CN115315510A - Chimeric antigen receptor-modified NK-92 cells targeting EGFR superfamily receptors - Google Patents

Abstract

The present invention provides genetically modified NK cells expressing chimeric antigen receptors that target EGFR superfamily receptors. The CAR may comprise an intracellular domain of fcsri γ, and the additional recombinant protein expressed by the genetically modified NK cells is CD16, an autocrine growth-stimulating cytokine, and optionally one of IL-12, a TGF- β trap, or a homing receptor. Also described are methods for treating a patient having or suspected of having a disease treatable with NK-92 cells, such as cancer, comprising administering the genetically modified NK cells to the patient.

Description

NK-92 Cells Modified by Chimeric Antigen Receptors Targeting EGFR Superfamily Receptors

sequence listing

The contents of the ASCII text file of the 61 kb Sequence Listing, entitled Seq_listing_20200127_b_ST25, was created on January 27, 2020 and filed electronically with this application via EFS-Web, and is incorporated by reference in its entirety.

technical field

The field of the invention is genetically modified immunocompetent cells expressing chimeric antigen receptors (CARs), in particular CARs with the Fcε receptor gamma (FcεRIγ) signaling domain targeting EGFR superfamily receptors Modified NK-92 cells.

Background technique

Natural killer (NK) cells are cytotoxic lymphocytes that constitute an important component of the innate immune system. In most cases, NK cells make up about 10%-15% of circulating lymphocytes and bind and kill target cells, which include virus-infected cells and many malignant cells. NK cell killing is not specific for a particular antigen and can occur without prior immune sensitization. Targeted cell killing is often mediated by cytolytic proteins, including perforin, granzyme, and granulysin.

Autologous NK cells have been used as therapeutic entities. To this end, NK cells are isolated from the peripheral blood lymphocyte fraction of whole blood, expanded in cell culture to obtain sufficient numbers of cells, and then reinfused into the subject. Autologous NK cells have been shown to be moderately effective in ex vivo and in vivo therapy, 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 are cytolytic, further limiting autologous NK cell therapy.

At least some of these difficulties can be overcome by using NK-92 cells, a cytolytic cancer cell line that is found in the blood of subjects with non-Hodgkin's lymphoma , and then immortalized in vitro (Gong et al., Leukemia 8:652-658 (1994)). Although NK-92 cells are derivatives of NK cells, NK-92 cells lack the major inhibitory receptors that normal NK cells have, while retaining most of the activating receptors. However, NK-92 cells will not attack normal cells, nor will they trigger immune rejection that is unacceptable to the human body. Because of these expected properties, NK-92 cells have been characterized in detail and explored as therapeutic agents for the treatment of certain cancers, eg as described in WO 1998/049268 or US 2002/068044.

The phenotypic changes that distinguish tumor cells from normal cells derived from the same tissue are usually associated with one or more changes in the expression of specific gene products, including loss of normal cell surface components or gain of other cell surface components (in the corresponding undetectable antigens in normal non-cancerous tissues). Antigens that are expressed in neoplastic or tumor cells but not in normal cells, or that are expressed in neoplastic cells at levels much higher than those found in normal cells, are called "tumor-specific antigens" or "tumor-specific antigens." related antigens". Such tumor-specific antigens can be used as markers of tumor phenotype. Tumor-specific antigens include cancer/testis-specific antigens (eg, MAGE, BAGE, GAGE, PRAME, and NY-ESO-1), melanocyte differentiation antigens (eg, tyrosinase, Melan-A/MART, gp100, TRP-1 and TRP-2), mutated or aberrantly expressed antigens (such as MUM-1, CDK4, β-catenin, gp100-in4, p15, and N-acetylglucosaminyltransferase V) and at higher levels in tumors Antigens expressed (eg CD19 and CD20).

Tumor-specific antigens have been used as targets for cancer immunotherapy. One such therapy exploits chimeric antigen receptors (CARs) expressed on the surface of immune cells, including T cells and NK cells, to improve cytotoxicity against cancer cells. A CAR comprises a single-chain variable fragment (scFv) linked to at least one intracellular signaling domain. scFvs recognize and bind antigens on target cells (eg, cancer cells) and trigger effector cell activation. The signaling domain contains an immunoreceptor tyrosine-based activation domain (ITAM), which is important for intracellular signaling through the receptor.

The first-generation CARs for T cells contained a cytoplasmic signaling domain. For example, one version of the first-generation CAR in T cells included a signaling domain from Fcε receptor gamma (FcεRIγ), which contained an ITAM, while another version contained a signaling domain from CD3ζ, which contained three ITAM. In vivo and in vitro studies have shown that CD3ζ CAR T cells are more effective in eradicating tumors than FcεRIγ CAR T cells (eg, Haynes et al., 2001, J. Immunology 166:182-187; Cartellieri et al. 2010, J. Biomed and Biotech [Journal of Biomedicine and Biotechnology], Volume 2010, Article ID 956304). Then further studies showed that the full activation and proliferation of such recombinant T cells required certain co-stimulatory signals, and the second and third generation CARs combined multiple signaling domains into a single CAR to enhance the recombinant CAR T cells. cell efficacy. Because of their suboptimal literature-reported effects in the T cells tested, first-generation CAR and FcεRIγ signaling domains were largely abandoned in favor of CD3ζ in combination with one or more additional signaling domains (e.g., Hermanson et al. and Kaufman 2015, Frontiers in Immunol. Volume 6, Article 195).

