NK-92 cells can be further modified to express a cytokine, such as IL-2, and / or a suicide gene.

[0035] As used herein, "non-irradiated NK-92 cells" refer to NK-92 cells that have not been irradiated. Irradiation renders the cells incapable of growth and proliferation. In some embodiments, it is contemplated that NK-92 cells for administration are irradiated at a treatment facility or elsewhere prior to treatment of a patient, as the time between irradiation and infusion should not exceed 4 hours to maintain optimal activity. Alternatively, NK-92 cells can be inactivated by another mechanism.

[0036] As used to describe the present invention, "inactivation" of NK-92 cells renders them incapable of proliferation. Inactivation can also refer to the death of NK-92 cells. It is envisioned that NK-92 cells can be inactivated after they have effectively purged an ex vivo sample of cells associated with pathology in therapeutic applications, or after they have resided in a mammalian body for a sufficient time to effectively kill many or all target cells present in the body. As a non-limiting example, inactivation can be induced by administering an inactivating agent to which NK-92 cells are sensitive.

[0037] The terms "cytotoxicity" and "cytolysis" used to describe the present invention are intended to be synonymous when used to describe the activity of effector cells such as NK cells. Generally, cytotoxic activity involves killing target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more specifically refers to the activity of the effector cell to dissolve the plasma membrane of the target cell, thereby destroying its physical integrity, which ultimately results in the death of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK cells is due to cytolysis.

[0038] The term "killing" in relation to a cell / cell population is intended to include any type of manipulation that leads to the death of that cell / cell population.

[0039] The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the defensive function of immune cells by binding to a portion of an antibody known as the Fc region. Binding of the Fc region of an antibody to a cell's Fc receptor (FcR) stimulates the cell's phagocytic or cytotoxic activity through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc-gamma receptors (FCγR) bind to IgG class antibodies. FCγRIII-A (also known as CD16) is a low-affinity Fc receptor that binds to IgG antibodies and activates ADCC. FCγRIII-A is typically found on NK cells. A representative polynucleotide sequence encoding a native form of CD16 is shown in SEQ ID NO:5.

[0040] The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted with non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to make up the double-stranded form.

[0041] A polynucleotide consists of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) is substituted for thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule.

[0042] As used herein, "percent identity" refers to the sequence identity between two peptides or two nucleic acid molecules. Percent identity can be determined by comparing positions in each sequence that can be aligned for comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. As used herein, the phrase "homologous" or "variant" nucleotide sequence or "homologous" or "variant" amino acid sequence refers to sequences characterized by at least a certain percentage of identity at the nucleotide or amino acid level. Homologous nucleotide sequences include sequences encoding naturally occurring allelic variants and mutants of the nucleotide sequences described herein. Homologous nucleotide sequences include nucleotide sequences encoding proteins of mammalian species other than humans. Homologous amino acid sequences include amino acid sequences containing conservative amino acid substitutions and whose polypeptides have the same binding and / or activity. In some embodiments, a homologous nucleotide or amino acid sequence has at least 60% or more identity with the comparison sequence, e.g., at least 70%, at least 80%, at least 85% or more. In some embodiments, a homologous nucleotide or amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a comparison sequence. In some embodiments, a homologous amino acid sequence has no more than 15, no more than 10, no more than 5, or no more than 3 conservative amino acid substitutions. Percent identity can be determined, for example, by the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.) using the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489) with default settings.

[0043] The term "express" refers to the production of a gene product. The term "transient" when referring to expression means that the polynucleotide is not integrated into the genome of the cell.

[0044] The term "cytokine" or "cytokines" refers to a general class of biological molecules that act on cells of the immune system. Exemplary cytokines for use in practicing the present invention include, but are not limited to, interferons and interleukins (IL), particularly IL-2, IL-12, IL-15, IL-18, and IL-21. In a preferred embodiment, the cytokine is IL-2.

[0045] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing an intact replicon such that it can replicate when placed into a permissive cell, for example, by the process of transformation. A vector can replicate in one cell type, such as bacteria, but has limited replication capacity in another cell type, such as mammalian cells. A vector can be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, either alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles containing DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, optionally contained within liposomes; and the use of ternary complexes containing viruses and polylysine DNA.

[0046] As used herein, the term "targeted" is intended to include, but is not limited to, directing a protein or polypeptide to an appropriate intracellular or extracellular destination. Targeting is typically achieved via a signal or targeting peptide, which is a stretch of amino acid residues in a polypeptide chain. Such signal peptides can be located anywhere within the polypeptide sequence but are often located at the N-terminus. Polypeptides can also be engineered to have a signal peptide at their C-terminus. Signal peptides can direct polypeptides to extracellular regions, to the plasma membrane, or to the Golgi, endosomes, endoplasmic reticulum (ER), or other intracellular compartments. For example, polypeptides with a specific amino acid sequence at their C-terminus (e.g., KDEL) are retained in or transported back into the ER lumen.

[0047] The term "suicide gene" allows for the negative selection of cells. Suicide genes are used as a safety system, allowing cells expressing the gene to be killed by the introduction of a selective agent. This is desirable when a recombinant gene mutates, resulting in uncontrolled cell proliferation. Many suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E. coli Deo gene (see also, for example, Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9). In one embodiment, the suicide gene is inducible caspase 9 (iCas9) (Di Stasi, (2011) "Inducible apoptosis as a safety switch for adoptive cell therapy." N Engl J Med 365: 1673-1683; see also Morgan, "Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic" Molecular Therapy (2012); 20: 11-13). The TK gene can be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.

[0048] The term "monoclonal antibody" as used herein refers to a pure, target-specific antibody produced from a single clone of cells capable of growing and proliferating indefinitely in culture. Monoclonal antibodies that can be used in accordance with the present invention include naked antibodies that bind to and block antigens on cancerous cells. In one embodiment, the naked monoclonal antibody is alemtuzumab, which binds to the CD52 antigen on lymphocytes. Monoclonal antibodies that can be used in accordance with the present invention also include conjugated monoclonal antibodies, such as tagged, labeled, or loaded antibodies. Specifically, the antibodies are tagged or loaded with drugs or toxins, or radiolabeled. Examples of such antibodies include, but are not limited to, ibritumomab, which targets the CD20 antigen, brentuximab, which targets the CD30 antigen, and trastuzumab, which targets the HER2 protein. Other monoclonal antibodies that can be used in accordance with the present invention are bispecific monoclonal antibodies, such as blinatumomab, which targets CD19 on lymphoma cells and CD3 on T cells.

[0049] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof (whether in vitro or in situ) amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0050] The term "treating" or "treatment" encompasses the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., preventing its onset; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. The term "administering" or "administration" of a monoclonal antibody or natural killer cells to a subject includes any route of introducing or delivering the antibody or cells to perform its intended function. Administration may be by any route suitable for delivering the cells or monoclonal antibody. Thus, delivery routes may include intravenous, intramuscular, intraperitoneal, or subcutaneous delivery. In some embodiments, the monoclonal antibody and / or NK-92 cells are administered directly to the tumor, for example, by injection into the tumor. Administration includes self-administration and administration by another.

