HK40077825A - Elimination of bcma-positive malignancies by car expressing nk cells - Google Patents

Description

CAR-expressing NK cell ablation of BCMA-positive malignancies

Sequence listing

The contents of an ASCII text file of the sequence Listing, named Seq _ listing _20200127_0019_ST25, size 59 KB, was created at 28 days 01/2020 and submitted electronically with the present application via the EFS-Web and incorporated by reference in its entirety.

Technical Field

The field of the invention is genetically modified immune competent cells expressing a Chimeric Antigen Receptor (CAR), in particular modified NK-92 cells expressing a CAR with an fcepsilon receptor gamma (fcepsilon RI gamma) signaling domain targeting the B Cell Maturation Antigen (BCMA) receptor.

Background

B Cell Maturation Antigen (BCMA) is a membrane receptor expressed only in plasmablasts and differentiated plasma cells, is essential for cell survival, but is not expressed in early B cells, hematopoietic stem cells, or other normal tissue cells. Surface expression of BCMA is increased in multiple myeloma plasma cell-like B cells and has also been detected in a subtype of hodgkin's disease. The very narrow expression profile of BCMA and its absence in normal tissues makes it a particularly promising therapeutic target, and a variety of therapeutic approaches have been and are being developed, such as antibody-drug conjugates (ADCs), bispecific T cell adaptors (bites), CAR-T cells (CAR-ts), bispecific molecules and bispecific/trispecific antibodies, and cancer vaccines. However, ADCs and bispecific/trispecific antibodies may be affected by poor tissue penetration. BiTE has a short serum half-life and has been shown to induce CRS and neurotoxicity. CAR-T cell therapy may be associated with serious adverse events (CRS and associated neurotoxicity). Due to the risk of GvHD associated with allogeneic T cell infusion, current CAR-T technology typically relies on engineered autologous T cells, which can lead to patient-to-patient variability and rule out patients who are unable to expand T cells.

Natural Killer (NK) cells are cytotoxic lymphocytes that constitute an important component of the innate immune system. In most cases, NK cells account for approximately 10% to 15% of circulating lymphocytes and bind to and kill target cells, including virally infected cells and many malignant cells. NK cell killing is not specific for a particular antigen and can occur without prior immunosensitization. Killing of target cells is typically mediated by cytolytic proteins, including perforin, granzyme and granulysin.

Autologous NK cells have been used as therapeutic entities. To do this, NK cells are isolated from the peripheral blood lymphocyte fraction of whole blood, expanded in cell culture to obtain a sufficient number of cells, which are then re-infused into the subject. Autologous NK cells have been shown to be of moderate effectiveness in ex vivo therapy and in vivo treatment at least in some cases. However, the isolation and growth of autologous NK cells requires a lot of time and cost. Furthermore, not all NK cells have a cytolytic effect, which further limits autologous NK cell therapy.

At least some of these difficulties can be overcome by using NK-92 cells, a cytolytic cancer cell line, which is found in the blood of subjects with non-hodgkin's lymphoma, and then immortalized in vitro (Gong et al, leukamia [ Leukemia ] 8. Although NK-92 cells are derivatives of NK cells, NK-92 cells lack the major inhibitory receptor possessed by normal NK cells, while retaining most of the activating receptor. However, NK-92 cells do not attack normal cells and do not elicit an immune rejection response unacceptable to humans. Because of these expected characteristics, NK-92 cells have been characterized in detail and explored for use as therapeutic agents for the treatment of certain cancers, for example, as described in WO 1998/049268 or US 2002/068044.

Phenotypic changes that distinguish tumor cells from normal cells derived from the same tissue are often associated with one or more changes in the expression of a particular gene product, including loss of normal cell surface components or acquisition of other cell surface components (antigens that are not detectable in the corresponding normal, non-cancerous tissue). Antigens expressed in neoplastic or tumor cells but not in normal cells or expressed at levels much higher than those found in normal cells are referred to as "tumor specific antigens" or "tumor associated antigens". Such tumor-specific antigens can be used as markers of tumor phenotype. Tumor specific antigens include cancer/testis specific antigens (e.g., MAGE, BAGE, GAGE, PRAME, and NY-ESO-1), melanocyte differentiation antigens (e.g., tyrosinase, melan-A/MART, gp100, TRP-1, and TRP-2), mutated or aberrantly expressed antigens (e.g., MUM-1, CDK4, β -catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V), and antigens expressed at higher levels in tumors (e.g., CD19 and CD 20).

Tumor specific antigens have been used as targets for cancer immunotherapy. One such therapy utilizes Chimeric Antigen Receptors (CARs) expressed on the surface of immune cells, including T cells and NK cells, to improve cytotoxicity against cancer cells. The CAR comprises a single chain variable fragment (scFv) linked to at least one intracellular signaling domain. The scFv recognizes and binds to an antigen on a target cell (e.g., a cancer cell) and triggers effector cell activation. The signaling domain contains an immunoreceptor tyrosine-based activation domain (ITAM), which is important for intracellular signaling through the receptor.

First generation CARs for T cells comprise a cytoplasmic signaling domain. For example, one version of the first generation CAR in a T cell includes a signaling domain from the fcepsilon receptor gamma (fcepsilon RI gamma) that comprises one ITAM, while another version comprises a signaling domain from CD3 ζ, wherein CD3 ζ comprises three ITAMs. In vivo and in vitro studies have shown that CD3 ζ CAR T cells are more effective at eradicating tumors than fcepsilon RI γ CAR T cells, e.g., haynes et al, 2001, j.immunology [ journal of immunology ]166:182 to 187; cartellieri et al, 2010, J.biomed and Biotech [ biomedical and Biotech ], vol.2010, article ID 956304). Further studies then suggest that some co-stimulatory signal is required for complete activation and proliferation of such recombinant T cells, and that second and third generation CARs incorporate multiple signaling domains into a single CAR to enhance the efficacy of recombinant CAR T cells. Because of their less than ideal effects reported in literature on T cells tested, the first generation CARs and fcepsilon RI γ signaling domains were largely abandoned, in turn using CD3 ζ in combination with one or more additional signaling domains (e.g., hermanson and Kaufman 2015, frontiers in Immunol. [ immune front ], volume 6, article 195).

Recently, selected CARs have also been expressed in NK cells. For example, CAR-modified NK-92 cells have used first generation CARs with only CD3 ζ intracellular signaling domains. These first generation CAR-NK cells have targeted multiple antigens, including CD19 and CD20 for B-cell lymphoma, erbB2 for breast, ovarian and squamous cell carcinoma, GD2 for neuroblastoma, and CD138 for multiple myeloma. Second generation CAR-NK cells from the NK-92 line have also been constructed against several antigens, including EpCAM for multiple cancers, HLA-A2EBNA3 complex for EB (Epstein-Barr) virus, CS1 for multiple myeloma, and ErbB2 for HER2 positive epithelial cancers. In the second generation NK-92CAR, the most common intracellular costimulatory domain used with CD3 ζ is CD28. However, since NK cells do not naturally express CD28, the potential role of the CD28 domain is not yet clear. Additional second generation CARs have combined a 4-1BB intracellular signaling domain with CD3zeta to improve NK cell persistence. Others compared the functionality of different intracellular domains on breast cancer cells using ErbB2scFv, CD28 and CD3 ζ, or 4-1BB and CD3 ζ fused to CD3 ζ alone. They found that both second generation constructs had better lethality compared to the first generation CAR, with 65% target lysis for CD28 and CD3 ζ, 62% for 4-1BB and CD3 ζ, and 51% for CD3 ζ alone. In recent studies, 4-1BB and CD28 intracellular domains were also compared for B cell malignancies using anti-CD 19CAR expressed on NK-92 cells. Still others have found that the CD3 zeta/4-1 BB construct is less effective than CD3 zeta/CD 28 in cell killing and cytokine production, highlighting the distinct effects of the CD28 and 4-1BB co-stimulatory domains.

Third generation NK-92 CARs were constructed from anti-CD 5scFv with CD3 ζ, CD28, and 4-1BB intracellular signaling domains and have demonstrated specific and potent anti-tumor activity against a variety of T cell leukemia and lymphoma cell lines as well as primary tumor cells. Such cells are also capable of inhibiting disease progression in xenografted mouse models of T-cell Acute Lymphoblastic Leukemia (ALL) cell lines and primary tumor cells (trans Res [ transformation medicine research ] 9 months 2017; 187. In a further example, WO 2016/201304 and WO 2018/076391 teach the use of a third generation CD3 ζ CAR expressed in NK cells and NK-92 cells.

However, NK cells (particularly NK-92 cells) are often difficult to genetically modify as evidenced by the multiple failures of engineering NK-92 cells to express Fc receptors. These difficulties are further exacerbated when NK-92 cells are transfected with multiple recombinant genes or relatively large recombinant nucleic acid payloads for heterologous expression. In addition, NK-92 cells also show a clear lack of predictability in the recombinant expression of foreign proteins (e.g., CD 16). At the functional level, most (if not all) CAR NK-92 cells require a high effector cell to target cell ratio, although targeted cytotoxicity is shown in most cases.

In addition, even if cytotoxic cells expressing the CAR are relatively effective in vitro, various suppressive or inhibitory factors associated with the tumor microenvironment in vivo may reduce or even eliminate the cytotoxicity of such cells. Also, despite the expression of CARs, such modified cells may not always be effective in targeting the tumor microenvironment.

Thus, although a large number of recombinant NK-92 cells are known in the art, all or almost all recombinant cells encounter various difficulties. Furthermore, although therapeutic approaches to BCMA are being developed, they also have various drawbacks. Thus, there remains a need for CAR-expressing NK-92 cells that express highly active CARs and target BCMA, are easy to culture in a simple and efficient manner, and have high cytotoxicity in the tumor microenvironment to eliminate BCMA positive malignancies in large quantities.

Disclosure of Invention

The inventors have found that Natural Killer (NK) cells (and in particular NK-92 cells) can be genetically modified to express a target-specific CAR and an additional recombinant protein to increase CAR-mediated cell killing, ADCC, and cytotoxicity and/or homing in the tumor microenvironment. In addition, such recombinant cells also express cytokines for stimulating autocrine growth, which advantageously facilitates clonal selection of modified cells.