More recently, selected CARs have also been expressed in NK cells. For example, CAR-modified NK-92 cells have used first-generation CARs with only the CD3ζ intracellular signaling domain. 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, and 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 various cancers, HLA-A2 EBNA3 complex for EB (Epstein-Barr) virus, for CS1 for multiple myeloma, and ErbB2 for HER2-positive epithelial cancer. In second-generation NK-92 CARs, the most common intracellular co-stimulatory domain used with CD3ζ was CD28. However, since NK cells do not naturally express CD28, the potential role of the CD28 domain is unclear. Additional second-generation CARs have combined the 4-1BB intracellular signaling domain with CD3ζ to improve NK cell persistence. Others compared the functionality of different intracellular domains on breast cancer cells using ErbB2 scFv, CD28 and CD3ζ, or 4-1BB and CD3ζ fused to CD3ζ alone. They found that both second-generation constructs had better lethality than the first-generation CARs, with 65% target cleavage for CD28 and CD3ζ, 62% cleavage for 4-1BB and CD3ζ, and 62% cleavage for CD3ζ alone. 51% on target. In a recent study, 4-1BB and CD28 intracellular domains were also compared for B-cell malignancies using an anti-CD19 CAR expressed on NK-92 cells. Still others found that the CD3ζ/4-1BB construct was less effective in cell killing and cytokine production than CD3ζ/CD28, highlighting the distinct roles of the CD28 and 4-1BB co-stimulatory domains.

The third-generation NK-92CAR was constructed from an anti-CD5 scFv with CD3ζ, CD28, and 4-1BB intracellular signaling domains and has demonstrated specificity against multiple T-cell leukemia and lymphoma cell lines as well as primary tumor cells and potent antitumor activity. Such cells were also able to suppress disease progression in xenograft mouse models of T-cell acute lymphoblastic leukemia (ALL) cell lines and primary tumor cells (Transl Res. 2017 Sep;187:32 -43). In a further example, WO 2016/201304 and WO 2018/076391 teach the use of third generation CD3ζ CAR expressed in NK cells and NK-92 cells.

However, NK cells, especially NK-92 cells, are often difficult to genetically modify, as evidenced by multiple failures to engineer 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 exhibited a marked lack of predictability in recombinant expression of foreign proteins (eg, CD16). At the functional level, most (if not all) CAR NK-92 cells require a highly efficient target cell ratio, despite exhibiting on-target cytotoxicity in most cases.

Furthermore, even though CAR-expressing cytotoxic cells are relatively potent in vitro, various repressors or suppressors associated with the tumor microenvironment in vivo can reduce or even eliminate the cytotoxicity of such cells. Similarly, despite expressing CARs, such modified cells may not always effectively target the tumor microenvironment.

Thus, although a large number of recombinant NK-92 cells are known in the art, all or nearly all of them suffer from various difficulties. Therefore, there remains a need for CAR-expressing NK-92 cells that express highly active CARs in significant amounts, can be easily cultured in a simple and efficient manner, and are highly cytotoxic in the tumor microenvironment.

Contents of the invention

The inventors have discovered that natural killer (NK) cells, particularly NK-92 cells, can be genetically modified to express target-specific CARs and additional recombinant proteins to increase the presence of cells in and/or homing to the tumor microenvironment. Environmental CAR-mediated cell killing, ADCC and cytotoxicity. In addition, such recombinant cells also express cytokines for autocrine growth stimulation, which advantageously facilitates clonal selection of the modified cells.

In one aspect of the present subject matter, the inventors contemplate a genetically modified NK cell (and in particular an NK-92 cell) expressing (i) a membrane-bound recombinant chimeric antigen receptor (CAR), which The membrane-bound recombinant chimeric antigen receptor comprises an extracellular binding domain, a hinge domain, a transmembrane domain and a signaling domain in a single polypeptide chain, wherein the extracellular binding domain specifically binds to EGFR superfamily receptors; (ii) recombinant CD16; (iii) an autocrine growth stimulating cytokine; and (iv) optionally one of IL-12, a TGF-beta trap, or a homing receptor.

In some embodiments, the extracellular binding domain comprises a scFv, and/or the signaling domain comprises a FcεRIγ signaling domain. Most typically, the EGFR superfamily receptor is HER-2 or EGFR. In a further contemplated aspect, the recombinant CD16 is a CD16158V mutant, and/or the autocrine growth stimulating cytokine is IL-2 or IL-15, which may additionally comprise an endoplasmic retention sequence. In still further contemplated embodiments, IL-12 is a single chain IL-12 heterodimer, and the TGF-beta well comprises a single chain dimer of the TGF-beta receptor II ectodomain, 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 receptors include CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6 , DARC and CXCL14 receptors.

Viewed from a different perspective, genetically modified NK cells are envisioned that include nucleic acid encoding (i) a membrane-bound recombinant chimeric antigen receptor (CAR) in A single polypeptide chain includes an extracellular binding domain, a hinge domain, a transmembrane domain and a signaling domain, wherein the extracellular binding domain specifically binds to EGFR superfamily receptors; (ii) recombinant CD16; (iii) an autocrine growth stimulating cytokine; and (iv) optionally one of IL-12, a TGF-beta trap, or a homing receptor. With regard to NK cells and the proteins expressed, the same considerations above apply.

In another aspect of the inventive subject matter, the inventors also contemplate methods of treating cancer in a patient in need thereof, wherein the patient receives a therapeutically effective amount of the genetically modified NK cells presented herein, thereby treating the cancer. The envisaged method may also include, if desired, 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 transplantation and tumor targeting cytokines. For example, contemplated cancers include lung, breast, thyroid, esophageal, gastric, gastroesophageal, or head and neck cancers. Most typically, about 1 x ¹⁰⁸ to about ¹ x 1011 cells/ ^m2 of patient body surface area are administered to the patient. Accordingly, the inventors also contemplate the use of genetically modified NK cells in the treatment of cancer.

Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of the preferred embodiments together with the accompanying drawings, wherein like reference numerals refer to like components.