[0051] NK-92 cells The NK-92 cell line is a unique cell line discovered to proliferate in the presence of interleukin-2 (IL-2). Gong et al., Leukemia 8:652-658 (1994). These cells have high cytolytic activity against a variety of cancers. The NK-92 cell line is a homogeneous cancer NK cell population with broad antitumor cytotoxicity and can be obtained in predictable yields after expansion. Phase I clinical trials have confirmed its safety profile.

[0052] The NK-92 cell line is CD56 brightIt has been found to exhibit CD1, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers. Furthermore, it does not display the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, or CD34 markers. Proliferation of NK-92 cells in culture is dependent on the presence of recombinant interleukin-2 (rIL-2), with doses as low as 1 IU / mL sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term proliferation, nor do other cytokines tested, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. NK-92 possesses high cytotoxicity even at a low effector:target (E:T) ratio of 1:1. Gong, et al., supra. NK-92 cells have been deposited with the American Type Culture Collection (ATCC) under the designation CRL-2407.

[0053] Previous studies of endogenous NK cells have shown that IL-2 (1000 IU / mL) is important for NK cell activation during transport, but that maintaining the cells at 37°C and 5% carbon dioxide is not necessary. Koepsell, et al., Transfusion 53:398-403 (2013).

[0054] Suicide gene The term "suicide gene" allows for negative selection of cells. Suicide genes are used as a safety system, allowing cells expressing the gene to be killed by introducing a selective agent. This is desirable when a recombinant gene mutates, resulting in uncontrolled cell proliferation. Many suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the Escherichia coli Deo gene (see, for example, Yazawa K, Fisher WE, Brunicardi FC: Current progress in suicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9). As used herein, a suicide gene is active in NK-92 cells. Typically, a suicide gene encodes a protein that does not adversely affect the cell but kills it in the presence of a specific compound. Thus, suicide genes are typically part of a system.

[0055] In one embodiment, the suicide gene is a thymidine kinase (TK) gene. The TK gene can be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing TK protein can be killed using ganciclovir.

[0056] In another embodiment, the suicide gene is cytosine deaminase, which is toxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez et al. "Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation." Blood 1998 Jul 15;92(2):672-82.

[0057] In another embodiment, the suicide gene is a cytochrome P450 that is toxic in the presence of ifosfamide or cyclophosphamide. See, e.g., Touati et al. "A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response." Curr Gene Ther. 2014;14(3):236-46.

[0058] In another embodiment, the suicide gene is iCas9. Di Stasi, (2011) "Inducible apoptosis as a safety switch for adoptive cell therapy." N Engl J Med 365: 1673-1683. See also Morgan, "Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic" Molecular Therapy (2012); 20:11-13. The iCas9 protein induces apoptosis in the presence of the small molecule AP1903. AP1903 is a biologically inert small molecule that has been shown to be well tolerated in clinical trials and has been used in adoptive cell therapy settings.

[0059] Fc receptors Fc receptors bind to the Fc portion of an antibody. Several Fc receptors are known, which differ by their preferred ligand, affinity, expression, and effect after binding to the antibody.

[0060] Table 1. Exemplary Fc receptors TIFF2026021400000002.tif212158TIFF2026021400000003.tif125158

[0061] In some embodiments, the NK-92 cells are modified to express an Fc receptor protein on the cell surface.

[0062] In some embodiments, the Fc receptor is CD16. In the present disclosure, specific amino acid residues of CD16 are designated with reference to SEQ ID NO:2 or with reference to SEQ ID NO:1, which differs by one position from SEQ ID NO:2. Thus, the amino acid residue at "position 158" of a CD16 polypeptide according to the present invention is the amino acid residue corresponding to position 158 of SEQ ID NO:2 (or SEQ ID NO:1) when the CD16 polypeptide and SEQ ID NO:2 are maximally aligned. In some embodiments, NK-92 cells are engineered to express human CD16 having a phenylalanine at position 158 of the mature form of the protein, e.g., having SEQ ID NO:1. In an exemplary embodiment, NK-92 cells are engineered to express a high-affinity form of human CD16 having a valine at position 158 of the mature form of the protein, e.g., having SEQ ID NO:2. Position 158 of the mature protein corresponds to position 176 of the CD16 sequence, including the native signal peptide. In some embodiments, the CD16 polypeptide is encoded by a polynucleotide that encodes the precursor (ie, having the native signal peptide) polypeptide sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0063] In some embodiments, a polynucleotide encoding a CD16 polypeptide has at least about 70% polynucleotide sequence identity to a polynucleotide sequence encoding a full-length naturally occurring CD16, including the signal peptide, having a phenylalanine at position 176 of full-length CD16 (corresponding to position 158 of the mature CD16 protein). In some embodiments, a polynucleotide encoding a CD16 polypeptide has at least about 70% polynucleotide sequence identity to a polynucleotide sequence encoding a full-length naturally occurring CD16, including the signal peptide, having a valine at position 176 (corresponding to position 158 of the mature protein). In some embodiments, a polynucleotide encoding CD16 has at least 70% identity to SEQ ID NO:5 and includes a codon encoding a valine at position 176 of the full-length CD16 polypeptide, including the signal peptide. In some embodiments, a polynucleotide encoding CD16 has at least 90% identity to SEQ ID NO:5 and includes a codon encoding a valine at position 176 of full-length CD16. In some embodiments, the polynucleotide encoding CD16 comprises SEQ ID NO:5, but includes a codon encoding valine at position 176 of full-length CD16.

[0064] In some embodiments, the CD16 polynucleotide encodes a polypeptide having at least 70%, 80%, 90%, or 95% identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 70% identity or at least 80% identity to SEQ ID NO:2 and comprising a valine at position 158 as determined with respect to SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 90% identity to SEQ ID NO:2 and comprising a valine at position 158 as determined with respect to SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide having at least 95% identity to SEQ ID NO:2 and comprising a valine at position 2 as determined with respect to SEQ ID NO:2. In some embodiments, the polynucleotide encodes SEQ ID NO:2. In some embodiments, the CD16 polynucleotide encodes the extracellular domain of CD16, with or without a signal sequence, or any other fragment of full-length CD16, or a chimeric receptor comprising at least a subsequence of CD16 fused to the amino acid sequence of another protein. In other embodiments, an epitope tag peptide, such as FLAG, myc, polyhistidine, or V5, can be added to the amino-terminal domain of the mature polypeptide to aid in cell surface detection by using an anti-epitope tag peptide monoclonal or polyclonal antibody.

[0065] In some embodiments, homologous CD16 polynucleotides can be about 150 to about 700, about 750, or about 800 polynucleotides in length, although CD16 variants having more than 700 to 800 polynucleotides are within the scope of the present disclosure.