In one aspect of the inventive subject matter, the inventors contemplate a genetically modified NK cell (and in particular NK-92 cell) that expresses: (i) A membrane-bound recombinant Chimeric Antigen Receptor (CAR) comprising in a single polypeptide chain an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, wherein the extracellular binding domain specifically binds to a BCMA receptor; (ii) recombinant CD16; (iii) an autocrine growth-stimulating cytokine; and (iv) optionally one of IL-12, TGF-beta trap, or homing receptor.

In some embodiments, the extracellular binding domain comprises a scFv and/or the signaling domain comprises an fcsri γ signaling domain. In a further contemplated aspect, the recombinant CD16 is CD16 ^158V The mutant, and/or autocrine growth-stimulating cytokine is IL-2 or IL-15, which may additionally comprise an endoplasmic retention sequence. In still further contemplated embodiments, the IL-12 is a single chain IL-12 heterodimer, and the TGF- β trap comprises a single chain dimer of the TGF- β receptor II extracellular domain and is preferably secreted. Preferred homing receptors include cell adhesion molecules, selectins, integrins, C-C chemokine receptors, or C-X-C chemokine receptors. For example, suitable homing receptorsReceptors including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, and CXCL 14.

Viewed from a different perspective, genetically modified NK cells are envisaged comprising a nucleic acid encoding: (i) A membrane-bound recombinant Chimeric Antigen Receptor (CAR) comprising in a single polypeptide chain an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, wherein the extracellular binding domain specifically binds to a BCMA receptor; (ii) recombinant CD16; (iii) an autocrine growth-stimulating cytokine; and (iv) optionally one of IL-12, TGF-beta trap, or homing receptor. With respect to NK cells and expressed proteins, the same considerations apply as described above.

In another aspect of the inventive subject matter, the inventors also contemplate a method of treating cancer in a patient in need thereof, wherein the patient receives a therapeutically effective amount of the genetically modified NK cell set forth herein, thereby treating cancer. Where desired, contemplated methods may further comprise the step of administering at least one additional therapeutic entity selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor targeting cytokines. For example, contemplated cancers include lung cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, or head and neck cancer. Most typically, about 1x10 is administered to the patient ⁸ To about 1x10 ¹¹ Individual cell/m ² The surface area of the patient's body. Thus, the inventors also contemplate the use of genetically modified NK cells in the treatment of cancer.

Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments and the accompanying drawings in which like numerals represent like components.

Drawings

Figure 1 is a schematic representation of an exemplary CAR construct. All CAR variants have an extracellular domain comprising an anti-BCMA receptor scFv region, a hinge region from CD8 (CD 8 hinge), a transmembrane domain from CD28 (CD 28 TM), and an intracellular domain from fcsry.

Fig. 2 is a schematic representation of the background of BCMA in normal body function in Plasma Cell (PC) and PC in Multiple Myeloma (MM) patients.

Figure 3 shows one embodiment of a schematic representation of a cloned BCMA t-haNK construct as described herein. BCMA VH and VL fragments can have SEQ ID nos: 39-42.

Figure 4 depicts exemplary flow cytometry analysis results of bcma.car and CD16 protein expression on aNK, haNK, and BCMA t-haNK cells, as shown in the upper panel. The lower panels show CAR and CD16 protein expression on BCMA t-haNK cell constructs with VH-VL scFv CAR and VL-VH scFv CAR, respectively.

Figure 5 depicts exemplary CAR cytotoxicity of BCMA t-haNK polyclonal cell populations. The left panel shows BCMA antigen specific cytotoxicity against SUP-B15BCMA +, while the right panel shows spontaneous cytotoxicity against K562.

Figure 6 depicts an exemplary ADCC assay for BCMA t-haNK polyclonal cell populations.

FIG. 7 depicts exemplary flow cytometry analysis results for BCMA t-haNK clones.

Figure 8 depicts exemplary BCMA CAR and CD16 expression analysis of BCMA t-haNK clones using flow cytometry. Clones 1-4 were from VH-VL pool #2, while clones 5-13 were from VH-VL pool #3.

Figure 9 depicts the results of another exemplary flow cytometry analysis of BCMA t-haNK clones.

Figure 10 depicts exemplary BCMA CAR and CD16 expression analysis of BCMA t-haNK clones using flow cytometry. Clone 15 was from VL-VH pool #1, clones 21-25 were from VL-VH pool #3, and clones 27-47 were from VH-VL pool #1.

Figure 11 depicts exemplary CARs and native cytotoxicity of BCMA tricistronic clones. The left panel shows the natural cytotoxicity of BCMA t-haNK cells on the K562 cell line. The right panel shows CAR-mediated cytotoxicity of BCMA t-haNK cells against SUP-B15BCMA + cell line.

FIG. 12 depicts exemplary ADCC activity of BCMA tricistronic selected clones on SUPB-15 BCMA-/CD20+ cell lines.

FIG. 13 depicts exemplary sequence validation of TGF-. Beta.trap/BCMA tetra-cistronic (quad) DNA clone # 11.

FIG. 14 depicts an exemplary expression analysis of BCMA TGF- β trap/BCMA tetra-cistronic (quad) t-haNK polyclonal cell populations.

Figure 15 depicts exemplary CAR and natural cytotoxicity of TGF- β trap/BCMA tetra-cistronic t-haNK cells. The left panel shows CAR-mediated cytotoxicity of TGF- β trap/BCMA Quad on the SUP-B15BCMA + cell line. The right panel shows the natural cytotoxicity of TGF-. Beta.trap/BCMA Quad on the K562 cell line.

FIG. 16 depicts exemplary ADCC of TGF-beta trap/BCMA tetra-cistronic (quad) t-haNK polyclonal cell population against the SUP B15-19KO/CD20 cell line.

Detailed Description

The inventors have found that genetically modified NK cells can have significant systemic toxicity, target-specific CAR-mediated cytotoxicity and ADCC (antibody-dependent cell-mediated cytotoxicity), and it was found that such genetically modified cells can be grown under autocrine growth stimuli that will also have a selective effect on successfully transfected cells. In addition, contemplated cells may be further expressed from recombinant nucleic acids that secrete IL-12 and/or TGF- β traps to reduce or eliminate the immunosuppressive environment. Additionally or alternatively, contemplated cells may express a homing receptor from a recombinant nucleic acid. Thus, modified cells can be generated by transfection with a tricistronic or a tetracistronic nucleic acid.

CARs combine an extracellular antigen-recognition moiety (typically derived from the variable domain of a particular antibody) with an intracellular signaling domain, which may be single or have additional co-stimulatory elements. Once a specific antigen is recognized, such CARs can trigger a cytolytic response. CAR is provided in FIG. 1 illustrative examples of (a).

The inventors have now developed CAR structures that specifically recognize BCMA. In a preferred embodiment, the CAR is a first generation CAR, as shown in figure 1. The CAR enables effector cells to specifically engage and kill BCMA-positive target cells. This DNA sequence encoding BCMA is typically integrated into the dicistronic CD16 (158V) -erlI-2 plasmid in a manner that allows for the simultaneous expression of three elements: (a) Car, which allows specific killing of BCMA positive cells; (b) CD16 (158V), which can achieve ADCC when combined with a therapeutic monoclonal antibody; and (c) erll 2, which allows the cells to expand and maintain the selective pressure of transgene expression in the absence of exogenous IL-2. CAR and CD16 (158V) are preferably separated by a 2A sequence that allows equimolar expression of 2 proteins from a single mRNA. CD16 (158V) and erli 2 are separated by an IRES sequence that allows for internal translation initiation.

The tricistronic plasmid designed as described above was transfected into aNK cells using electroporation. Stable expression of the tricistronic transgene was ensured by growth in the absence of IL-2. CAR and CD16 (158V) expression can be measured as described in more detail below. The inventors also showed that the cell population so produced was able to grow in the absence of IL-2 with > 80% purity and simultaneously express high levels of CAR and CD16.

Currently, specific targeting of BCMA-positive malignancies is achieved by antibody-drug conjugates (ADCs), bispecific T cell adaptors (bites), CAR T cells (CAR ts), bispecific molecules, and bispecific/trispecific antibodies. Antibody-based molecules (ADC, bispecific/trispecific Ab) have strong specificity but do not enter all parts of the body, especially the brain. BiTE has better diffusion properties than Ab, but exhibits a very short serum half-life, which may require their continuous infusion to achieve long-term activity. They have also been shown to be associated with significant risk of CRS and neurotoxicity. Furthermore, they are functionally dependent on the patient's cytotoxic cells (T cells and NK cells), which may exhibit highly impaired activity, depending on the patient's treatment history and patient-to-patient variability. The cell therapy products and methods disclosed herein overcome the above disadvantages because these products and methods are directed specifically to BCMA, allow passage through the cerebral blood barrier, and are independent of the patient's cells.

BCMA-targeted CAR-T cells have been designed, but currently rely on engineering and expansion of the patient's own T cells (autologous). This may lead to differences in expansion yield and T cell subset composition between patients. The disclosure herein overcomes these deficiencies because the cell therapy products presented herein include clonal cell lines without expansion limitations and without batch-to-batch differences and are therefore useful for all patients.

Allogeneic CAR-T therapy using engineered T cells from healthy donors requires further cell engineering to eliminate the risk of causing graft versus host disease (GvHD). The parental cell lines disclosed herein do not cause GvHD. CAR-T infusion often leads to serious adverse events such as CRS and neurotoxicity, which, although treatable by proper care, can still be fatal. Infusion of the parental cell line (aNK) of the present invention did not cause any serious adverse events. Successful response to CAR-T therapy is often accompanied by expansion and long-term persistence of CAR-T cells, which may cause long-term toxicity due to on-target/off-tumor activity. The cell therapy products disclosed herein do not transplant, do not have a long life span, and therefore do not cause long lasting toxicity.