Description of drawings

Figure 1 is a schematic diagram of an exemplary CAR construct. All CAR variants have cells comprising an anti-EGFR superfamily receptor scFv region (e.g., αEGFR-scFv, αHER2-scFv), a hinge region from CD8 (CD8 hinge), and a transmembrane domain from CD28 (CD28TM) ectodomain. The intracellular domain of CD19CAR was varied as indicated.

Figure 2 is a schematic representation of an exemplary tricistronic recombinant nucleic acid encoding a HER2.CAR, followed by a P2A sequence, followed by a sequence encoding a CD16 high affinity variant. The CD16 sequence is followed by the IRES sequence, which in turn is followed by the sequence encoding erIL-2.

3 is another schematic diagram of an exemplary tricistronic recombinant nucleic acid encoding HER2.CAR, a high-affinity variant of CD16, and erIL-2 for generating recombinant NK cells.

Figure 4 depicts exemplary FACS results from polyclonal pools of cells transfected with tricistronic recombinant nucleic acids displaying CD16 and HER2.CAR expression.

Figure 5 depicts exemplary results of CAR-mediated cytotoxicity of NK cells transfected with the tricistronic recombinant nucleic acid of Figure 3.

FIG. 6 depicts exemplary results of ADCC of NK cells transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

Figure 7 depicts exemplary FACS results of NK cells transfected with tricistronic recombinant nucleic acids exhibiting CD16 and HER2.CAR expression from selected clonal cell lines.

FIG. 8 depicts exemplary results of natural cytotoxicity of NK cells from selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

FIG. 9 depicts exemplary results of CAR-mediated cytotoxicity of NK cells from selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

FIG. 10 depicts further exemplary results of CAR-mediated cytotoxicity of NK cells from selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

FIG. 11 depicts further exemplary results of ADCC of NK cells from selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

FIG. 12 depicts exemplary results of erIL-2 release from NK cells of selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

FIG. 13 depicts exemplary in vivo results of MDA-MB-453 tumor volume changes using NK cells transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

14 depicts exemplary in vivo results of BT-474 tumor volume changes using NK cells transfected with the tricistronic recombinant nucleic acid of FIG. 3 .

Figure 15 is a schematic diagram of an exemplary tricistronic recombinant nucleic acid encoding EGFR2.CAR, followed by a P2A sequence, followed by a sequence encoding a high affinity variant of CD16. The CD16 sequence is followed by the IRES sequence, which in turn is followed by the sequence encoding erIL-2.

16 is another schematic diagram of an exemplary tricistronic recombinant nucleic acid encoding EGFR2.CAR, a high affinity variant of CD16, and erIL-2 for generating recombinant NK cells.

Figure 17 depicts exemplary FACS results from polyclonal pools of cells transfected with tricistronic recombinant nucleic acids exhibiting CD16 and EGFR2.CAR expression.

18 depicts exemplary results of polyclonal collections of cells for natural cytotoxicity of NK cells transfected with the tricistronic recombinant nucleic acid of FIG. 16 .

19 depicts exemplary results of a polyclonal pool of cells for CAR-mediated cytotoxicity of NK cells transfected with the tricistronic recombinant nucleic acid of FIG. 16 .

FIG. 20 depicts exemplary results for a cellular polyclonal pool of ADCC of NK cells transfected with the tricistronic recombinant nucleic acid of FIG. 16 .

Figure 21 depicts exemplary results of native cytotoxicity of NK cells transfected with the tricistronic recombinant nucleic acid of Figure 16 from selected cell lines.

Figure 22 depicts exemplary results of CAR-mediated cytotoxicity of NK cells transfected with the tricistronic recombinant nucleic acid of Figure 16 from selected cell lines.

Figure 23 depicts further exemplary results of CAR-mediated cytotoxicity of NK cells transfected with the tricistronic recombinant nucleic acid of Figure 16 from selected cell lines

FIG. 24 depicts further exemplary results of ADCC of NK cells from selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 16 .

25 depicts exemplary results of erIL-2 release from NK cells of selected clonal cell lines transfected with the tricistronic recombinant nucleic acid of FIG. 16 .

26 is a schematic diagram of exemplary tetracistronic recombinant nucleic acids encoding TGF-beta Trap, CAR, high affinity variants of CD16, and erIL-2 for generating recombinant NK cells.

FIG. 27 depicts exemplary results for expression of TGF-β well in NK cells transfected with the tetracistronic recombinant nucleic acid of FIG. 26 .

28 is a schematic diagram of exemplary tetracistronic recombinant nucleic acids encoding IL-12 heterodimers, CAR, high affinity variants of CD16, and erIL-2 for generating recombinant NK cells.

29 is a graph showing IL-12 heterodimers and the corresponding expression levels of IL-12 heterodimers in NK cells transfected with the tetracistronic recombinant nucleic acid of FIG. 28 .

30 is a schematic diagram of exemplary tetracistronic recombinant nucleic acids encoding CCR7, CAR, high affinity variants of CD16, and erIL-2 for generating recombinant NK cells.

Detailed ways

The inventors have found that genetically modified NK cells can have significant general toxicity, target-specific CAR-mediated cytotoxicity, and ADCC (antibody-dependent cellular cytotoxicity), and that such genetically modified cells can be expressed in autocrine Growth under a growth stimulus will also confer selectivity against successfully transfected cells. In addition, contemplated cells may further express secreted IL-12 and/or TGF-beta traps from recombinant nucleic acids to reduce or eliminate the immunosuppressive environment. Additionally or alternatively, contemplated cells may express homing receptors from recombinant nucleic acids. Thus, modified cells can be generated by transfection with tricistronic or tetracistronic nucleic acids.