[0066] Homologous polynucleotide sequences include those that encode polypeptide sequences that encode variants of CD16. Homologous polynucleotide sequences also include naturally occurring allelic variations relative to SEQ ID NO:5. Transfection of NK-92 cells with any polynucleotide encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, a naturally occurring variant thereof, or a sequence at least 70% identical, or at least 80%, 90%, 95% identical to SEQ ID NO:1 or SEQ ID NO:2, is within the scope of the present disclosure. In some embodiments, the homologous polynucleotide sequence encodes conservative amino acid substitutions in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, NK-92 cells are transfected with a degenerate homologous CD16 polynucleotide sequence that differs from the native polynucleotide sequence but encodes the same polypeptide.

[0067] In another example, a cDNA sequence having a polymorphism that alters the CD16 amino acid sequence, such as an allelic variation between individuals that exhibit genetic polymorphisms in the CD16 gene, is used to modify NK-92 cells. In another example, a CD16 gene from another species having a polynucleotide sequence that differs from the sequence of SEQ ID NO:5 is used to modify NK-92 cells.

[0068] In some examples, mutant polypeptides are generated using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al., 1985), or other known techniques can be performed on cloned DNA to generate CD16 mutants (Ausubel, 2002; Sambrook and Russell, 2001).

[0069] In some embodiments, polynucleotides encoding CD16 are mutated to change the amino acid sequence encoding CD16 without altering the function of CD 16. For example, polynucleotide substitutions resulting in amino acid substitutions at "non-essential" amino acid residues can be made in SEQ ID NO:1 or SEQ ID NO:2.

[0070] Conservative substitutions in SEQ ID NO:1 or SEQ ID NO:2, in which an amino acid of one class is replaced with another amino acid of the same class, fall within the scope of the disclosed CD16 variants, as long as the substitution does not substantially alter the activity of the CD16 polypeptide. Conservative substitutions are well known to those skilled in the art. Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, for example, β-sheet or α-helical conformation, (2) charge, (3) hydrophobicity, or (4) the bulkiness of the side chains of the target site, may alter the function or immunological identity of the CD16 polypeptide. Non-conservative substitutions involve exchanging a member of one of these classes for another class. Substitutions can be introduced into conservative substitution sites, more preferably into non-conserved sites.

[0071] In some embodiments, the CD16 polypeptide variant is at least 200 amino acids in length and has at least 70% amino acid sequence identity, or at least 80%, or at least 90% identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the CD16 polypeptide variant is at least 225 amino acids in length and has at least 70% amino acid sequence identity, or at least 80%, or at least 90% identity to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the CD16 polypeptide variant has a valine at position 158 as determined with reference to SEQ ID NO:2.

[0072] In some embodiments, the nucleic acid encoding a CD16 polypeptide can encode a CD16 fusion protein. A CD16 fusion polypeptide comprises any portion of CD16 or the entire CD16 fused to a non-CD16 polypeptide. Fusion polypeptides are easily produced using recombinant methods. For example, a polynucleotide encoding a CD16 polypeptide, such as SEQ ID NO:1 or SEQ ID NO:2, is fused in frame with a non-CD16-encoding polynucleotide (e.g., a polynucleotide sequence encoding a signal peptide of a heterologous protein). In some embodiments, fusion polypeptides can be produced in which the heterologous polypeptide sequence is fused to the C-terminus of CD16 or positioned internally within CD16. Typically, up to about 30% of the CD16 cytoplasmic domain can be replaced. Such modifications can enhance expression or increase cytotoxicity (e.g., ADCC response). In other examples, chimeric proteins replace portions of the CD16 cytoplasmic domain with domains from other lymphocyte activation receptors, including but not limited to Ig-a, Ig-B, CD3-e, CD3-d, DAP-12, and DAP-10.

[0073] Fusion genes can be synthesized by conventional techniques, such as automated DNA synthesizers and PCR amplification, which uses anchor primers that generate complementary overhangs between two consecutive gene fragments that are subsequently annealed and reamplified to generate a chimeric gene sequence (Ausubel, 2002). Many vectors are commercially available that facilitate subcloning CD16 in frame with the fusion moiety.

[0074] cytokines The cytotoxicity of NK-92 cells is dependent on the presence of cytokines, such as interleukin-2 (IL-2). The exogenous IL-2 required to maintain and expand NK-92 cells in commercial-scale cultures is costly. Administering IL-2 to human subjects in amounts sufficient to sustain NK-92 cell activation would cause adverse side effects.

[0075] In some embodiments, the FcR-expressing NK-92 cells are further modified to express at least one cytokine and a suicide gene. In certain embodiments, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof. In a preferred embodiment, the cytokine is IL-2. In certain embodiments, the IL-2 is a mutant that targets the endoplasmic reticulum, and the suicide gene is iCas9.

[0076] In one embodiment, IL-2 is expressed with a signal sequence that targets IL-2 to the endoplasmic reticulum. In some embodiments, the polynucleotide encoding IL-2 encodes a polypeptide having the sequence of SEQ ID NO:7. Without being bound by theory, targeting IL-2 to the endoplasmic reticulum can allow expression of IL-2 at levels sufficient for autocrine activation without releasing IL-2 extracellularly. See Konstantinidis et al. "Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 cells" Exp Hematol. 2005 Feb;33(2):159-64. Continuous activation of FcR-expressing NK-92 cells can be prevented, for example, by the presence of a suicide gene.

[0077] immunotherapy Antibodies can be used to target infected cells or cells expressing cancer-associated markers. Many antibodies have been approved for the treatment of cancer alone.

[0078] Table 2: Exemplary therapeutic monoclonal antibodies TIFF2026021400000004.tif55157TIFF2026021400000005.tif229157TIFF2026021400000006.tif54157

[0079] Antibodies can treat cancer through a number of mechanisms: antibody-dependent cellular cytotoxicity (ADCC) occurs when immune cells such as NK cells bind to antibodies, which are then attached to target cells via Fc receptors such as CD16.

[0080] Thus, in some embodiments, NK-92 cells expressing CD16 are administered to a patient along with antibodies against specific cancer-associated proteins.

[0081] Administration of FcR-expressing NK-92 cells can be simultaneous with or sequential to administration of a monoclonal antibody. Genetically modifying NK-92 cells to express FcR enables the cells to recognize antibody-coated target cells and induce NK cell-mediated ADCC, resulting in rapid NK cell activation. In some embodiments, the FcR-expressing NK-92 cells are administered to a subject after the subject has been treated with a monoclonal antibody. In some embodiments, the FcR-expressing NK-92 cells are administered within 24 hours, 18 hours, 12 hours, 8 hours, or 6, 5, 4, 3, 2, or 1 hour after administration of the monoclonal antibody. In some embodiments, the FcR-expressing NK-92 cells are administered 24 to 72 hours after administration of the antibody. In some embodiments, the FcR-expressing NK-92 cells are administered within 1, 2, 3, or 4 days or more after administration of the antibody.

[0082] In some embodiments, the FcR-expressing NK-92 cells and monoclonal antibodies are administered intravenously. In some embodiments, the FcR-expressing NK-92 cells are infused directly into the bone marrow.