It is further understood that fcsry-containing CARs have not been used in NK-92 cells, other NK cell lines, or endogenous NK cells, as other signaling domains (e.g., CD3 ζ) are considered more effective, particularly in combination with other signaling domains (in second and third generation CARs). Notably, the inventors have found that NK-92 cells expressing a first generation CAR comprising an intracellular domain from fcsri γ (the domain having only one ITAM domain) have the same or higher cytotoxic activity against cancer cells expressing an antigen recognized by the CAR compared to NK-92 cells expressing a CAR having a CD3zeta signaling domain with three ITAM domains, even wherein the ITAM domains are combined with other signaling domains (i.e., a second or third generation CAR). The inventors have also unexpectedly found that CARs comprising an intracellular domain from fcsri γ are expressed at higher levels on the NK-92 cell surface than other CARs, particularly CARs comprising a CD3zeta signaling domain. Cytotoxic effects can even be further enhanced by expression and secretion of IL-12 and/or by expression and secretion or presentation of TGF- β trap, IL-12 reducing immunosuppression in the tumor microenvironment (e.g., stimulation of exocrine via NK cells, modulation of MDSCs, etc.). Alternatively or additionally, cytotoxic effects may also be enhanced by expression of one or more homing receptors for attracting and/or retaining NK cells in the tumor microenvironment.

Thus, in some aspects of the inventive subject matter, a genetically modified NK-92 cell or NK cell line is engineered to express a Chimeric Antigen Receptor (CAR), in particular a CAR that specifically binds BCMA, on the cell surface. Most typically, the CAR comprises an intracellular domain from the fcepsilon receptor gamma (fcepsilon RI gamma), similar to the first generation construct shown in figure 1. However, in further contemplated embodiments, the CAR can further comprise a T Cell Receptor (TCR) CD3 ζ (CD 3 zeta) intracellular domain alone or in combination with additional components known from second and third generation CAR constructs (e.g., CD28, CD134, CD137, and/or ICOS). As will be readily appreciated, the CAR can be transiently or stably expressed by NK-92 cells from a recombinant DNA or RNA molecule. An exemplary CAR construct suitable for use herein is shown in figure 1.

Thus, in one aspect of the inventive subject matter, an NK cell, an NK-92 cell, or NK/NK-92 cell line expresses a Chimeric Antigen Receptor (CAR) on the surface of an NK-92 cell comprising an FceRI γ cytoplasmic domain (e.g., having the amino acid sequence of SEQ ID NO: 1). Alternatively or additionally, the CAR can further comprise a cytoplasmic domain of CD3 ζ (e.g., having the amino acid sequence of SEQ ID NO:7, which can be encoded by the nucleic acid of SEQ ID NO:8 (codon optimized)). In another aspect, an NK or NK-92 cell line transformed with a nucleic acid encoding a Chimeric Antigen Receptor (CAR) is contemplated. For example, preferred nucleic acids encode the cytoplasmic domain of Fc ε RI γ (e.g., comprising or consisting of SEQ ID NO: 2). As discussed in more detail below, the CAR can target BCMA.

In further contemplated embodiments, the NK, NK-92, or other NK cell may be modified to express an autocrine growth-stimulating cytokine or variant thereof. For example, a suitable cytokine may be transiently or stably expressed by a recombinant cell, and the cytokine may include an endoplasmic retention signal. Most typically (but not necessarily) the retention signal reduces the amount of cytokine secreted and thus can act as an endocrine growth stimulator without producing the systemic effects otherwise encountered with cytokine expression. Advantageously, the nucleic acid sequence encoding the autocrine growth stimulating cytokine or variant thereof is located on the same recombinant nucleic acid, typically as part of a tricistronic or a tetracistronic configuration. Thus, recombinant cells transfected with a tricistronic or tetracistronic nucleic acid can be easily selected and propagated because they are independent of exogenous IL-2 that would otherwise be required.

In addition, it is generally preferred that the genetically modified NK cells will also express recombinant CD16 or high affinity variants thereof (e.g., CD 16) ^158V ) To confer target-specific ADCC to the cell. Advantageously, this co-expression with the CAR is believed to further increase cytotoxicity to tumor cells. In this context, it is understood that unmodified NK cells do not typically express CD16 and exhibit cytotoxicity only as part of the innate immune system.

It has recently been discovered that various factors present in the tumor microenvironment may reduce or even completely eliminate the efficacy of immunotherapy. For example, immunosuppressive factors include certain cytokines (e.g., TGF- β) and various suppressor cells (e.g., MDSC), among others. Thus, the genetically modified NK cells may further express one or more recombinant proteins to counteract immunosuppressive factors. For example, as described in more detail below, genetically modified NK cells may express TGF- β traps to reduce TGF- β mediated effects in the tumor microenvironment, and/or may express IL-12 to inhibit MDSCs. Additionally or alternatively, the genetically modified NK cells may also express one or more homing receptors against the tumor microenvironment (or other desired tissue), thereby increasing the number of therapeutic cells in the tumor microenvironment, thereby enhancing the therapeutic effect.

In another aspect of the inventive subject matter, the inventors also contemplate a method of treating cancer in a patient in need thereof, the method comprising the steps of: administering to the patient a therapeutically effective amount of a modified NK/NK-92 cell or NK/NK-92 cell line engineered to express a Chimeric Antigen Receptor (CAR) as described herein. Viewed from a different perspective, the inventors also contemplate a modified NK/NK-92 cell or NK/NK-92 cell line expressing a Chimeric Antigen Receptor (CAR), preferably comprising a cytoplasmic domain of fcsry, for use in treating a tumor in a subject. In some embodiments, the use comprises administering to a subject an effective amount of a modified cell or cell line described herein to treat a tumor. In yet other embodiments, an in vitro method of killing a tumor cell is contemplated and may include the step of contacting the tumor cell with a modified NK-92 cell or NK-92 cell line described herein. In some embodiments, the modified NK-92 cell or NK-92 cell line expresses a CAR that binds to an antigen on a tumor cell. In some embodiments, the CAR preferably comprises an intracellular domain from the fcepsilon receptor gamma (fcepsilon RI gamma). Alternatively or additionally, the CAR comprises a T Cell Receptor (TCR) CD3 ζ (CD 3 zeta) intracellular domain.

With respect to suitable NK cells, it should be noted that all NK cells are considered suitable for use in the present invention, and thus include primary NK cells (preserved, expanded and/or fresh cells), immortalized secondary NK cells, autologous or heterologous NK cells (stock solution, preserved, fresh, etc.) and modified NK cells, as described in more detail below. In some embodiments, preferably, the NK cells are NK-92 cells. The NK-92 cell line is a unique cell line that was found to proliferate in the presence of interleukin 2 (IL-2) (see, for example, gong et al, leukemia [ Leukemia ] 8. NK-92 cells are cancerous NK cells with broad anti-tumor cytotoxicity and predictable yields after expansion in a suitable medium. Advantageously, NK-92 cells have high cytolytic activity against a variety of cancers.

Primitive NK-92 cell lines express CD56 ^bright 、CD2、CD7、CD11aCD45, and CD54 surface markers, but CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers are not shown. The growth of such NK-92 cells in culture depends on the presence of interleukin 2 (e.g., rIL-2), with doses as low as 1IU/mL being sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, and various other cytokines have not been detected, including IL-1 α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. NK-92 typically has higher cytotoxicity compared to primary NK cells even at relatively low effector to target (E: T) ratios (e.g., 1: 1). Representative NK-92 cells were deposited at the American Type Culture Collection (ATCC) and designated CRL-2407.

Thus, suitable NK cells may have one or more modified KIRs that are mutated to reduce or eliminate interaction with MHC class I molecules. Of course, it should be noted that one or more KIRs can also be deleted or inhibited from expression (e.g., by miRNA, siRNA, etc.). Most typically, more than one KIR will be mutated, deleted or silenced, and especially contemplated KIRs include those with two or three domains, with short or long cytoplasmic tails. Viewed from a different perspective, a modified, silenced, or deleted KIR will include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR DL5B, KIR DS 2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1. Such modified cells can be prepared using protocols well known in the art. Alternatively, such cells are also commercially available as aNK cells (' activated natural killer cells) from santksast (NantKwest) (see URL www.nantkwest.com). Such cells can then be further genetically modified to CARs, as described in more detail below.

In another aspect of the inventive subject matter, the genetically engineered NK cell can also be an NK-92 derivative modified to express a high affinity Fc γ receptor (CD 16). The sequences of high affinity variants of Fc γ receptors are well known in the art (see, e.g., blood 2009113, SEQ ID NOs. It is believed that expression of such receptors allows for specific targeting of tumor cells using antibodies specific for the patient's tumor cells (e.g., neo-epitopes), specific tumor types (e.g., HER2/neu, PSA, PSMA, etc.), or cancer-associated antibodies (e.g., CEA-CAM). Advantageously, such antibodies are commercially available and can be used in conjunction with cells (e.g., binding to Fc γ receptors). Alternatively, such cells are also commercially available as haNK cells from yersinit. Such cells can then be further genetically modified to CARs, as described in more detail below.

Thus, NK cells suitable for use herein include NK-92 cells (which may be transfected with a tricistronic or tetracistronic construct encoding CAR, CD16 or a variant thereof, a cytokine or a variant thereof, and optionally one of IL-12, TGF- β trap and homing receptor), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof or a cytokine or a variant thereof (which may be transfected with a nucleic acid encoding CAR and CD16 or a variant thereof or a cytokine or a variant thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof and a cytokine or a variant thereof (which may be transfected with a nucleic acid encoding CAR). As previously mentioned, any NK cell contemplated herein can express one of IL-12, TGF-beta trap, and a homing receptor from the same or different recombinant nucleic acid.

The genetic modification of NK cells contemplated herein can be performed in a variety of ways, and all known ways are considered suitable for use herein. Moreover, it should be recognized that NK cells can be transfected with DNA or RNA, and the particular choice of transfection will depend at least in part on the type of recombinant cell contemplated and the efficiency of transfection. For example, where stable transfection of NK cells is contemplated, linearized DNA may be introduced into the cells for integration into the genome. On the other hand, where transient transfection is contemplated, circular DNA or linear RNA (e.g., with poly A) may be used ⁺ Tail mRNA).

Similarly, it will be appreciated that the manner of transfection will depend, at least in part, on the type of nucleic acid used. Thus, viral transfection, chemical transfection, and mechanical transfection methods are all considered suitable for use herein. For example, in one embodiment, the vector described herein is a transient expression vector. The exogenous transgene introduced using such vectors is not integrated into the nuclear genome of the cell; thus, without vector replication, the exogenous transgene would degrade or dilute over time.