It should be further understood that FcεRIγ-containing CARs have not been used in NK-92 cells, other NK cell lines, or endogenous NK cells, as other signaling domains (e.g., CD3ζ) are thought to be more effective, especially when combined with additional Signaling domains (in second- and third-generation CARs) when combined. Remarkably, the inventors have found that NK-92 cells expressing a first-generation CAR comprising an intracellular domain from FcεRIγ, which has only one ITAM domain, were compared to expressing a CAR with a CD3ζ signaling domain ( This domain has three ITAM domains, and even NK-92 cells in which these ITAM domains are combined with other signaling domains (i.e., second-generation or third-generation CARs) are less sensitive to CARs expressing CARs. Cancer cells that recognize the antigen have the same or higher cytotoxic activity. The inventors also unexpectedly found that CARs comprising the intracellular domain from FcεRIγ were expressed on the surface of NK-92 cells at higher levels than other CARs, especially CARs comprising the CD3ζ signaling domain. Cytotoxic effects can even be expressed and secreted by IL-12 (which reduces immunosuppression in the tumor microenvironment (e.g., via exocrine stimulation of NK cells, regulation of MDSCs, etc.)) and/or by expression of TGF-β traps and It is further enhanced by secretion or presentation. Alternatively or additionally, cytotoxicity may also be enhanced by the expression of one or more homing receptors to attract and/or retain NK cells in the tumor microenvironment.

Thus, in some aspects of the inventive subject matter, genetically modified NK-92 cells or NK cell lines are engineered to express a chimeric antigen receptor (CAR) on the cell surface, specifically binding to EGFR superfamily receptors CARs such as EGFR or HER2. Most typically, CARs comprise an intracellular domain derived from Fcε receptor gamma (FcεRIγ). However, in a further contemplated embodiment, the CAR may also comprise the T cell receptor (TCR) CD3zeta (CD3ζ) intracellular domain, alone or in combination with additional known from 2nd and 3rd generation CAR constructs. Combinations of components (eg, CD28, CD134, CD137 and/or ICOS). As will be readily understood, the CAR can be expressed transiently or stably by NK-92 cells from recombinant DNA or RNA molecules. Exemplary CAR constructs suitable for use herein are shown in Figure 1.

Thus, in one aspect of the inventive subject matter, the NK cell, NK-92 cell or NK/NK-92 cell line is expressed in an NK- 92 cells express chimeric antigen receptor (CAR) on their surface. Alternatively, or in addition, the CAR may also comprise the cytoplasmic domain of CD3ζ (eg, having the amino acid sequence of SEQ ID NO: 7, which may be encoded by the nucleic acid of SEQ ID NO: 8 (codon optimized)). In another aspect, a NK or NK-92 cell line transformed with a nucleic acid encoding a chimeric antigen receptor (CAR) is contemplated. For example, a preferred nucleic acid encodes the cytoplasmic domain of FcεRIγ (eg, comprises or consists of SEQ ID NO: 2). As discussed in more detail below, CARs can target EGFR superfamily receptors such as EGFR or HER2.

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

Additionally, it is generally preferred that the genetically modified NK cells also express recombinant CD16 or a high affinity variant thereof (eg, CD16 ^158V ) to confer target-specific ADCC on the cells. Advantageously, such co-expression with CAR is believed to further increase cytotoxicity against tumor cells. In this context, it is understood that unmodified NK cells generally do not express CD16 and only exhibit cytotoxicity as part of the innate immune system.

Recently, it has 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 (eg, TGF-β) and various suppressive cells (eg, MDSCs), among others. Therefore, the genetically modified NK cells can further express one or more recombinant proteins to resist immunosuppressive factors. For example, and as described in more detail below, genetically modified NK cells can express TGF-β traps to reduce TGF-β-mediated effects in the tumor microenvironment and/or can express IL-12 to inhibit MDSCs. Additionally, or alternatively, the genetically modified NK cells can also express one or more homing receptors to the tumor microenvironment (or other desired tissue), thereby increasing the number of therapeutic cells in the tumor microenvironment and The therapeutic effect is thus enhanced.

In another aspect of the present subject matter, the inventors also contemplate a method of treating cancer in a patient in need thereof, the method comprising the step 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. From a different point of view, the inventors also contemplate a modified NK/NK-92 cell or NK/NK-92 cell line expressing a chimeric antigen receptor (CAR), preferably a cell comprising FcεRIγ A plasma domain, for treating cancer 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 tumor cells is contemplated and may include the step of contacting the tumor cells 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 the tumor cell. In some embodiments, the CAR preferably comprises an intracellular domain from Fcε receptor gamma (FcεRIγ). Alternatively, or in addition, the CAR comprises a T cell receptor (TCR) CD3ζ (CD3zeta) intracellular domain.

With regard 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), secondary NK cells that have been immortalized, autologous or Xenogeneic NK cells (stock, 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, eg, Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are cancerous NK cells that exhibit broad antitumor cytotoxicity and predictable yields when expanded in appropriate media. Advantageously, NK-92 cells have high cytolytic activity against various cancers.

The original NK-92 cell line expresses CD56 ^bright , CD2, CD7, CD11a, CD45, and CD54 surface markers, but does not display CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers things. Growth of such NK-92 cells in culture is dependent on the presence of interleukin 2 (eg, rIL-2), with doses as low as 1 IU/mL sufficient to maintain proliferation. IL-7 and IL-12 do not support long-term growth, and multiple other cytokines have not been tested, including IL-1α, IL-6, tumor necrosis factor α, interferon α, and interferon γ. Compared to primary NK cells, NK-92 is generally more cytotoxic even at relatively lower effector-to-target (E:T) ratios (eg, 1:1). Representative NK-92 cells were deposited with the American Type Culture Collection (ATCC) under the designation CRL-2407.