[0083] In one aspect of the present invention, FcR-expressing NK-92 cells are administered to a subject with leukemia in combination with a therapeutic monoclonal antibody, e.g., alemtuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered simultaneously with alemtuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered after the subject has been treated with alemtuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered within 24 hours, 18 hours, 12 hours, 8 hours, or within 6, 5, 4, 3, 2, or 1 hour of administration of alemtuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered 24 to 72 hours or later after administration of alemtuzumab.

[0084] In a further aspect, FcR-expressing NK-92 cells are administered in combination with trastuzumab to a subject with cancer, such as breast cancer or gastric cancer. In some embodiments, the FcR-expressing NK-92 cells are administered simultaneously with trastuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered after trastuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered within 24 hours, 18 hours, 12 hours, 8 hours, or 6, 5, 4, 3, 2, or 1 hour after administration of trastuzumab. In some embodiments, the FcR-expressing NK-92 cells are administered 24 to 72 hours or later after administration of trastuzumab.

[0085] In a further aspect, FcR-expressing NK-92 cells are administered in combination with brentuximab to a subject with Hodgkin's lymphoma. In some embodiments, the FcR-expressing NK-92 cells are administered simultaneously with brentuximab. In some embodiments, the FcR-expressing NK-92 cells are administered after brentuximab. In some embodiments, the FcR-expressing NK-92 cells are administered within 24 hours, 18 hours, 12 hours, 8 hours, or 6, 5, 4, 3, 2, or 1 hour after administration of brentuximab. In some embodiments, the FcR-expressing NK-92 cells are administered 24 to 72 hours or later after administration of brentuximab.

[0086] In a further aspect, FcR-expressing NK-92 cells are administered to a subject with multiple myeloma in combination with daratumumab. In some embodiments, the FcR-expressing NK-92 cells are administered simultaneously with daratumumab. In some embodiments, the FcR-expressing NK-92 cells are administered after daratumumab. In some embodiments, the FcR-expressing NK-92 cells are administered within 24 hours, 18 hours, 12 hours, 8 hours, or 6, 5, 4, 3, 2, or 1 hour after administration of daratumumab. In some embodiments, the FcR-expressing NK-92 cells are administered 24 to 72 hours or later after administration of daratumumab.

[0087] Transgene expression Transgenes (e.g., CD16 and IL-2) can be engineered into expression plasmids by any mechanism known to those of skill in the art. The transgenes can be engineered into the same expression plasmid or can be different. In a preferred embodiment, the transgenes are expressed on the same plasmid.

[0088] Transgenes can be introduced into NK-92 cells using transient transfection methods known in the art, including, for example, electroporation, lipofection, nucleofection, or "gene guns."

[0089] Various vectors can be used to express CD16 and IL-2. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a plasmid vector. Other viral vectors that can be used include adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, pox viral vectors, etc.

[0090] The NK-92 cells are administered to the individual in absolute numbers of cells, e.g., the individual is administered from about 1,000 cells / infusion to about 10 billion cells / infusion, e.g., about, at least about, or at most about 1 x 10 cells per infusion. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 In other embodiments, the NK-92 cells are administered to the individual in a relative number of cells, e.g., from about 1,000 to about 10 billion cells per kilogram of the individual, e.g., about, at least about, or at most about 1 x 10 cells per kilogram of the individual. 8 , 1×10 7 , 5×10 7 , 1×10 6 , 5×10 6 , 1×10 5 , 5×10 5 , 1×10 4 , 5×10 4 , 1×10 3 , 5×10 3 (etc.) NK-92 cells, or any range between any two numbers (including the endpoints), may be administered. In some embodiments, about 1 billion to about 3 billion NK-92 cells are administered to a patient. In other embodiments, the total dose is administered per m of body surface area. 2For example, m 2 11 x 10 per 11 , 1×10 10 , 1×10 9 , 1×10 8 , 1×10 7 The average person has a height of 1.6-1.8m 2 is.

[0091] The NK-92 cells, monoclonal antibodies described below and / or other anti-cancer agents can be administered once to a cancer or virally infected patient, or multiple times, for example, once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours, or once every 1, 2, 3, 4, 5, 6, or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks, or any range between any two numbers (endpoints inclusive), throughout the treatment period. [Example]

[0092] The following examples are for illustrative purposes only and should not be construed as limiting the invention as claimed. There are various alternative techniques and procedures available to those skilled in the art which will similarly enable one to successfully practice the intended invention.

[0093] Example 1: Preparation of CD16 recombinant retrovirus CD16 cDNA X52645.1, encoding the low-affinity mature form of the transmembrane immunoglobulin gamma Fc region receptor III-A (FcγRIII-A or CD16) (SEQ ID NO:1) [phenylalanine-158 (F158), complete sequence: SwissProt P08637 (SEQ ID NO:3)], or a polymorphic variant encoding a higher affinity mature form of the CD16 receptor [valine-158 (F158V) (SEQ ID NO:2), complete sequence: SwissProt VAR_008801 (SEQ ID NO:4)], was subcloned into the bicistronic retroviral expression vector pBMN-IRES-EGFP (obtained from G. Nolan, Stanford University, Stanford, CA) using the BamHI and NotI restriction sites according to standard procedures.

[0094] The recombinant vector was mixed with 10 μL of PLUS™ Reagent (Invitrogen; Carlsbad, CA) and diluted to 100 μL with prewarmed serum-free Opti-MEM® (Invitrogen; MEM, minimum essential medium). This was further diluted by adding 8 μL of Lipofectamine™ (Invitrogen) in 100 μL of prewarmed serum-free Opti-MEM® and incubated at room temperature for 15 minutes. The mixture was then brought to a total volume of 1 mL by adding prewarmed serum-free Opti-MEM®. Phoenix-Amphotropic packaging cells (obtained from G. Nolan, Stanford University, Stanford, CA; (Kinsella and Nolan, 1996)) were grown in 6-well plates to 70–80% confluence and washed with 6 mL of prewarmed serum-free Opti-MEM® medium (Invitrogen). After removing the medium, 1 mL of a solution of the recombinant vector in Lipofectamine™ PLUS™ Reagent was added to each well, and the cells were incubated at 37°C under a 7% CO2 / balance air atmosphere for at least 3 hours. 4 mL of pre-warmed RPMI medium containing 10% fetal bovine serum (FBS) was added to each well, and the cells were incubated overnight at 37°C under a 7% CO2 / balance air atmosphere. The medium was then removed, the cells were washed with 6 mL of pre-warmed serum-free Opti-MEM®, and 2 mL of serum-free Opti-MEM® was added. The cells were incubated for an additional 48 hours at 37°C under a 7% CO2 / balance air atmosphere.

[0095] The virus-containing supernatant was collected in a 15 mL plastic centrifuge tube and centrifuged at 1300 rpm for 5 minutes to remove cells and cell debris, and the supernatant was transferred to another 15 mL plastic centrifuge tube. Just before use, 20 μL of PLUS™ reagent was added to the virus suspension, and the mixture was incubated at room temperature for 15 minutes. 8 μL of Lipofectamine™ was added to the mixture, and the mixture was incubated at room temperature for another 15 minutes.