In another embodiment, the vector will preferably allow stable transfection of cells. In one embodiment, the vector allows for the incorporation of one or more transgenes into the genome of a cell. Preferably, such vectors have a positive selection marker, and suitable positive selection markers include any gene that allows cells to grow under conditions that would kill cells that do not express the gene. Non-limiting examples include antibiotic resistance, e.g., geneticin (Neo gene from Tn 5). Alternatively, or in addition, the vector is a plasmid vector. In one embodiment, the vector is a viral vector. As will be appreciated by those skilled in the art, any suitable carrier may be used and is well known in the art.

In still other embodiments, the cell is transfected with an mRNA encoding a protein of interest (e.g., CAR). Transfection of the mRNA results in transient expression of one or more proteins. In one embodiment, mRNA is transfected into NK-92 cells immediately prior to administration of the cells. In one embodiment, immediately "before" the cells are administered means between about 15 minutes and about 48 hours before administration. Preferably, mRNA transfection is performed from about 5 hours to about 24 hours prior to administration. In at least some embodiments described in more detail below, mRNA transfected NK cells resulted in unexpectedly consistent and strong expression of CAR on a high proportion of transfected cells. Moreover, such transfected cells also exhibit high specific cytotoxicity at a relatively low ratio of effector cells to target cells.

With respect to contemplated CARs, it is noted that NK/NK-92 cells will be genetically modified to express the CAR as a membrane bound protein, thereby exposing a portion of the CAR on the cell surface while maintaining a signaling domain in the intracellular space. Most typically, a CAR will include at least the following elements (in order): an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain (preferably, but not necessarily, an fceri γ domain).

In preferred embodiments, the cytoplasmic domain of the CAR comprises or consists of the signaling domain of fcsri γ. For example, the fcsri γ signaling domain comprises SEQ ID NO:1 or consists essentially of or consists of the amino acid sequence of 1. In some embodiments of the present invention, the, the fceri γ cytoplasmic domain is the only signaling domain. However, it is to be understood that additional elements may also be included, such as other signaling domains (e.g., CD28 signaling domain, CD3zeta signaling domain, 4-1BB signaling domain, etc.). These additional signaling domains may be located downstream of the fcsri γ cytoplasmic domain and/or upstream of the fcsri γ cytoplasmic domain.

In some embodiments, the fcsri γ signaling domain comprises a sequence identical to SEQ ID NO:1, or consists essentially of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

As described above, in some embodiments, the cytoplasmic domain of the CAR comprises a signaling domain of CD3zeta (CD 3 zeta). In one embodiment, the cytoplasmic domain of the CAR consists of the signaling domain of CD3 ζ. In one embodiment, the CD3zeta signaling domain comprises SEQ ID NO:7 or consists essentially of or consists of the amino acid sequence of seq id no. In some embodiments, the CD3zeta signaling domain comprises a sequence identical to SEQ ID NO:7, or consists essentially of, or consists of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

The CAR may comprise any suitable transmembrane domain. In one aspect, the CAR comprises a transmembrane domain of CD28. In one embodiment, the CD28 transmembrane domain comprises SEQ ID NO:4 or consists essentially of or consists of the amino acid sequence of seq id no. In some embodiments, the CD28 transmembrane domain comprises a sequence identical to SEQ ID NO:4, or consists essentially of, or consists of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto. In one embodiment, the transmembrane domain is selected from a CD28 transmembrane domain, a 4-1BB transmembrane domain, or an fcepsilonri γ transmembrane domain.

The CAR can comprise any suitable hinge region. In one aspect, the CAR comprises a hinge region of CD 8. In one embodiment, the CD8 hinge region comprises SEQ ID NO:3 or consists essentially of or consists of the amino acid sequence of 3. In one embodiment, the CD8 hinge region comprises an amino acid sequence identical to SEQ ID NO:3, or consists essentially of or consists of an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology thereto.

Most typically, but not necessarily, the extracellular binding domain of the CAR will be an scFv or other natural or synthetic binding moiety that specifically binds to the B cell maturation antigen. Particularly suitable binding moieties include small antibody fragments with single, dual or multiple target specificity, beta barrel domain binders, phage display fusion proteins, and the like. However, in other embodiments, suitable extracellular binding domains will specifically bind to tumor-specific antigens, tumor-associated antigens, or patient-specific and tumor-specific antigens. In a particularly preferred embodiment, the antigen envisaged comprises BCMA. Other contemplated antigens may include CD19, CD20, GD2, CD30, FAP, CD33, CD123, PD-L1, IGF1R, CSPG, or B7-H4. As non-limiting examples, in US 2013/0189268; WO 1999024566A1; US 7098008; and WO 2000020460, each of which is incorporated herein by reference in its entirety.

Thus, contemplated CARs will typically have a structure with an extracellular binding domain (directly) coupled to a hinge domain (directly) coupled to a transmembrane domain, which in turn is (directly) coupled to a (e.g., fceri γ) signaling domain. In still further contemplated aspects, contemplated CARs can further comprise one or more signaling domains in addition to or in place of the fcsri γ signaling domain, and in particular contemplated signaling domains comprise a CD3zeta signaling domain, a 4-1BB signaling domain, and a CD28 signaling domain. In one example, contemplated CARs can comprise a polypeptide having the sequence of SEQ ID NO:38 or a nucleotide sequence identical to SEQ ID NO:38 a BCMA CAR having a sequence of at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity. In another example, contemplated CARs can comprise a VH scFv having BCMA VL-VH and SEQ ID NO:39, or the nucleic acid sequence of SEQ ID NO:41, or a polypeptide sequence substantially identical to SEQ ID NO:39 or SEQ ID NO:41 a BCMA CAR having a sequence of at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity. In another example, contemplated CARs can comprise a VH-VL scFv having BCMA and SEQ ID NO:40, or the nucleic acid sequence of SEQ ID NO:42, or a polypeptide sequence substantially identical to SEQ ID NO:40 or SEQ ID NO:42 a BCMA CAR having a sequence of at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity.

In another example, contemplated CARs may thus include a polypeptide having the sequence of SEQ ID NO:39-42 or a sequence identical to SEQ ID NO:39 or SEQ ID NO:40 or SEQ ID NO:41 or SEQ ID NO:42, coupled to a hinge domain (e.g., a CD8 hinge shown in SEQ ID NO: 3), which in turn is coupled to a transmembrane domain (e.g., a CD28TM shown in SEQ ID NO: 4), which is coupled to a signaling domain (e.g., an Fc epsilon RI gamma signaling domain shown in SEQ ID NO:1, a CD28 signaling domain shown in SEQ ID NO:5, a 4-1BB signaling domain shown in SEQ ID NO:6, and/or a CD3zeta signaling domain shown in SEQ ID NO: 7).

With respect to the construction of contemplated CARs, it should be recognized that CARs can be engineered in a variety of ways as described below, for example, WO 2014/039523; US 2014/0242701; US 2014/0274909; US 2013/0280285 and WO 2014/099671, each of which is incorporated herein by reference in their entirety.

From a different perspective, contemplated CARs target antigens associated with a particular cancer type, and more particularly cancers that overexpress BCMA, such as multiple myeloma or hodgkin's lymphoma.

In still further contemplated aspects, the NK cell can be further genetically modified to express one or more cytokines, and particularly autocrine growth-stimulating cytokines, to provide a selectable marker, wherein the cytokine and the CAR encode the same recombinant nucleic acid and/or render the recombinant cell independent of exogenous IL-2. Thus, in some embodiments, the NK-92 cell is modified to express at least one cytokine. In particular, at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21 or variants thereof. In a preferred embodiment, the cytokine is IL-2 or a variant thereof, and particularly preferred variants include an endoplasmic retention signal (e.g., human IL-2 as shown in SEQ ID NO:9, optionally with an ER retention signal as shown in SEQ ID NO: 10). For example, the IL-2 gene is cloned and expressed with a signal sequence that directs IL-2 to the endoplasmic reticulum. This allows IL-2 to be expressed at levels sufficient for autocrine activation, but without extracellular release of IL-2 (e.g., exp Hematol [ Experimental hematology ]2005 month 2; 33 (2): 159-64). Alternatively, the expression of the cytokine (and in particular IL-15) may also be such that the amount of cytokine expressed is sufficient to provide an autocrine growth signal to the recombinant cell, but also to allow at least some of the expressed IL-15 to be released from the cell, thereby providing an immunostimulatory signal. For example, the use of a signal peptide and endoplasmic retention sequence of human IL-15 sequences to achieve such expression. Exemplary DNA and protein sequences of endoplasmic retention IL-15 are set forth in SEQ ID NO:36 and SEQ ID NO: shown at 37.

Contemplated cells may also express suicide genes, as expected. The term "suicide gene" refers to a transgene that allows negative selection of cells expressing the suicide gene. 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 in cases where the recombinant gene causes a mutation that results in uncontrolled cell growth or the cell itself is capable of such growth. A number of 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 E.coli (Escherichia coli) gpt gene, and the E.coli Deo gene. Typically, the proteins encoded by suicide genes have no adverse effect on the cells, but kill the cells in the presence of specific compounds. Thus, suicide genes are typically part of the system.

In one embodiment, the suicide gene is active in NK-92 cells. In one embodiment, the suicide gene is a Thymidine Kinase (TK) gene. The TK gene may be a wild type or mutant TK gene (e.g. TK30, TK75, sr39 TK). Ganciclovir (ganciclovir) can be used to kill cells expressing the TK protein. In another embodiment, the suicide gene is cytosine deaminase, which is toxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez et al, "cytokine amine adaptive vector and 5-fluorocytose selective reduction noise cells 1 mileon-fold w hen the y conjugate acidic cells: a potential purposing method for autologous transplantation, [ cytosine deaminase adenoviral vectors and 5-fluorocytosine selectively reduce breast cancer cells by 100 ten thousand fold when contaminating hematopoietic cells: potential methods of clearance for autografting ] "Blood. [ Blood ]1998, 7/15; 92 (2): 672-82. In a further embodiment, the suicide gene is cytochrome P450, which is toxic in the presence of ifosfamide or cyclophosphamide. See, e.g., toutati et al, "A suicide Gene therapy combining the improvement of cyclophosphamide cytotoxicity and the reduction of an anti-tumor immune response ]" Curr Gene Ther. [ current Gene therapy ]2014;14 (3): 236-46. In yet another embodiment, the suicide gene is iCasp9.Di Stasi, (2011) "inductive apoptosis as a safety switch for adaptive cell therapy" [ safety switch for induced apoptosis as adoptive cell therapy ] "N Engl J Med [ new england journal of medicine ]365:1673-1683. See also Morgan, "Live and Let Die: a New Suicide Gene Therapy Moves to the clinical [ live or dead: a novel suicide gene Therapy entry clinical ] "Molecular Therapy [ Molecular Therapy ] (2012); 20:11-13.iCasp9 induces apoptosis in the presence of small molecule AP 1903. AP1903 is a biologically inert small molecule that has been shown to be well tolerated in clinical studies and has been used in the context of adoptive cell therapy.