Accordingly, suitable NK cells may have one or more modified KIRs 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 their expression inhibited (eg, by miRNA, siRNA, etc.). Most typically, more than one KIR will be mutated, deleted or silenced, and particularly contemplated KIRs include those with two or three domains, with short or long cytoplasmic tails. Viewed differently, a modified, silenced or deleted KIR would include KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, 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 NantKwest (see URL www.nantkwest.com). These cells can then be additionally genetically modified for CARs, as described in more detail below.

In another aspect of the inventive subject matter, the genetically engineered NK cells may also be derivatives of NK-92 modified to express the high affinity Fc gamma receptor (CD16). Sequences of high-affinity variants of Fcγ receptors are well known in the art (see, e.g., Blood [blood] 2009 113:3716-3725; or SEQ ID NO: 11 (with a V at amino acid position 158) and SEQ ID NO: 12 ( There is a codon for V at amino acid position 158)) and all means of production and expression are considered suitable for use herein. Expression of this receptor is believed to allow the use of antibodies specific to the patient's tumor cells (e.g., neoepitopes), specific tumor types (e.g., her2neu, PSA, PSMA, etc.), or antibodies associated with cancer ( For example, CEA-CAM) specifically targets tumor cells. Advantageously, such antibodies are commercially available and can be used in conjunction with cells (eg, to bind to Fcγ receptors). Alternatively, such cells are also commercially available as haNK cells from Nant Guist. These cells can then be additionally genetically modified for CARs, as described in more detail below.

Accordingly, NK cells suitable for use herein include NK-92 cells (which may encode CARs, CD16 or variants thereof, and cytokines or variants thereof, and optionally IL-12, TGF-β traps, and homing receptors Transfection with a tricistronic or tetracistronic construct of one of the above), genetically modified NK cells or NK-92 cells expressing CD16 or a variant thereof or a cytokine or a variant thereof (which may encode a CAR and CD16 or its variants or cytokines or variants thereof), and genetically modified NK cells or NK-92 cells expressing CD16 or its variants and cytokines or variants thereof (which can encode CAR nucleic acid transfection). Any of the NK cells contemplated herein can express one of IL-12, a TGF-beta trap, and a homing receptor from the same or different recombinant nucleic acids, as previously described.

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

Similarly, it will be appreciated that the manner of transfection will depend, at least in part, on the type of nucleic acid used. Therefore, viral transfection, chemical transfection, mechanical transfection methods are all considered suitable for use herein. For example, in one embodiment, the vectors described herein are transient expression vectors. Exogenous transgenes introduced using such vectors are not integrated into the nuclear genome of the cell; thus, in the absence of vector replication, the exogenous transgenes will degrade or dilute over time.

In another embodiment, the vector will preferably allow stable transfection of cells. In one embodiment, the vector allows the incorporation of one or more transgenes into the genome of the 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, eg, geneticin (Neo gene from Tn5). 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 suitable carriers are well known in the art.

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

Regarding the envisioned CAR, it was noted that NK/NK-92 cells would 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 the signaling domain in the intracellular space. Most typically, a CAR will comprise 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 FcεRIγ domain).

In a preferred embodiment, the cytoplasmic domain of the CAR comprises or consists of the signaling domain of FcεRIγ. For example, the FcεRIγ signaling domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:1. In some embodiments, the FcεRIγ cytoplasmic domain is the only signaling domain. However, it should be understood that additional elements may also be included, such as other signaling domains (eg, CD28 signaling domain, CD3ζ signaling domain, 4-1BB signaling domain, etc.). These additional signaling domains may be located downstream of the FcεRIγ cytoplasmic domain and/or upstream of the FcεRIγ cytoplasmic domain.

In some embodiments, the FcεRIγ signaling domain comprises at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequence set forth in SEQ ID NO: 1 or consists of or consists essentially of the amino acid sequence of

As noted above, in some embodiments, the cytoplasmic domain of the CAR comprises the signaling domain of CD3ζ (CD3zeta). In one embodiment, the cytoplasmic domain of the CAR consists of the signaling domain of CD3ζ. In one embodiment, the CD3ζ signaling domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:7. In some embodiments, the CD3ζ signaling domain comprises at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequence set forth in SEQ ID NO:7 or consists of or consists essentially of the amino acid sequence of

The CAR can comprise any suitable transmembrane domain. In one aspect, the CAR comprises the transmembrane domain of CD28. In one embodiment, the CD28 transmembrane domain comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to the amino acid sequence of SEQ ID NO: 4 The amino acid sequence either consists of or consists essentially of. In one embodiment, the transmembrane domain is selected from CD28 transmembrane domain, 4-1BB transmembrane domain or FcεRIγ transmembrane domain.

The CAR can include any suitable hinge region. In one aspect, the CAR comprises the hinge region of CD8. In one embodiment, the CD8 hinge region comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:3. In one embodiment, the CD8 hinge region comprises at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology to the amino acid sequence set forth in SEQ ID NO:3 The amino acid sequence either consists of or consists essentially of.

Most typically, but not necessarily, the extracellular binding domain of the CAR will be a scFv or other natural or synthetic binding moiety that specifically binds an antigen of interest such as EGFR superfamily receptors (and in particular EGFR or HER2). Particularly suitable binding moieties include small antibody fragments with single, dual or multiple target specificities, beta barrel domain binders, phage display fusion proteins, and the like. However, in other embodiments, a suitable extracellular binding domain will specifically bind a tumor-specific antigen, a tumor-associated antigen, or a patient-specific antigen and a tumor-specific antigen. For example, contemplated antigens include CD19, CD20, GD2, HER-2, CD30, EGFR, FAP, CD33, CD123, PD-L1, IGF1R, CSPG4 or B7-H4. Other tumor-specific antigens are described, by way of non-limiting examples, in US2013/0189268, WO 1999024566A1, US 7098008 and WO2000020460, each of which is incorporated herein by reference in its entirety.