[0096] Example 2: Cloning of the IL-2 gene and the TK suicide gene into a CD16 recombinant retrovirus A recombinant retrovirus incorporating the gene for IL-2 expression was prepared using a thymidine kinase (TK) gene and a KDEL-tagged construct that produces ER-resident IL-2 (Konstantinidis et al. 2005 Experimental Hematology 33: 159-64), and the corresponding cDNA was ligated into the CD16 pBMN-IRES-EGFP vector (Miah and Campbell 2010 Methods Mol. Biol. 612: 199-208).The pBMN-IRES-EGFP vector was then transfected into the Phoenix-Amphotropic packaging cell line in the presence of Lipofectamine™ Plus.

[0097] Example 3: Retroviral transduction of NK-92 cells with TK, CD16, and IL-2 NK-92 cells cultured in A-MEM (Sigma; St. Louis, MO) supplemented with 12.5% ​​FBS, 12.5% ​​fetal horse serum (FHS), and 500 IU rhIL-2 / mL (Chiron; Emeryville, CA) were collected by centrifugation at 1300 rpm for 5 minutes, and the cell pellet was resuspended in 10 mL of serum-free Opti-MEM® medium. 4An aliquot of the cell suspension containing 100 cells was sedimented at 1300 rpm for 5 minutes, the cell pellet resuspended in 2 mL of the retroviral suspension described in Example 1, and the cells were plated onto 12-well culture plates. The plates were centrifuged at 1800 rpm for 30 minutes and incubated at 37°C in a 7% CO2 / balance air atmosphere for 3 hours. This cycle of centrifugation and incubation was repeated twice. The cells were diluted with 8 mL of α-MEM, transferred to a T-25 flask, and incubated at 37°C in a 7% CO2 / balance air atmosphere until the cells became confluent. The transduced cells were harvested, resuspended in serum-free Opti-MEM® medium, and sorted using a fluorescence-activated cell sorter (FACS) based on the level of EGFP expression; EGFP is co-expressed with CD16 and is a surrogate marker for CD16. Cell surface expression of CD16 was confirmed by immunostaining the transduced cells with an anti-CD16 antibody. Cell surface expression of IL-2 was measured by immunostaining with purified rat anti-human IL-2 antibody, and intracellular localization of IL-2 was confirmed by immunostaining with rabbit anti-calreticulin ER marker. Transduced cells, designated NK-92-TK-CD16-IL2, were assayed for cell surface expression of CD16 and intracellular expression of IL-2 prior to use. The cells were assayed for TK expression by testing for susceptibility to gancylovir.

[0098] Example 4: Growth of NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells in the presence or absence of exogenous IL-2 NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells were first cultured in the presence of exogenous IL-2 (1,200 IU / mL) for 4–5 weeks, then transferred to IL-2-free medium and cultured in the absence of exogenous IL-2. Proliferation of these cells was then assayed.

[0099] Surface expression of CD16 and IL-2 is measured by flow cytometry. Flow cytometry analysis performed after 24 hours of incubation of NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells in the absence of exogenous IL-2 shows similar cytotoxic activity in NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells, but NK-92-TK-CD16-IL-2 cells show increased surface expression of CD16 and much lower surface expression of IL-2 compared to unmodified NK-92 cells.

[0100] These results were confirmed by experiments to determine whether NK-92-TK-CD16-IL-2 cells support the proliferation of bystander, unmodified NK-92 cells, in which unmodified NK-92 cells are mixed with NK-92-TK-CD16-IL-2 and co-cultured in the absence of exogenous IL-2. These experiments demonstrate that NK-92-TK-CD16-IL-2 does not support the proliferation of unmodified NK-92 cells due to minimal release of IL-2 into the culture medium. Indeed, unmodified NK-92 cells cease proliferation after 48 hours of incubation in the absence of exogenous IL-2. In contrast, proliferation of NK-92-TK-CD16-IL-2 cells is still observed after 72 hours of incubation.

[0101] Overall, these results indicate that endoplasmic reticulum (ER)-IL-2 stimulates proliferation of NK-92-TK-CD16-IL-2 cells when maintained in an environment without exogenous IL-2.

[0102] Example 5: Systemic toxicity and proliferation of NK-92-TK-CD16-IL-2 cells is effectively abrogated by a suicide gene Endogenous expression of IL-2 may lead to the potential development of killer cell mutants with autonomous proliferation. Therefore, the in vivo proliferation of NK-92-TK-CD16-IL-2 cells, NK-92-TK-CD16-IL-2 cells, and unmodified NK-92 cells was evaluated. SCID mice were sublethally irradiated (250 rads) and divided into two groups. 15–20 days later, when tumors became palpable (0.5–0.8 cm in diameter), NK-92-TK-CD16-IL-2 cells were intravenously injected into the first group of irradiated mice, and unmodified NK-92 cells were intravenously injected into the second group of mice. No exogenous cytokines were administered to the mice. Localization and proliferation were monitored using fluorescence-activated cell sorting (FACS) to detect EGFP expression. Both groups of mice demonstrated targeted localization and proliferation 24 hours after injection. After 24 hours, the unmodified NK-92 cells in the control mice stopped proliferating, while the NK-92-TK-CD16-IL-2 cells continued to proliferate. Forty-eight hours after injection, apoptosis of the unmodified NK cells was observed in the control mice, while exponential growth of the NK-92-TK-CD16-1L-2 cells was observed in the test mice. In the control mice, tumors rapidly grew to a diameter of more than 1.2 cm, and the mice were euthanized.

[0103] Mice from the test group with smaller tumors or complete tumor regression were separated into two groups to evaluate the functionality of the suicide gene. The first group of mice was treated with two or three intraperitoneal doses of ganciclovir (50 μg) every other day. The second group of mice was treated with a placebo. Ganciclovir administration to the mice resulted in a significant decrease in NK-92-TK-CD16-IL-2 cells within 24 to 72 hours, with the cells returning to pre-expansion levels. In placebo-treated mice, NK-92-TK-CD16-IL-2 cell proliferation continued to expand over time.

[0104] These results indicate that the presence of the TK gene ensures that NK-92-TK-CD16-IL-2 remains sensitive to ganciclovir and prevents exponential growth of NK-92-TK-CD16-IL-2 cells. This combination of TK and IL-2 on the same retroviral vector, when integrated into the chromosome of NK-92 cells, provides improved biosafety. Because the cells are dependent on IL-2, there is strong selection for retaining the TK-CD16-IL-2 sequence. Thus, the cells are sensitive to ganciclovir. Cells that lose the TK gene and become resistant to ganciclovir will also lose the IL-2 gene required for their proliferation.

[0105] Example 6: Cytotoxic activity of NK-92-TK-CD16-IL-2 against different leukemia cell lines NK-92-TK-CD16-IL-2 effector cells were washed by suspending in α-MEM (without IL-2) and sedimenting at 1300 rpm for 5 minutes. The cell pellet was suspended in α-MEM, the cells were counted, and 1 x 10 5 cells / mL (effector-to-target cell ratio (E:T) = 1:1), 5 × 10 5 / mL (E:T=5:1), 1×10 6 / mL (E:T=10:1), 2 x 10 6 Prepare aliquots at a cell concentration of 0.1% / mL (E:T = 20:1) or as appropriate depending on the measurements to be performed.