Where the modified NK cells are further engineered to express IL-12, it is generally preferred that IL-12 is expressed as a single chain heterodimer, where the p35 and p40 components are linked together by a flexible linker (in either orientation, p 35-linker-p 40 or p 40-linker-p 35). Furthermore, it is generally preferred that the heterodimer will be secreted and may therefore include a signal peptide for protein export. Thus, a suitable IL-12 sequence as contemplated herein may comprise a sequence identical to SEQ ID NO:26 (p 35 nucleotide sequence) or SEQ ID NO:28 (p 40 nucleotide sequence) a nucleic acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. IL-12 as contemplated herein may also comprise a sequence identical to SEQ ID NO:27 (p 35 amino acid sequence, isoform 1 precursor) or SEQ ID NO:29 (p 40 amino acid sequence, precursor) having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Thus, the single chain p40_ p35 sequence of IL-12 may comprise a sequence identical to SEQ ID NO:30, or may comprise a polypeptide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:31, or a polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. Most preferably, but not necessarily, the nucleic acid encoding the IL-12 single-chain heterodimer will be part of a polycistronic nucleic acid sequence (e.g., present as a tetra-cistron sequence with CAR, CD16, and erll-2).

Where the modified NK cells are further engineered to express a TGF- β trap, it is generally preferred that the TGF- β trap is a single chain dimer of the extracellular domain of the TGF β RII molecule, and most preferably comprises a single chain dimer of the extracellular domain of TGF- β receptor II. Thus, a suitable TGF- β trap consists of a sequence identical to SEQ ID NO:32 (TGFBRII extracellular domain) or SEQ ID NO:34 (TGF-beta trap sequence) a polynucleotide sequence encoding at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. TGF- β traps contemplated herein may further comprise a sequence identical to SEQ ID NO:33 (TGFBRII extracellular domain) or SEQ ID NO:35 (TGF-beta trap sequence) amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Other suitable TGF- β traps include those described in mol. Canc. Ther. [ molecular cancer therapeutics ]2012, volume 11 (7), 1477-1487. Most preferably, but not necessarily, the nucleic acid encoding the TGF- β trap will be part of a polycistronic nucleic acid sequence (e.g., present as a tetra-cistronic sequence with CAR, CD16, and erll-2).

Where the modified NK cell is further engineered to express a homing receptor, it should be noted that the term "homing receptor" refers to a receptor that will activate a cellular pathway that directly or indirectly leads to cell migration to a target cell or tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissue via the high endothelial venules. Homing receptors can also be used by cells to migrate toward chemical gradient sources, such as chemokine gradient sources. Examples of homing receptors include G protein-coupled receptors, such as chemokine receptors, including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, and DARC; a cytokine receptor; cell adhesion molecules, such as selectins, including L-selectin (CD 62L); integrins, such as α 4 β 7 integrin, LPAM-1 and LFA-1. Homing receptors typically bind to cognate ligands on target tissues or cells. In some embodiments, the homing receptor binds to addressin (addressin) on the endothelium of the venules, e.g., mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1).

In some exemplary embodiments, the chemokines and homing receptors contemplated herein may comprise a sequence identical to SEQ ID NO:13 (CCR 7 nucleotide sequence), or SEQ ID NO:14 (CCL 19 nucleotide sequence), or SEQ ID NO:15 (CCL 21 nucleotide sequence) or SEQ ID NO:16 (CXCR 2 nucleotide sequence), or SEQ ID NO:17 (CXCR 2 amino acid sequence), or SEQ ID NO:18 (CXCL 4 nucleotide sequence), or SEQ ID NO:19 (CXCL 4 amino acid sequence), or SEQ ID NO:20 (CD 62L nucleotide sequence), or SEQ ID NO:21 (CD 62L amino acid sequence), or SEQ ID NO:22 (IL-8 nucleotide sequence), or SEQ ID NO:23 (IL-8 amino acid sequence), or SEQ ID NO:24 (CXCL 1 nucleotide sequence), or SEQ ID NO:25 (CXCL 1 amino acid sequence) a polypeptide sequence or polynucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Most preferably, but not necessarily, the nucleic acid encoding the homing receptor will be part of a polycistronic nucleic acid sequence (e.g., as a tetra-cistronic sequence with CAR, CD16, and erll-2).

It should be noted, of course, that all recombinant proteins can be expressed from a single recombinant sequence. However, it is generally preferred that in the case of expression of multiple recombinant sequences (e.g., CAR, CD16, cytokine, TGF- β trap), the coding regions may be arranged in polycistronic units having at least two or at least three or at least four coding regions encoding the recombinant protein. Thus, the transgene can be engineered into an expression vector by any mechanism known to those skilled in the art. Where multiple transgenes are inserted into a cell, the transgenes may be engineered into the same expression vector or into different expression vectors. In some embodiments, the cells are transfected with mRNA encoding the transgenic protein to be expressed. In some embodiments, the cells are transfected with DNA encoding the transgenic protein to be expressed. The transgene, mRNA and DNA can be introduced into NK-92 cells using any transfection method known in the art, including but not limited to infection, viral vector, electroporation, lipofection, nuclear transfection or "gene gun".

Thus, in a preferred embodiment, it is noted that genetically modified NK cells (particularly cells expressing CAR and CD16 or variants thereof) will exhibit three different cell killing patterns: general cytotoxicity mediated by activating receptors (e.g., NKG2D receptor), ADCC mediated by antibodies binding to target cells, and CAR-mediated cytotoxicity. Therapeutic effects in the tumor microenvironment can be further enhanced, if desired, via expression and secretion of IL-12 (e.g., as a single chain heterodimer), via expression and presentation/secretion of TGF- β traps (e.g., as a single chain dimer of the TGF- β receptor II ectodomain), or via expression of homing receptors (e.g., CCR 7). It is apparent that contemplated genetically modified cells can be used to treat a variety of diseases, and in particular, a variety of cancers and viral infections in which diseased cells present disease-specific or disease-associated antigens. Thus, the inventors contemplate methods of treating patients using modified NK or NK-92 cells as described herein. In one embodiment, the patient has a cancer (e.g., a tumor), and the modified NK-92 cell or cell line expresses a CAR that is specific for an antigen expressed on the surface of a cell from the cancer or tumor. As described above, in some embodiments, the cancer may be multiple myeloma or hodgkin's lymphoma.

Contemplated modified NK or NK-92 cells can be administered to an individual in absolute numbers of cells. For example, about 1000 cells/injection up to about 100 hundred million cells/injection may be administered to an individual, e.g., about, at least about, or up to about 1x10 per injection 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.) modified NK-92 cells, or any range between any two values, inclusive. In other embodiments, the modified NK-92 cells can be administered to an individual in a relative amount of cells, e.g., can be administered to the individual from about 1000 cells per kilogram of the individual to up to about 100 hundred million cells per kilogram of the individual, e.g., about, at least about, or up to about 1x10 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.) modified NK-92 cells, or any range between any two values, inclusive. In other embodiments, the total dose may be in m ² Body surface area, including per m ² About 1X10 ¹¹ 、1×10 ¹⁰ 、1×10 ⁹ 、1×10 ⁸ 、1×10 ⁷ Or any range between any two values (inclusive). Human averages about 1.6 to about 1.8m ² . In a preferred embodiment, about 10 to about 30 million NK-92 cells are administered to a patient.

The modified NK-92 cells and optional other anti-cancer agents may be administered once to a patient having cancer or an infected virus during therapy, or may be administered multiple times, e.g., 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 values (inclusive).

In one embodiment, the agent that triggers death of the modified NK cells is administered to the patient where the modified NK cells express a suicide gene. In one embodiment of the present invention, the agent is administered at a time point after administration of the modified NK cells sufficient for the NK cells to kill the target cells.

In one embodiment, the modified NK cells are irradiated prior to administration to the patient. Irradiation of NK cells is described, for example, in U.S. patent No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, modified NK cells that have not been engineered to express a suicide gene are irradiated.

Furthermore, it will be understood that contemplated methods of treatment will also include the administration of other immunotherapeutic entities, and particularly preferred immunotherapeutic entities include viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., non-pyrogenic e.coli expressing one or more cancer-specific antigens), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), and antibodies (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell transplants (e.g., allogeneic or autologous), and tumor-targeting cytokines (e.g., NHS-IL12, IL-12 conjugated to tumor-targeting antibodies or fragments thereof).

After reading this description, it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, not all embodiments of the invention are described herein. It is to be understood that the embodiments presented herein are presented by way of example only, and not limitation. Also, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below.

Before aspects of the present subject matter are disclosed and described in greater detail, it is to be understood that the aspects described below are not limited to particular compositions, methods of making such compositions, or uses thereof, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

Examples of the invention

The following examples are for illustrative purposes only and should not be construed as limiting the claimed invention. Those skilled in the art may use a variety of alternative techniques and procedures that will similarly allow one to successfully carry out the intended invention.

Example 1: b Cell Maturation Antigen (BCMA) CAR constructs

Unless otherwise indicated in the examples below, the inventors used NK-92 cells for total transfection of cells with recombinant nucleic acid. Furthermore, and also unless otherwise indicated, all recombinant nucleic acids are linearized DNA constructs encoding a tricistronic or a tetracistronic configuration.

B cell maturation antigen (BCMA or BCM), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF 17), is a protein encoded by the TNFRSF17 gene in humans. This receptor is preferentially expressed in mature B lymphocytes and has been shown to bind specifically to the tumor necrosis factor (ligand) superfamily and cause activation of NF- κ B and MAPK 8/JNK. In normal body functions, BCMA expression in Plasma Cells (PCs) supports survival of long-lived PCs, production of antibodies, and class switching of immunoglobulins. In Multiple Myeloma (MM), BCMA promotes the proliferation and survival of MM cells, associated with an immunosuppressive BM microenvironment, and elevated levels of sbbcma are associated with disease progression and poor outcome. This background for BMCA is further illustrated in fig. 2.