Thus, a contemplated CAR will generally have the structure of an extracellular binding domain (directly) coupled to a hinge domain coupled (directly) to a transmembrane domain that is coupled (e.g., FcεRIγ) signaling domain (direct) coupling. In still further contemplated aspects, contemplated CARs may include one or more signaling domains in addition to or in place of the FcεRIγ signaling domain, and in particular contemplated signaling domains include CD3ζ signaling domains, 4-1BB signaling domain and CD28 signaling domain. For example, a contemplated CAR polypeptide may thus include a scFv-amino acid sequence of SEQ ID NO: 41 (EGFR scFv-amino acid sequence; derived from a scFv of SEQ ID NO: 40 or Nucleic acid encoding of a sequence having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to SEQ ID NO: 40) and SEQ ID NO: 43 (HER2 /neu scFv-amino acid sequence; having SEQ ID NO: 42 or having at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to SEQ ID NO: 42 Any one of the binding domains of the nucleic acid encoding of the sequence), the hinge domain is then coupled to the transmembrane domain (such as CD28TM shown in SEQ ID NO: 4), the transmembrane domain is coupled to the signal Transduction domain (for example, FcεRIγ signaling domain shown in SEQ ID NO: 1, CD28 signaling domain shown in SEQ ID NO: 5, 4-1BB signaling domain shown in SEQ ID NO: 6, and/or the CD3ζ signaling domain shown in SEQ ID NO:7) coupling.

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

Viewed from a different perspective, envisioned CARs target antigens associated with specific cancer types, more particularly cancers that overexpress EGFR and/or HER2, such as lung cancer (e.g., small cell) and breast cancer (e.g., TBNC), Thyroid cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, head and neck cancer, etc.

In yet a further contemplated aspect, the NK cells may be further genetically modified to express one or more cytokines, and particularly autocrine growth stimulating cytokines, thereby providing a selection marker, wherein the cytokine and the CAR are expressed in the same recombinant nucleic acid Encodes and/or renders recombinant cells independent of exogenous IL-2. Accordingly, in some embodiments, NK-92 cells are modified to express at least one cytokine. In particular, the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21 or variants thereof. In preferred embodiments, the cytokine is IL-2 or a variant thereof, and particularly preferred variants include an endoplasmic retention signal (e.g., the human IL-2 polypeptide shown in SEQ ID NO: 9, optionally Has an ER retention signal 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 results in IL-2 expression sufficient for autocrine activation, but does not release IL-2 extracellularly (eg, Exp Hematol. [Experimental Hematology] 2005 Feb;33(2):159-64). Alternatively, expression of the cytokine (and particularly IL-15) can also be such that the cytokine is expressed in an amount sufficient to provide an autocrine growth signal to the recombinant cell, but also allows at least some of the expressed IL-15 to be released from the cell, thereby Provides immunostimulatory signals. For example, such expression can be achieved using a human IL-15 sequence comprising both a signal peptide and an endoplasmic retention sequence. Exemplary DNA and protein sequences of endosomally retained IL-15 are shown in SEQ ID NO: 38 and SEQ ID NO: 39, respectively.

Contemplated cells can also express suicide genes, if desired. The term "suicide gene" refers to a transgene that allows negative selection of cells expressing the suicide gene. A suicide gene is used as a safety system, allowing cells expressing the gene to be killed by introducing a selection agent. This is ideal where the recombinant gene causes a mutation that results in uncontrolled cell growth, or where 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 Escherichia coli gpt gene, and the Escherichia coli Deo genes. Typically, suicide genes encode proteins that have no adverse effect on cells, but kill 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 the thymidine kinase (TK) gene. The TK gene can be a wild type or a mutant TK gene (eg, tk30, tk75, sr39tk). Cells expressing TK proteins can be killed using ganciclovir. In another embodiment, the suicide gene is cytosine deaminase, which is toxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez et al. "Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method forautologous transplantation." [Cytosine deaminase adenoviral vector and 5-fluorocytosine Pyrimidines selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential clearance method for autologous transplantation] Blood. 1998 Jul 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, eg, Touati et al. "A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response. Curr Gene Ther. [Current Gene Therapy] 2014;14(3):236-46. In yet another embodiment, the suicide gene is iCasp9. Di Stasi, (2011) "Inducibleapoptosis as a safety switch for adoptive cell therapy." N Engl J Med 365: 1673-1683. See also Morgan, "Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic" Molecular Therapy (2012); 20:11-13 . iCasp9 induces apoptosis in the presence of the small molecule AP1903. AP1903 is a biologically inert small molecule that has been shown to be well tolerated in clinical studies and has been used in the context of adoptive cell therapy.

When 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 in which the p35 and p40 components pass through a flexible linker (in either orientation, p35-linker -p40 or p40-linker-p35) are linked together. Furthermore, it is generally preferred that the heterodimer will be secreted, and thus may include a signal peptide for protein export. Accordingly, suitable IL-12 sequences contemplated herein may comprise sequences having at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleic acid sequences. IL-12 contemplated herein may also comprise at least 80%, 85%, 90 Amino acid sequences that are %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. Thus, the IL-12 single chain p40_p35 sequence may comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of SEQ ID NO: 31 % or 99% identity of a polypeptide sequence, or may comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% to SEQ ID NO: 32 , 98% or 99% identical polynucleotide sequences. Most preferably, but not necessarily, the nucleic acid encoding the IL-12 single-stranded heterodimer will be part of a polycistronic nucleic acid sequence (e.g., present as a tetracistronic sequence with CAR, CD16 and erIL-2) .