[0106] The cytotoxic activity of NK-92-TK-CD16-IL-2 effector cells against K562, Daudi, TF-1, AML-193, and SR-91 cells was measured (Gong et al. (1994)). K562 (erythroleukemia) and Daudi (Burkitt's) lymphoma cell lines were obtained from ATCC. They were maintained in continuous suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). TF-1 is a myelomonocytic cell line that requires the presence of medium containing 2 ng / mL human GM-CSF (Kitamura et al., J. Cell Physiol. 140:323-334 (1989)). AML-193 is a myeloid cell line that is maintained in the presence of 10% 5637-conditioned medium (Lange et al., Blood 70:192-199 (1987)). Both TF-1 and AML-193 cells were obtained from Dr. D. Hogge of the Terry Fox Laboratory at the University of British Columbia (Vancouver, BC). SR-91 is a cell line with early progenitor characteristics established by Gong et al. (1994) from a patient with acute lymphoblastic leukemia (ALL) (Klingemann et al., Leuk. Lymphoma, 12, 463-470 (1994)). It is resistant to the cytotoxicity of both NK cells and activated NK (A-NK) cells. SR-91 is also maintained in RPMI 1640 / 10% FCS. This cell line can be made sensitive to killing by NK-92 by treatment with cytokines.

[0107] The cytotoxic activity of NK-92-TK-CD16-IL-2 effector cells against these target cells is measured in triplicate by a standard 4-hour 51Cr release assay. Briefly, 1 × 10 6NK-92-TK-CD16-IL-2 cells were labeled with 100 μL of 51Cr (specific activity 1 mCi / mL) and incubated for 1 hour at 37°C. Effector cells were counted by trypan blue exclusion and mixed with target cells to achieve effector:target ratios of 10:1, 3:1, 1:1, and 0.3:1. CellGro medium was used as a negative control, and for a positive control, cells were incubated with 1% Triton X. After 4 hours of incubation at 37°C in a V-bottom 96-well plate, 70 μL of supernatant was aspirated from each well and counted using a Packard Cobra Auto-Gamma 5000 series counting system (Meriden, CT, USA). The percentage of spontaneous release was calculated using the following formula: % specific 51Cr release = (sample release - spontaneous release) / (maximum release - spontaneous release) × 100.

[0108] The cytotoxic activity of NK-92-TK-CD16-IL-2 cells against K562 and Daudi cells was significantly higher than that of unmodified NK-92 cells. The cytolytic activity of NK-92-TK-CD16-IL-2 cells against TF-1 and AML-193 cells was less potent, but still higher than that of unmodified NK-92 cells. SR-91 cells were resistant to the cytotoxic effects of both NK-92-TK-CD16-IL-2 and unmodified NK-92 cells. This lack of cytotoxic activity against SR-91 cells is consistent with the lack of adhesion molecules necessary to mediate initial binding with NK-92 cells.

[0109] Example 7: Cytolysis of human primary leukemia cells by NK-92-TK-CD16-IL-2 cells Samples were obtained with informed consent from patients with newly diagnosed or relapsed leukemia during routine diagnostic blood tests or bone marrow (BM) aspirates. Blast-enriched mononuclear cells were isolated by Ficoll Hypaque (Pharmacia, Piscataway, NJ) density gradient separation and washed with RPMI 1640 medium. NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells were maintained in culture in α-MEM medium supplemented with 12.5% ​​FCS and 12.5% ​​horse serum. The cytotoxic activity of NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells against leukemia samples was then compared using a standard 4-hour chromium release assay.

[0110] The cytolytic activity of NK-92-TK-CD16-IL-2 cells against leukemic targets is significantly greater than that of unmodified NK-92 cells. The NK-92-TK-CD16-IL-2 cells of the present invention are surprisingly and significantly more effective at lysing patient-derived tumor cells than unmodified NK-92 cells, and exert their effect in a shorter time.

[0111] Example 8: Anti-leukemic effect of NK-92-TK-CD16-IL-2 cells in a human leukemia xenograft SCID mouse model To study the in vivo tumoricidal ability of NK-92-TK-CD16-IL-2 cells, leukemia cells from T-lineage acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and pre-B-cell ALL patients are subcutaneously (SC) inoculated and propagated in SCID mice. Leukemia cells (passage 1) recovered from the leukemia nodules of the mice are used in these experiments. Each group of SCID mice is inoculated with 5 x 10 cells from passage 1 in 0.2 mL PBS. 6 leukemia cells were inoculated intraperitoneally (IP), and 24 hours later, 2 × 10 cells were cultured in 0.4 mL PBS. 7NK-92-TK-CD16-IL-2 cells are administered via L.P. injection. Animals receive a single dose or a series of five doses of NK-92-TK-CD16-IL-2 cells administered on days 1, 3, 5, 7, and 9, with or without exogenous IL-2.

[0112] All human leukemias grow vigorously in SCID mice. Leukemia cells from T-ALL, AML, and pre-B-ALL patients are highly sensitive to NK-92-TK-CD16-IL-2 cells and unmodified NK-92 cells in vitro.

[0113] Treatment with NK-92-TK-CD16-IL-2 cells significantly prolonged the lifespan and survival of mice compared with treatment with unmodified NK-92 cells. Several animals receiving five infusions of NK-92-TK-CD16-IL-2 cells survived without signs of leukemia six months after inoculation. While NK-92-treated mice showed initial improvement, leukemia cells were observed in a small number of mice by six months.

[0114] These results demonstrate that in vivo treatment of leukemic tumors with NK-92-CD16-IL-2 cells is highly effective, resulting in improved survival and health.

[0115] Example 9: ADCC-mediated cell lysis The activity of several antibodies, which are highly selective and effective antitumor agents, depends, at least in part, on the binding of natural killer cells to the Fc (constant) portion of the antibody, resulting in tumor cell lysis via the antibody-dependent cellular cytotoxicity (ADCC) mechanism. NK-92 cells retain almost all of the activating receptors and cytolytic pathways associated with NK cells, but they do not express the CD16 receptor and therefore cannot lyse target cells via the ADCC mechanism. Transgenic insertion of CD16 expression into NK-92 cells allows them to act via the ADCC mechanism if the cells have sufficient binding affinity for an effective antibody.

[0116] The effects of conjugation to various antibodies are assessed in FcR-expressing NK-92 cells administered to subjects with leukemia 24 to 72 hours after treatment with alemtuzumab. The cytotoxic effects of antibody conjugation on FcR-expressing NK-92 cells and target cancer cells are compared to the cytotoxic effects of unmodified NK-92 cells. Antibodies and corresponding target cancer cells are selected according to Table 2 and assayed.