To generate bcma. Car t-haNK cells, recombinant DNA molecules were assembled as schematically depicted in fig. 3, wherein the tricistronic configuration included a sequence encoding bcma. Car, followed by a P2A sequence, followed by a sequence encoding CD16 (or CD 16) ^158V ) Followed by an IRES sequence element upstream of the sequence encoding erll-2. Car, CD16, then results from the formation of primary transcripts of the tricistronic nucleic acid ^158V And erll-2 as recombinant polypeptides. FIG. 3 depicts schematically and exemplarily two linearized recombinant nucleic acids for transfecting NK-92 cells. Car constructs can comprise a VH chain followed by a VL, or a VL followed by a VH, as shown in figure 3. Typically, VH and VL are linked through a 15 amino acid linker (e.g., (G) ₄ S) ₃ ) And (4) connecting.

All transfections are general following the standard protocol: NK-92 cells were grown in X-Vivo10 medium (Longsha (Lonza), basel, switzerland) supplemented with 5% human AB serum (Valley Biomedical, wechester, varginia) and 500IU/mL IL-2 (Prospec, israel Lei Huo Watt). Using Neon ^TM Electroporation apparatus (Life Technologies, calif.) according to the manufacturer's NK-92 cell parameters (1250V, 10ms,3 pulses) in 100 μ 1 volumes per 10 μ 1 ⁶ 2 μ g DNA per cell cells were electroporated with either tricistronic or tetracistronic DNA. The electroporated cells were transferred to IL-2 free medium (supra).

Selection of recombinant cells and clones is based on continuing cell culture in the absence of exogenously added IL-2, as all recombinant cells include recombinant autocrine growth promoting cytokines (e.g., IL-2, erlI-2, IL-15, or erlI-15).

Car and CD16 expression on NK-92 cell surface by flow cytometry using biotinylated anti-scFv antibodies and labeling with allophycocyaninStreptavidin and a fluorescently labeled anti-CD 16 antibody. Car and CD16 expression (polyclonal) are shown in figure 4, as well as aNK (no CD16 expressed, no bcma.car expressed) and haNK (CD 16 expressed, but no bcma.car expressed) controls. Car and CD16 polyclonal cell cultures as can be readily seen from figure 4 ^158V All had significant and strong expression. Furthermore, it should be noted that BCMA. Car is expressed at relatively the same level regardless of whether the BCMA construct is used in VL-VH or VH-VL orientation.

As can be seen from the results depicted in fig. 5, bcma.car t-haNK cells have strong and target-specific CAR-mediated cytotoxicity. The left panel of figure 5 is CAR-mediated toxicity to SUP-B15BCMA + target cells, while the right panel shows spontaneous cytotoxicity to K562 target cells. As shown in FIG. 6, SUP-B15 is used ^HER-2-/CD20+ Cells were tested for ADCC with rituximab as target-specific antibody and herceptin as control antibody. Again, as can be seen from the exemplary results depicted in fig. 6, the polyclonal bcma. Car t-haNK cells have a strong and target-specific ADCC approaching about 55%, without significant off-target toxicity. The results shown in fig. 4-6 are for the transfection pool of bcma. Car t-haNK cells (pools VH-VL #1 to 3 and VL-VH #1 to 3). After successful results from these pools, the inventors cloned from bcma. Car t-haNK cells in these pools. The results of these clones are described below.

As shown in fig. 7 and fig. 8, respectively, a number of individual clones from bcma. Car t-haNK cell populations were prepared after dilution propagation. Figure 7 depicts CAR expression on transfection pools VH-VL #1 to 3 and VL-VH # 2. Clones were prepared from these transfection pools by limiting dilution and CAR expression analysis of the clones was performed as shown in figure 8. Expression analysis was performed via FACS using the same procedure as described above. aNK and hanK cells were used as controls. Car and CD16 as can be seen from the exemplary results of fig. 8, it was observed that most of the individual clones had significant and strong bcma ^158V Expression of both. However, some clones (# 2, #4, and # 14) did not have significant and strong bcma ^158V Or expression of both.

In another example, single clones from bcma. Car t-haNK cell populations were also prepared after dilution propagation as shown in figure 9 and figure 10, respectively. FIG. 9 depicts CAR expression on transfection pools VL-VH #1 to 3 and VH-VL #2, respectively. Expression analysis was performed via FACS using the same procedure as described above. aNK and hanK cells were used as controls. Again, as can be seen from the exemplary results of fig. 10, it was observed that all clones obtained by limiting dilution had significant and strong bcma.car and CD16 ^158V Expression of both. Note that clone #15 was from VL-VH pool #1; clones 21-25 were from VL-VH pool #3; and clones 27-47 were from VH-VL pool #1.

CAR t-haNK tricistronic cell clones were tested for CAR and native cytotoxicity as shown in figure 11. As can be seen from fig. 11, most BCMA tricistronic clones maintained the CAR and native cytotoxicity seen in the transfection pool. Car t-haNK tricistronic cloning ADCC was performed on SUP-B15-19KO/CD20 cells as shown in figure 12. Figure 12 demonstrates that most BCMA tricistronic clones maintained ADCC as seen in the transfection pool. In addition, the growth rates of the clones are shown in table 1 below. As can be seen from table 1, the growth rate of most BCMA tricistronic clones was comparable to that of aNK cells.

Table 1.

	Average growth hr (-1)
		aNK	38.5
BCMA.21	40.7
		BCMA.22	41.1
BCMA.24	56.4
		BCMA.25	51.2
BCMA.27	41.3
		BCMA.29	44.9
BCMA.33	43.3
		BCMA.34	58.1
BCMA.35	34.9
		BCMA.46	42.5

Example 2: tetra cistronic constructs

Although the above examples were performed with a tricistronic construct, it will be appreciated that the same construct may also be implemented in a tetracistronic construct to express IL-12, TGF- β traps or homing receptors, thereby reducing immunosuppression in the tumor microenvironment and enriching the tumor microenvironment with NK cells so modified. An exemplary tetra-cistronic construct is schematically shown in fig. 13, in which the nucleic acid sequence encoding the TGF- β trap is located upstream of the tri-cistronic construct as discussed above. The P2A sequence may be located between the TGF- β trap and the CAR to ensure coordinated expression while producing different proteins. The expression of the TGF-. Beta.trap was then tested in ELISA to determine that the tetra-cistronic construct did produce a functional TGF-. Beta.trap. Notably, the expression level of TGF- β trap was significant in all recombinant clones with the tetra-cistronic construct.

Car and CD16 expression on the cell surface of NK-92 cells with a tetra-cistronic vector was determined by flow cytometry using biotinylated anti-scFv antibodies and streptavidin labeled with allophycocyanin and fluorescently labeled anti-CD 16 antibodies. Car and CD16 expression (polyclonal) are shown in figure 14, as well as aNK (no CD16 expressed, no bcma.car expressed) and haNK (CD 16 expressed, but no bcma.car expressed) controls.

As can be seen from the results depicted in fig. 15, bcma.car t-haNK cells with a tetra-cistronic vector have strong and target-specific CAR-mediated cytotoxicity. Use of SUP-B15 ^19KO/CD20+ Cells were tested for ADCC with rituximab as target-specific antibody and herceptin as control antibody. Again, as can be seen from the exemplary results depicted in fig. 16, polyclonal bcma. Car t-haNK cells have close to about 60% strong and target-specific ADCC, with no significant off-target toxicity.

Car constructs as presented herein can be combined with monoclonal antibodies targeting different antigens expressed on solid tumors, thereby reducing the risk of relapse due to antigen loss. In addition, the above-described tetracistronic vectors confer enhanced homing properties to chemokine receptors. Car t-haNK cells described herein can be further modified to express molecules that additionally manipulate/down-regulate inhibitory factors of the tumor microenvironment. For example, expression of chemokine receptor CXCR4 on the cell surface of bcma. Car t-haNK cells can direct cells to the bone marrow compartment where most MM cells reside. TGF β trap molecules that secrete molecules that can sequester TGF β in the tumor microenvironment or bone marrow compartment can counteract the immunosuppressive effects of TGF β. It will also counteract the stimulatory effect of TGF β on osteoclasts, which are responsible for the deterioration of bone tissue (a marker of MM progression).

Of course, it will be recognized that for all nucleic acid sequences provided herein, the corresponding encoded proteins are also within the scope of the explicit contemplation herein. Likewise, for all amino acid sequences, the corresponding nucleic acid sequences are also within the scope of the present disclosure (using any codons).

All patent applications, publications, references, and sequence accession numbers cited in this specification are hereby incorporated by reference in their entirety.

Unless defined otherwise, 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.

In this specification and the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

the terminology used 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" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

It is to be understood that all values (e.g., pH, temperature, time, concentration, amount, and molecular weight, including ranges) recited herein include normal variations in measured values encountered by one of ordinary skill in the art. Accordingly, the values described herein include variations of +/-0.1% to 10%, e.g., +/-0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. It is to be understood that, although not always explicitly stated, all numbers may be preceded by the term "about". Thus, the term about includes variations of +/-0.1% to 10%, e.g., +/-0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. It is also to be understood that the reagents described herein are exemplary only, and that equivalents thereof are known in the art, although not always explicitly stated.

As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed include the endpoints of the ranges, and all values between the endpoints of the ranges. All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range may be readily considered to be fully described, and the same range may be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those of skill in the art, all languages, such as "at most," "at least," and the like, include the recited numerical values and refer to ranges that may be subsequently subdivided into sub-ranges as discussed above. Finally, as understood by those of skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to a group having 1,2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1,2, 3, 4, or 5 cells, and so forth.

It is also to be understood that, although not always explicitly indicated, the reagents described herein are exemplary only and that equivalents thereof are known in the art.

"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.

The term "comprising" is intended to mean that the compositions and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean to exclude other elements of any significance to the combination. For example, a composition consisting essentially of the elements defined herein shall not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "consisting of" shall mean excluding more than trace amounts of other ingredients and the substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this disclosure.