When the modified NK cells are further engineered to express the TGF-β trap, it is generally preferred that the TGF-β trap is a single-chain dimer of the extracellular domain of the TGFβRII molecule, and most preferably comprises the TGF-β receptor II Single-chain dimers of extracellular domains. Thus, a suitable TGF-beta trap consists of at least 80%, 85%, 90%, 91%, 92%, A polynucleotide sequence encoding of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. TGF-beta wells contemplated herein may also comprise at least 80%, 85%, 90%, 91%, Amino acid sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. Other suitable TGF-β traps include those described in Mol.Canc.Ther. [Molecular Cancer Therapeutics] 2012, Vol. 11, No. 7, 1477-1487. Most preferably, but not necessarily, the TGF-beta trap-encoding nucleic acid will be part of a polycistronic nucleic acid sequence (eg, present as a tetracistronic sequence with CAR, CD16 and erIL-2).

When the modified NK cells are further engineered to express a homing receptor, it should be noted that the term "homing receptor" refers to a receptor that activates a cellular pathway that directly or indirectly leads to the migration of the cell to a target cell or tissue. For example, homing receptors expressed by leukocytes are used by leukocytes and lymphocytes to enter secondary lymphoid tissues via high endothelial venules. Homing receptors can also be used by cells to migrate towards a chemical gradient source, such as a chemokine gradient source. 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; cytokine receptors; cell adhesion molecules, such as selectins, including L-selectin (CD62L); integrins, such as α4β7 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 an addressin on the venule endothelium, such as mucosal vascular addressing cell adhesion molecule 1 (MAdCAM-1).

In some exemplary embodiments, the chemokines and homing receptors contemplated herein may comprise a combination of SEQ ID NO: 13 (CCR7 nucleotide sequence), or SEQ ID NO: 14 (CCL19 nucleotide sequence), or SEQ ID NO: 15 (CCL21 nucleotide sequence) or SEQ ID NO: 16 (CXCR2 nucleotide sequence), or SEQ ID NO: 17 (CXCR2 amino acid sequence), or SEQ ID NO: 18 (CXCL14 nucleotide sequence) , or SEQ ID NO: 19 (CXCL4 amino acid sequence), or SEQ ID NO: 20 (CD62L nucleotide sequence), or SEQ ID NO: 21 (CD62L amino acid sequence), or SEQ ID NO: 22 (IL-8 core nucleotide sequence), or SEQ ID NO: 23 (IL-8 amino acid sequence), or SEQ ID NO: 24 (CXCL1 nucleotide sequence), or SEQ ID NO: 25 (CXCL1 amino acid sequence) has at least 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical polypeptide or polynucleotide sequences. Most preferably, but not necessarily, the nucleic acid encoding the homing receptor will be part of a polycistronic nucleic acid sequence (eg, present as a tetracistronic sequence with CAR, CD16 and erIL-2).

Of course, it should be noted 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, cytokines, TGF-beta wells), the coding region may be arranged in a sequence with at least two, or at least three, or at least four sequences encoding recombinant proteins. in polycistronic units of the coding region. Thus, transgene engineering can be converted into an expression vector by any mechanism known to those skilled in the art. Where multiple transgenes are inserted into a cell, the transgenes can be engineered into the same expression vector or into different expression vectors. In some embodiments, cells are transfected with mRNA encoding the transgenic protein to be expressed. In some embodiments, cells are transfected with DNA encoding the transgenic protein to be expressed. Transgenes, 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 vectors, electroporation, lipofection, nucleofection, or "gene gun."

Therefore, in a preferred embodiment, it should be noted that genetically modified NK cells (especially cells expressing CAR and CD16 or variants thereof) will exhibit three distinct modes of cell killing: NKG2D receptor)-mediated general cytotoxicity, ADCC mediated by antibodies binding to target cells, and CAR-mediated cytotoxicity. If desired, the therapeutic effect in the tumor microenvironment can be via the expression and secretion of IL-12 (e.g., as a single-chain heterodimer), via the TGF-β trap (e.g., as a single chain of the TGF-β receptor II ectodomain). Expression and presentation/secretion of chain dimers) or via expression of homing receptors (eg, CCR7) are further increased. Clearly, envisioned genetically modified cells are useful in the treatment of various diseases, and are a variety of cancers and viral infections in which diseased cells present disease-specific or disease-associated antigens. Accordingly, the inventors contemplated methods of treating patients using the modified NK or NK-92 cells described herein. In one embodiment, the patient has cancer (eg, a tumor), and the modified NK-92 cell or cell line expresses a CAR specific for an antigen expressed on the surface of a cell from the cancer or tumor. As noted above, in some embodiments, the cancer is lung cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, or head and neck cancer.

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 10 billion cells/injection can be administered to an individual, such as about, at least about, or at most about 1×10 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 may be administered to an individual in relative numbers of cells, for example, from about 1000 cells per kilogram of individual up to about 10 billion cells per kilogram of individual, such as per kilogram of individual About, at least about, or at most about 1×10 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 can be calculated in m ² body surface area, including about 1×10 ¹¹ , 1×10 ¹⁰ , 1×10 ⁹ , 1×10 ⁸ , 1×10 ⁷ cells/m ² , or these Any range (including endpoints) between any two of the numbers. The average person is about 1.6 to about 1.8 m ² . In preferred embodiments, about 1 billion to about 3 billion NK-92 cells are administered to the patient.

The modified NK-92 cells, and optionally other anticancer agents, can be administered once to a patient with cancer or a virus infection, or can be administered multiple times, for example, 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 every 1, 2, 3, 4, 5, 6, or 7 Once a day, 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, where the modified NK cells express a suicide gene, an agent that triggers the death of the modified NK cells is administered to the patient. In one embodiment, the agent is administered at a time point subsequent to 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 a patient. Irradiation of NK cells is described, for example, in US Patent No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, the modified NK cells that have not been engineered to express the suicide gene are irradiated.