[0117] Selected target cells [ 51 Label with sodium chromate. 51 Aliquots of [Cr]-labeled target cells were further incubated with selected antibodies at multiple concentrations ranging from 0.01 μg to 5 μg / mL for 15 min at room temperature, washed with α-MEM, and resuspended in 1 × 10 cells before use. 5 Adjust the cell concentration to 1 x 10 cells / mL. Mix 100 µL of the selected type of target cells and 100 µL of effector cells. 5 / mL (E:T=1:1), 5×10 5 / mL (E:T=5:1), 1×10 6 / mL (E:T=10:1), 2 x 10 6 Add cells to each well of a 96-well V-bottom plate at a cell concentration of 100 cells / mL (E:T = 20:1), or as appropriate for the assay being performed. Prepare three to six replicate wells for each E:T ratio being evaluated. Allocate at least six wells for each of the spontaneous lysis control (effector cells replaced with 100 µL of α-MEM) and total release control (effector cells replaced with 100 µL of 2% Triton X-100 surfactant / α-MEM). Allocate three additional wells at each E:T ratio for "non-ADCC" controls in which target cells were not exposed to antibody. Allocate six or more additional wells for procedural controls and the use of unmodified NK-92 effector cells that do not express CD16 as an internal standard. Then, centrifuge the plate at 500 rpm for 3 minutes and incubate at 37°C in a 7% CO2 / balance air atmosphere for 4 hours. At the end of the incubation period, the plates were centrifuged at 1500 rpm for 8 minutes to determine the cytotoxicity. 51Collect 100 mL of supernatant from each well for counting in a gamma counter as a measure of [Cr] release, after which the percentage of specific lysis is calculated.

[0118] These assays are repeated using FcR-expressing NK-92 cells expressing various surface levels of CD16.

[0119] FcR-expressing NK-92 cells exhibit high cytotoxic activity against target cancer cells in the presence of selected antibodies. Unmodified NK-92 cells exhibit lower cytotoxic activity against target cancer cells. These results demonstrate that FcR-expressing NK-92 cells are capable of acting via the ADCC mechanism, thus providing enhanced therapeutic effects against tumor cells in the presence of antibodies.

[0120] Example 10: Anti-leukemia effect of combined use of FcR-expressing NK-92 cells and gemtuzumab in a human leukemia xenograft SCID mouse model To study the in vivo tumoricidal ability of FcR-expressing NK-92 cells, leukemia cells from acute myeloid leukemia (AML) patients are adoptively grown and expanded in SCID mice by SC inoculation. Leukemia cells (passage 1) recovered from the leukemia nodules of the mice are used in these experiments. Each group of SCID mice is inoculated with 5 x 10 1-passage leukemia cells in 0.2 mL PBS. 6 leukemia cells were inoculated IP, and 2 x 10 cells were cultured in 0.4 mL PBS for 24 h. 7 FcR-expressing NK-92 cells and gemtuzumab are administered by LP injection. Animals receive a single dose or a series of five doses of FcR-expressing NK-92 cells, with or without gemtuzumab, administered on days 1, 3, 5, 7, and 9. Control animals are treated with unmodified NK-92 cells, with or without gemtuzumab.

[0121] Human leukemia grows aggressively in SCID mice. Mice treated with FcR-expressing NK-92 cells in combination with gemtuzumab exhibited tumor regression, with a greater antitumor effect than mice treated with FcR-expressing NK-92 cells alone without gemtuzumab, and greater than mice treated with NK-92 alone.

[0122] These results demonstrate that in vivo treatment of leukemia tumors with FcR-expressing NK-92 cells in combination with a monoclonal antibody such as gemtuzumab is highly effective, resulting in improved survival and health.

[0123] Example 11: Construction of plasmid expression vectors for CD16 and endoplasmic reticulum-targeted IL-2 The Gene String program from GeneArt (Life Technologies) was used to de novo design a plasmid backbone, the minimal structure of which contains a colE1 bacterial replication origin, an ampicillin resistance cassette, and a mammalian expression cassette consisting of the EF1α promoter and SV40 polyadenylation site flanked by multiple cloning sites (MCS).

[0124] The mammalian expression cassette is flanked by BamH1 sites, which allow not only linearization of the plasmid but also removal of all non-eukaryotic sequences.

[0125] The transgene expressed is the human CD16 158V sequence, followed by the IRES sequence itself, followed by the ERIL-2 sequence (IL-2 KDEL) to target IL-2 to the endoplasmic reticulum. Both the CD16 and ERIL-2 sequences were codon-optimized by GeneArt to maximize expression in humans. The transgene can be excised using EcoRI and NotI. The resulting mRNA is a bicistronic transcript under the control of the EF1α promoter, with ERIL-2 translated independently of CD16 under the control of the IRES sequence. A schematic diagram of this plasmid is shown in Figure 1. This plasmid was used to transfect NK-92 cells.

[0126] Example 12: Transfection of NK-92.W cells with plasmid expression vectors NK-92.W cells are the parent cell line for most clinical trials to date. One vial from Bioreliance's Working Cell Bank (WCB, pl5 11 / 30 / 00) was thawed into a T25 flask containing 12 ml of X-Vivo10 5% HS + 500 IU / ml rhIL-2 and passaged every 2–4 days (×2–×4 dilution into fresh X-Vivo10 5% HS + IL-2, for a total of 18–20 splits / passage).

[0127] NK-92.W cells for transfection were spun at 500g for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in 15 ml of D-PBS 1x and centrifuged at 500g for 10 minutes. The pellet was resuspended in Buffer R (Neon kit, Invitrogen) at a cell density of 10e7 cells / ml. NK-92.W cells were electroporated with the pNEUKv1 CD16(158V)-ERIL2 plasmid using a Neon electroporator (5 μg of DNA per 10e6 cells in 100 μl of Buffer R; 1250V / 10 ms / 3 pulses with 3 ml of Buffer E2 in the electroporation tube). Electroporated cells were incubated overnight in IL-2-containing medium (6-well plate, 4 ml medium / well) and transferred to IL-2-free medium (one PBS spin / wash) on October 16, 2014. CD16 expression was assayed using an anti-CD16 antibody (clone 3G8, mouse IgG1k) conjugated to APC-Cy7 (Bd Pharmingen). After just over 2 weeks, approximately 78% of cells were CD16 positive (right peak, Figure 2a). Approximately 90% of cells were positive by approximately 4 weeks (right peak, Figure 2b).

[0128] NK-92.W CD16(158V)-ERIL2 cells were frozen (5 vials of approximately 1 x 10e6 cells per vial) on December 15, 2014. Freezing medium is 10% DMSO, 50% HS, 40%.

[0129] Frozen NK-92.W FcR-ERIL2 cells were evaluated. Cells were thawed and cultured in X-Vivo10 5% HS medium without IL-2 in T25 flasks. CD16 (158V) expression was monitored over time by flow cytometry (Attune) using the anti-CD16 antibody clone 3G8 conjugated to APC-Cy7, using the same settings to allow comparison of MFI between assays. CD16 expression was stable over time (Figure 3).