As used herein, "immunotherapy" refers to NK-92 cells that use either modified or unmodified, naturally occurring or modified NK cells or T cells, alone or in combination, whether used alone or in combination, and are capable of inducing cytotoxicity upon contact with a target cell.

As used herein, a "Natural Killer (NK) cell" is a cell of the immune system that kills a target cell without stimulation by a specific antigen, and is not limited according to the Major Histocompatibility Complex (MHC) class. The target cell may be a tumor cell or a cell carrying a virus. NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.

The term "endogenous NK cells" is used to refer to NK cells derived from a donor (or patient), as opposed to the NK-92 cell line. Endogenous NK cells are typically a heterogeneous population of cells in which NK cells have been enriched. Endogenous NK cells may be used for autologous or allogeneic treatment of a patient.

The term "NK-92" refers to natural killer cells derived from a unique cell line described by Gong et al (1994) which is highly potent and owned by Guigsite corporation (hereinafter "NK-92 ^TM Cells "). Immortalized NK cell lines were originally obtained from patients with non-hodgkin's lymphoma. Unless otherwise stated, the term "NK-92 ^TM "refers to the original NK-92 cell line as well as to NK-92 cell lines that have been modified (e.g., by introduction of a foreign gene). NK-92 ^TM Cells and exemplary and non-limiting modifications thereof are described in U.S. Pat. nos. 7,618,817;8,034,332;8,313,943;9,181,322;9,150,636; and published U.S. application Ser. No. 10/008,955, each of which is incorporated by reference herein in its entirety, and including wild type NK-92 ^TM 、NK-92 ^TM -CD16、NK-92 ^TM -CD16-γ、NK-92 ^TM -CD16-ζ、NK-92 ^TM -CD16(F176V)、NK-92 ^TM MI and NK-92 ^TM And CI. NK-92 cells are known to those of ordinary skill in the art, and such cells are readily available from NantKwest, inc.

The term "aNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al (1994), owned by Guidiford corporation (hereinafter "aNK ^TM Cells "). The term "haNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al (1994), owned by guy yerite, modified to express CD16 on the cell surface (hereinafter "CD16+ NK-92) ^TM Cell "or"Cells "). In some embodiments, CD16+ NK-92 ^TM Cells contain a high affinity CD16 receptor on the cell surface. The term "taNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al (1994), owned by guy et al, modified to express a chimeric antigen receptor (hereinafter "CAR modified NK-92) ^TM Cell "or"Cells "). The term "t-haNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al (1994), owned by kuntkitty corporation, modified to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter "CAR modified CD16+ NK-92) ^TM Cell "or" t-haNK ^TM Cells "). In some embodiments, t-haNK ^TM Cells express the high affinity CD16 receptor on the cell surface.

"modified NK-92 cells" refers to NK-92 cells expressing exogenous genes or proteins, such as Fc receptors, CAR, cytokines (e.g., IL-2 or IL-15), and/or suicide genes. In some embodiments, the modified NK-92 cell comprises a vector encoding a transgene, such as an Fc receptor, a CAR, a cytokine (e.g., IL-2 or IL-15), and/or a suicide gene. In one embodiment, the modified NK-92 cell expresses at least one transgenic protein.

As used herein, "unirradiated NK-92 cells" are NK-92 cells that have not been irradiated. Irradiation renders the cells incapable of growth and proliferation. It is envisioned that NK-92 cells will be irradiated at other time points prior to treatment device or patient treatment, as the time between irradiation and infusion should not exceed four hours to maintain optimal activity. Alternatively, NK-92 cells may be prevented from proliferating by another mechanism.

As used herein, "inactivation" of NK-92 cells renders them incapable of growing. Inactivation may also be associated with the death of NK-92 cells. It is envisioned that NK-92 cells may be inactivated after having been effectively cleared of an ex vivo sample of cells associated with pathology in therapeutic applications, or left in a mammal for a sufficient period of time to effectively kill many or all of the target cells in the body. As a non-limiting example, inactivation may be induced by administration of an inactivating agent to which NK-92 cells are sensitive.

As used herein, the terms "cytotoxic" and "cytolytic" when used to describe the activity of effector cells (e.g., NK-92 cells) are intended to be synonymous. Typically, cytotoxic activity involves killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis more particularly refers to the activity of an effector to lyse the plasma membrane of a target cell, thereby disrupting its physical integrity. This results in killing of the target cells. Without wishing to be bound by theory, it is believed that the cytotoxic effect of NK-92 cells is due to cytolysis.

The term "killing" with respect to a cell/cell population is intended to include any type of manipulation that will result in the death of that cell/cell population.

The term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contributes to the protective function of immune cells by binding to a portion of an antibody called the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates phagocytic or cytotoxic activity of the cell via antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcR is classified according to the type of antibody it recognizes. For example, fc-gamma receptors (Fc γ R) bind IgG class antibodies. FC γ RIII-a is a low affinity FC receptor that binds to IgG antibodies and activates ADCC. FC γ RIII-A is commonly found on NK cells. NK-92 cells do not express FC γ RIII-A. The Fc-epsilon receptor (Fcepsilon R) binds to the Fc region of IgE antibodies.

As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen-binding domain fused to an intracellular signaling domain. The CAR can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen-binding domain is an scFv specific for an antigen found on a cell of interest. Based on the specificity of the scFv domain, CAR-expressing NK-92 cells are targeted to cells expressing certain antigens on the cell surface. The scFv domains can be engineered to recognize any antigen, including tumor-specific antigens and virus-specific antigens. For example, CD19 CARs recognize CD19, CD19 being a cell surface marker expressed by certain cancers.

The term "tumor-specific antigen" as used herein refers to an antigen that is present on a cancer cell or neoplastic cell but is not detectable on normal cells derived from the same tissue or lineage as the cancer cell. As used herein, a tumor-specific antigen also refers to a tumor-associated antigen, i.e., an antigen that is expressed at a higher level on cancer cells as compared to normal cells derived from the same tissue or lineage as the cancer cells.

As used herein, the term "virus-specific antigen" refers to an antigen that is present on a virus-infected cell but is not detectable on normal cells derived from the same tissue or lineage as the virus-infected cell. In one embodiment, the virus-specific antigen is a viral protein expressed on the surface of an infected cell.

The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modification of the nucleotide structure, if present, may be performed before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of a polynucleotide of the invention encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (a); cytosine (C); guanine (G); thymine (T); when the polynucleotide is RNA, uracil (U) replaces thymine. Thus, the term "polynucleotide sequence" is a letter representation of a polynucleotide molecule.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

As used herein, "percent identity" refers to sequence identity between two peptides or between two nucleic acid molecules. The percent identity can be determined by comparing the position in each sequence, which can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules have identity at that position. Homologous nucleotide sequences include sequences that encode naturally occurring allelic variants and mutations 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 that contain conservative amino acid substitutions and polypeptides having the same binding and/or activity. 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. In some embodiments, the nucleotide or amino acid sequence has at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or more percent identity to a sequence described herein. In some embodiments, the nucleotide or amino acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence described herein. Percent identity can be determined, for example, by the Gap program (Wisconsin Sequence Analysis Package) for UNIX, 8 th edition, genetics Computer Group (Genetics Computer Group), university Research Park (University Research Park, madison division, wisconsin) using default settings using Smith and Waterman algorithms (adv.appl.math. [ advanced mathematics ],1981,2, 482-489). Algorithms suitable for determining percent sequence identity include the BLAST and BLAST 2.0 algorithms as described in Altschul et al (nuc. Acids Res. [ nucleic acids research ] 25-3389-402, 1977), and Altschul et al (j.mol.biol. [ journal of molecular biology ] 215. Software for BLAST analysis is publicly available through the National Center for Biotechnology Information (see ncbi. Nlm. Nih. Gov website). BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10,m =5,n = -4, and a comparison of the two strands. For amino acid sequences, the BLASTP program defaults to a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, proc. Natl. Acad. Sci. Usa [ proceedings of the american academy of science ]89, 10915, 1989) alignment (B) of 50 and an expectation (E) of 10, m =5, n = 4.

In some embodiments, the nucleic acid sequence is codon optimized for expression in a particular species, e.g., mouse sequences (expression of a protein encoded by a codon optimized nucleic acid sequence) may be codon optimized for expression in humans. Thus, in some embodiments, a codon optimized nucleic acid sequence has at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or more percent identity to a nucleic acid sequence described herein. In some embodiments, a codon optimized nucleic acid sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence described herein.

The term "expression" refers to the production of a gene product (e.g., a protein). The term "transient" when referring to expression means that the polynucleotide is not incorporated into the genome of the cell. The term "stable" when referring to expression means that the polynucleotide is incorporated into the genome of the cell, or that a positive selection marker (i.e., an exogenous gene expressed by the cell that has a benefit under certain growth conditions) is used to maintain the expression of the transgene.

The term "cytokines (or cytokins)" refers to the general class of biomolecules that affect cells of the immune system. Exemplary cytokines 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.

As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising an intact replicon, such that the vector can be replicated when placed in a permissive cell, e.g., by a transformation process. A vector may replicate in one cell type (e.g., bacterial), but has limited or no ability to replicate in another cell (e.g., mammalian). The vector may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers, such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, in some cases in liposomes; and the use of a ternary complex comprising a virus and polylysine-DNA. In one embodiment, the vector is a viral vector, e.g., an adenovirus. Viral vectors are well known in the art.

As used herein, the term "targeted" when referring to protein expression is intended to include, but is not limited to, directing a protein or polypeptide to an appropriate destination within a cell or outside thereof. Targeting is typically achieved by a signal or targeting peptide that is a stretch of amino acid residues in the polypeptide chain. These signal peptides may be located anywhere within the polypeptide sequence, but are typically located at the N-terminus. The polypeptide may also be engineered to have a signal peptide at the C-terminus. The signal peptide can direct the polypeptide to undergo extracellular cleavage, localization to the plasma membrane, golgi apparatus, endosomes, endoplasmic reticulum, and other cellular compartments. For example, a polypeptide having a particular amino acid sequence at its C-terminus (e.g., KDEL) is retained in or transported back into the ER lumen.