[0130] Example 13: Evaluation of ADCC activity ADCC activity was first tested against the CD20+ cell line DoHH2 in combination with rituximab. This test was repeated over time (n=9) and also against the Her2 / Neu+ cell line SKOV3 in combination with Herceptin (n=5). The results are shown in Figure 4. Modified NK-92.W cells expressing CD16 and endoplasmic reticulum-targeted IL-2 (labeled HaNK.12 / 15 in Figure 4) showed enhanced ADCC activity against SKOV-3 cells when used with Herceptin and against DoHH2 cells when combined with rituximab. haNK.12 / 15 cells did not show ADCC activity in controls (DoHH2 cells, Herceptin antibody; SKOV-3 cells / rituximab). Unmodified NK-92.2 cells also did not show ADCC activity when administered with the antibody.

[0131] Thus, Examples 11-13 demonstrate that NK-92 cells modified to express CD16 and IL-2 using a plasmid vector exhibited enhanced ADCC activity when used in combination with a monoclonal antibody.

[0132] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art, which are to be included within the spirit and scope of this application and the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0133] Exemplary Sequences SEQ ID NO: 1 Amino acid sequence of low affinity immunoglobulin gamma Fc region receptor III-A (mature form). The phenylalanine at position 158 is underlined. TIFF2026021400000007.tif73149 SEQ ID NO:2 Amino acid sequence of high affinity mutant F158V immunoglobulin gamma Fc region receptor III-A (mature form). The valine at position 158 is underlined. TIFF2026021400000008.tif73149 SEQ ID NO:3 Amino acid sequence of low affinity immunoglobulin gamma Fc region receptor III-A (precursor form). Position 176 of the precursor form corresponds to position 158 of the mature form. The Phe at position 176 is underlined. TIFF2026021400000009.tif80150 SEQ ID NO:4 Amino acid sequence of high affinity mutant immunoglobulin gamma Fc region receptor III-A (precursor form). Position 176 of the precursor form corresponds to position 158 of the mature form. Val at position 176 is underlined. TIFF2026021400000010.tif80150 SEQ ID NO: 5 Polynucleotide encoding low affinity immunoglobulin gamma Fc region receptor III-A (precursor) (encodes phenylalanine at position 158) TIFF2026021400000011.tif66145 SEQ ID NO:6 Wild type IL-2 TIFF2026021400000012.tif52150 SEQ ID NO:7 IL-2-ER TIFF2026021400000013.tif52150

[0134] Array information SEQUENCE LISTING <110> IMMUNITYBIO, INC. <120> GENETICALLY MODIFIED NK-92 CELLS AND MONOCLONAL ANTIBODIES FOR THE TREATMENT OF CANCER <150> US 62 / 139,258 <151> 2015-03-27 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 236 <212> PRT <213> Homo sapiens <400> 1 Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp 1 5 10 15 Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala 20 25 30 Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu 35 40 45 Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp 50 55 60 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp 65 70 75 80 Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro 85 90 95 Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His Ser 100 105 110 Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys 115 120 125 Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala 130 135 140 Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe Gly Ser 145 150 155 160 Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu 165 170 175 Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser 180 185 190 Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr 195 200 205 Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp 210 215 220 His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 225 230 235 <210> 2 <211> 236 <212> PRT <213> Homo sapiens <400> 2 Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp 1 5 10 15 Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala 20 25 30 Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu 35 40 45 Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp 50 55 60 Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp 65 70 75 80 Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro 85 90 95 Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys His Ser 100 105 110 Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys 115 120 125 Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala 130 135 140 Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser 145 150 155 160 Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu 165 170 175 Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser 180 185 190 Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr 195 200 205 Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp 210 215 220 His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 225 230 235 <210> 3 <211> 254 <212> PRT <213> Homo sapiens <400> 3 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 4 <211> 254 <212> PRT <213> Homo sapiens <400> 4 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 5 <211> 765 <212> DNA <213> Homo sapiens <400> 5 atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60 gaagatctcc caaaggctgt ggtgtcctg gagcctcaat ggtacagggt gctcgagaag 120 gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180 tttcacaatg agagcctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240 gtcgacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300 cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360 gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420 tatttacaga atggcaaagg caggaagtat tttcatcata attctgactt ctacattcca 480 aaagccacac tcaaagacag cggctcctac ttctgcaggg ggctttttgg gagtaaaaat 540 gtgtcttcag agactgtgaa catcaccatc actcaggtt tggcagtgtc aaccatctca 600 tcattctttc cacctgggta ccaagctct tctgcttgg tgatgtact cctttttgca 660 gtggacacag gactatattt ctctgtgaag acaacattc gaagctcaac agagactgg 720 aaggaccata atttaatg gagaaggac cctcagaca atga 765 <210> 6 <211> 153 <212> PRT <213> Homo sapiens <400> 6 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Lew Lys Pro Lew Glu Glu Val Lew Asn Lew Wing Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 7 <211> 160 <212> PRT <213> Homo sapiens <400> 7 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr Gly Ser Glu Lys Asp Glu Leu 145 150 155 160

Claims

1. A combination therapeutic agent for treating cancer in a subject in need thereof, comprising a monoclonal antibody having a cytotoxic effect and genetically modified NK-92 cells, wherein the genetically modified NK-92 cells express a CD16 polypeptide having a valine at position 158 of the mature form of the CD16 polypeptide; and have been genetically modified using a plasmid expression vector to generate stable NK-92 cells that express interleukin-2 (IL-2) targeted to the endoplasmic reticulum (ER); and the expression vector further comprises, in a 5' to 3' direction: a polynucleotide encoding the CD16 polypeptide, an IRES, and a polynucleotide encoding the ER-targeted IL-2.

2. 2. The combination therapy of claim 1, wherein the CD16 polypeptide comprises a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 and comprises a valine at position 158.

3. The combination therapy of claim 1, wherein the CD16 polypeptide comprises the amino acid sequence of SEQ ID NO:

2.

4. 10. The combination treatment of claim 1, wherein the genetically modified NK-92 cells are further modified to express a suicide gene.

5. 5. The combination therapeutic agent of claim 4, wherein the suicide gene is inducible caspase-9.

6. The combination therapeutic agent according to any one of claims 1 to 5, wherein the cancer is multiple myeloma, leukemia, non-Hodgkin's lymphoma, metastatic breast cancer, gastric cancer, or non-small cell lung cancer.

7. The combination therapeutic agent according to any one of claims 1 to 6, wherein the monoclonal antibody is a naked monoclonal antibody that induces ADCC.

8. 8. The combination therapy of claim 7, wherein the monoclonal antibody is alemtuzumab, rituximab, trastuzumab, avelumab, daratumumab, or elotuzumab.

9. 9. The combination therapy of any one of claims 1-8, wherein the monoclonal antibody and the genetically modified NK-92 cells are administered to the subject simultaneously, or wherein the subject is administered the monoclonal antibody followed by treatment with the genetically modified NK-92 cells.

10. 10. The combination therapy of claim 9, wherein the monoclonal antibody is injected intravenously into the subject.

11. 11. The combination treatment of claim 9 or 10, wherein the genetically modified NK-92 cells are infused into the bone marrow.

Images