As used herein, the term "targeting" when referring to a target of a tumor refers to the ability of NK-92 cells to recognize and kill tumor cells (i.e., target cells). Herein, the term "targeted" refers to the ability of a CAR expressed by, for example, NK-92 cells to recognize and bind to a cell surface antigen expressed by a tumor.

The term "transfection" as used herein refers to the insertion of a nucleic acid into a cell. Any means of allowing nucleic acids to enter the cell may be used for transfection. The DNA and/or mRNA may be transfected into the cell. Preferably, the transfected cells express the gene product (i.e., protein) encoded by the nucleic acid.

The term "suicide gene" refers to a transgene that allows for the negative selection of cells expressing the transgene. Suicide genes are used as a safety system, allowing cells expressing the gene to be killed by the introduction of a selective agent. A variety of suicide gene systems have been identified, including the herpes simplex virus Thymidine Kinase (TK) gene, cytosine deaminase gene, varicella zoster virus thymidine kinase gene, nitroreductase gene, E.coli gpt gene, and E.coli (E.coli) Deo gene (see, e.g., yazawa K, fisher W E, brunicardi F C: current progress in suicide gene therapy for cancer, recent J.Surg. [ J.world J. [ J.surgery ] 2002.7 months; 26 (7): 783-9). In one embodiment, the suicide gene is a Thymidine Kinase (TK) gene. The TK gene may be a wild type or mutant TK gene (e.g. TK30, TK75, sr39 TK). Ganciclovir can be used to kill cells expressing the TK protein.

The claims (modification according to treaty clause 19)

1. A genetically modified NK cell expressing:

(i) A membrane-bound recombinant Chimeric Antigen Receptor (CAR) comprising in a single polypeptide chain an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, wherein the extracellular binding domain specifically binds to a BCMA receptor;

(ii) Recombinant CD16;

(iii) An autocrine growth-stimulating cytokine; and

(iv) Optionally one of IL-12, TGF-beta trap, or homing receptor.

2. The genetically modified NK cell of claim 1, wherein the NK cell is an NK-92 cell.

3. The genetically modified NK cell of any one of the preceding claims, wherein the extracellular binding domain comprises a scFv.

4. The genetically modified NK cell of claim 1, wherein the signaling domain comprises an fcsri γ signaling domain or a CD3 ζ signaling domain.

5. The genetically modified NK cell of claim 1, wherein the recombinant CD16 is CD16 ^158V And (3) mutants.

6. The genetically modified NK cell of claim 1, wherein the autocrine growth stimulating cytokine is IL-2 or IL-15.

7. The genetically modified NK cell of claim 6, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence.

8. The genetically modified NK cell of claim 1, wherein the IL-12 is a single chain IL-12 heterodimer.

9. The genetically modified NK cell of claim 1, wherein the TGF- β trap comprises a single-chain dimer of the TGF- β receptor II extracellular domain.

10. The genetically modified NK cell of claim 9, wherein the TGF- β trap is a secreted form of a single chain dimer of the TGF- β receptor II extracellular domain.

11. The genetically modified NK cell of claim 1 wherein the homing receptor is a cell adhesion molecule, a selectin, an integrin, a C-C chemokine receptor, or a C-X-C chemokine receptor.

12. The genetically modified NK cell of claim 11, wherein the homing receptor is selected from the group consisting of: receptors for CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, and CXCL 14.

13. A genetically modified NK cell comprising:

a recombinant nucleic acid encoding:

(i) A membrane-bound recombinant Chimeric Antigen Receptor (CAR) comprising in a single polypeptide chain an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, wherein the extracellular binding domain specifically binds to a BCMA receptor;

(ii) Recombinant CD16;

(iii) An autocrine growth-stimulating cytokine; and

(iv) Optionally one of IL-12, TGF-beta trap, or homing receptor.

14. The genetically modified NK cell of claim 13, wherein the recombinant nucleic acid is a polycistronic RNA.

15. The genetically modified NK cell of claim 13, wherein the NK cell is an NK-92 cell.

16. The genetically modified NK cell of any one of claims 13-15, wherein the extracellular binding domain comprises a scFv.

17. The genetically modified NK cell of claim 13, wherein the signaling domain comprises an fcsri γ signaling domain or a CD3 ζ signaling domain.

18. The genetically modified NK cell of claim 13, wherein the recombinant CD16 is CD16 ^158V And (3) mutants.

19. The genetically modified NK cell of claim 13, wherein the autocrine growth stimulating cytokine is IL-2 or IL-15.

20. The genetically modified NK cell of claim 19, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence.

21. The genetically modified NK cell of claim 13, wherein the IL-12 is a single chain IL-12 heterodimer.

22. The genetically modified NK cell of claim 13, wherein the TGF- β trap comprises a single-chain dimer of the TGF- β receptor II extracellular domain.

23. The genetically modified NK cell of claim 22, wherein the TGF- β trap is a secreted form of a single chain dimer of the TGF- β receptor II extracellular domain.

24. The genetically modified NK cell of claim 13 wherein the homing receptor is a cell adhesion molecule, a selectin, an integrin, a C-C chemokine receptor, or a C-X-C chemokine receptor.

25. The genetically modified NK cell of claim 24, wherein the homing receptor is selected from the group consisting of: receptors for CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, and CXCL 14.

26. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of any one of the genetically modified NK cells of claim 1 or claim 13, thereby treating the cancer.

27. The method of claim 26, further comprising the step of administering at least one additional therapeutic entity selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor targeting cytokines.

28. The method of claim 26, wherein the cancer is multiple myeloma or hodgkin's lymphoma.

29. The method of claim 26, wherein about 1x10 is administered to the patient ⁸ To about 1x10 ¹¹ Cell/m ² The surface area of the patient's body.

30. Use of the genetically modified NK cell of claim 1 or claim 13 for the treatment of cancer.

Claims (30)

1. A genetically modified NK cell expressing:

(i) A membrane-bound recombinant Chimeric Antigen Receptor (CAR) comprising in a single polypeptide chain an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, wherein the extracellular binding domain specifically binds to a BCMA receptor;

(ii) Recombinant CD16;

(iii) An autocrine growth-stimulating cytokine; and

(iv) Optionally one of IL-12, TGF-beta trap, or homing receptor.

2. The genetically modified NK cell of claim 1, wherein the NK cell is an NK-92 cell.

3. The genetically modified NK cell of any one of the preceding claims, wherein the extracellular binding domain comprises a scFv.

4. The genetically modified NK cell of any one of the preceding claims, wherein the signaling domain comprises an fceri γ signaling domain or a CD3 ζ signaling domain.

5. The genetically modified NK cell of any one of the preceding claims, wherein the recombinant CD16 is CD16 ^158V And (3) mutants.

6. The genetically modified NK cell of any one of the preceding claims, wherein the autocrine growth-stimulating cytokine is IL-2 or IL-15.

7. The genetically modified NK cell of claim 6, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence.

8. The genetically modified NK cell of any one of the preceding claims, wherein the IL-12 is a single chain IL-12 heterodimer.

9. The genetically modified NK cell of any one of the preceding claims, wherein the TGF- β trap comprises a single-chain dimer of the TGF- β receptor II extracellular domain.

10. The genetically modified NK cell of claim 9, wherein the TGF- β trap is a secreted form of a single chain dimer of the TGF- β receptor II extracellular domain.

11. The genetically modified NK cell of any one of the preceding claims, wherein the homing receptor is a cell adhesion molecule, a selectin, an integrin, a C-C chemokine receptor, or a C-X-C chemokine receptor.

12. The genetically modified NK cell of claim 11, wherein the homing receptor is selected from the group consisting of: receptors for CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, and CXCL 14.

13. A genetically modified NK cell comprising:

a recombinant nucleic acid encoding:

(i) A membrane-bound recombinant Chimeric Antigen Receptor (CAR) comprising in a single polypeptide chain an extracellular binding domain, a hinge domain, a transmembrane domain, and a signaling domain, wherein the extracellular binding domain specifically binds to a BCMA receptor;

(ii) Recombinant CD16;

(iii) An autocrine growth-stimulating cytokine; and

(iv) Optionally one of IL-12, TGF-beta trap, or homing receptor.

14. The genetically modified NK cell of claim 13, wherein the recombinant nucleic acid is a polycistronic RNA.

15. The genetically modified NK cell of claim 13, wherein the NK cell is an NK-92 cell.

16. The genetically modified NK cell of any one of claims 13-15, wherein the extracellular binding domain comprises a scFv.

17. The genetically modified NK cell of any one of claims 13-16, wherein the signaling domain comprises an fceri γ signaling domain or a CD3 ζ signaling domain.

18. The genetically modified NK cell of any one of claims 13-17, wherein the recombinant CD16 is CD16 ^158V And (3) mutants.

19. The genetically modified NK cell of any one of claims 13-18, wherein the autocrine growth-stimulating cytokine is IL-2 or IL-15.

20. The genetically modified NK cell of claim 19, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence.

21. The genetically modified NK cell of any one of claims 13-20, wherein the IL-12 is a single chain IL-12 heterodimer.

22. The genetically modified NK cell of any one of claims 13-21, wherein the TGF- β trap comprises a single-chain dimer of the TGF- β receptor II extracellular domain.

23. The genetically modified NK cell of claim 22, wherein the TGF- β trap is a secreted form of a single chain dimer of the TGF- β receptor II extracellular domain.

24. The genetically modified NK cell of any one of claims 13-23, wherein the homing receptor is a cell adhesion molecule, a selectin, an integrin, a C-C chemokine receptor, or a C-X-C chemokine receptor.

25. The genetically modified NK cell of claim 24, wherein the homing receptor is selected from the group consisting of: receptors for CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC, and CXCL 14.

26. A method of treating cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of any one of the genetically modified NK cells of claims 1-25, thereby treating the cancer.

27. The method of claim 26, further comprising the step of administering at least one additional therapeutic entity selected from the group consisting of: viral cancer vaccines, bacterial cancer vaccines, yeast cancer vaccines, N-803, antibodies, stem cell transplants, and tumor targeting cytokines.

28. The method of claim 26 or 27, wherein the cancer is multiple myeloma or hodgkin's lymphoma.

29. The method of any one of claims 26-28, wherein about 1x10 is administered to the patient ⁸ To about 1x10 ¹¹ Cell/m ² The surface area of the patient's body.

30. The genetically modified NK cell of any one of claims 1-25 for use in the treatment of cancer.