Furthermore, it should be understood that the envisaged methods of treatment will also include the administration of other immunotherapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., , nonpyrogenic Escherichia coli expressing one or more cancer-specific antigens), yeast cancer vaccine, N-803 (also known as ALT-803, ALTOR Biosciences), and antibody (e.g., binding to tumor-associated antigens or patient-specific tumor neoantigens), stem cell transplantation (e.g., allogeneic or autologous) and tumor-targeting cytokines (e.g., NHS-IL12, IL-12 combined with tumor-targeting antibodies or their fragment coupling).

After reading this description, it will become apparent to a person 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 will be understood that the embodiments presented herein are presented by way of example only, and not limitation. Likewise, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention, as described 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 these may, of course, vary. It is also to be understood that terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting thereof.

example

The following examples are for illustrative purposes only and should not be construed as limitations on the claimed invention. Those skilled in the art can employ various alternative techniques and procedures that would similarly allow one to successfully carry out the contemplated invention.

Example 1: HER2.CAR t-haNK cells

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

To generate HER2.CAR t-haNK cells, recombinant DNA molecules were assembled as schematically depicted in FIG. CD16 ^158V ), and this in turn is followed by an IRES sequence element upstream of the sequence encoding erIL-2. Unless otherwise stated, HER2.CAR has a structure as exemplarily shown in FIG. 1 (however, has an Fcε signal sequence) and has a nucleic acid sequence of SEQ ID NO:30. Of course, it should be noted that other suitable sequences for generating HER2.CAR may have at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of SEQ ID NO: 30 sequence identity. Primary transcripts of the tricistronic nucleic acid then lead to the formation of HER2.CAR, CD16 ^158V and erIL-2 as recombinant polypeptides. Figure 3 schematically and schematically depicts the linearized recombinant nucleic acid used to transfect NK-92 cells.

All transfections generally followed standard protocols: NK-92 cells were treated with 5% human AB serum (Valley Biomedical, Winchester, VA) supplemented with 500 IU/mL IL-2 (Israel Grow in X-Vivo10 medium (Lonza, Basel, Switzerland) from Prospec, Rehovot, Israel. Using the Neon ^TM electroporation device (Life Technologies, Carlsbad, CA), according to the manufacturer's NK-92 cell parameters (1250V, 10ms, 3 pulses), and press Cells were electroporated with tricistronic or tetracistronic DNA at ⁵ μg DNA per 106 cells in a volume of 100 μl. Electroporated cells were transferred to medium (same as above) without exogenously added IL-2.

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

HER2.CAR and CD16 expression on the surface of NK-92 cells was determined by flow cytometry using biotinylated soluble HER2 protein and APC-labeled streptavidin and fluorescently labeled anti-CD16 antibody. Exemplary results for HER2.CAR and CD16 expression (polyclonal) are shown in Figure 4 along with aNK (CD16 not expressed) and haNK (CD16 expressed) controls. As can be easily seen in Figure 4, the polyclonal cell cultures had significant and strong expression for both HER2.CAR and CD16 ^158V . In this context, it should be noted that HER2.CAR was expressed at significantly higher levels, with HER2.CAR having an FcεRIγ signaling moiety compared to the other signaling moieties (data not shown).

From the results depicted in Figure 5, it can be seen that HER2.CAR t-haNK cells have strong and target-specific CAR-mediated cytotoxicity. Here, SKBR3 cells and SUP-B15 ^HER-2+ cells were compared with HER2.CAR t-haNK cells at different effector-to-target cell ratios (e.g., between 0.075 and 10) in flow cytometry-based in vitro Cytotoxicity assays were co-incubated and target specificity compared to aNK cells as indicated. As can be seen, CAR-mediated cytotoxicity using the polyclonal HER2.CAR t-haNK cell population was >60% for SKBR3 cells and nearly 90% for SUP-B15 ^HER-2+ cells.

In a similar manner, ADCC was tested using SUP-B15 ^HER-2-/CD20+ cells and rituximab as target-specific antibody and Herceptin as control antibody. Again, polyclonal HER2.CAR t-haNK cells had potent and nearly 60% target-specific ADCC, as can be seen from the exemplary results depicted in Figure 6, without significant off-target toxicity.

A number of individual clones from the HER2.CAR t-haNK cell population were then prepared after dilution propagation and expression analysis was performed again via FACS using the same procedure as above. aNK and haNK cells were used as controls. Again, all individual clones were observed to have significant and strong expression of both HER2.CAR and CD16 ^158V , as can be seen from the exemplary results in FIG. 7 .

The native cytotoxicity of individual HER2.CAR t-haNK cell clones was tested using K562 cells, and the results were compared with untransfected aNK cells, as shown in Figure 8. Here, specific lysis at the indicated ratios of effector cells to target cells was lower than control aNK lysis, however, only about 10%-20% lower. On the other hand, using SUP-B15 ^HER-2+ cells, CAR-mediated cytotoxicity was again substantial and target-restricted, and was shown in Figure 9 to be specific over a wide range of effector-to-target ratios. Exemplary results of HER2.CAR t-haNK cell clones. Likewise, where SKBR3 cells were used as target cells, again significant CAR-mediated cytotoxicity was observed for all tested HER2.CAR t-haNK cell clones, as exemplarily shown in FIG. 10 . Selected HER2.CAR t-haNK cell clones were also tested for ADCC against SUP-B15 ^HER-2-/CD20+ cells using rituximab as target-specific antibody and Herceptin as control antibody. As can be seen from Figure 11, all HER2.CAR t-haNK cell clones again had significant, potent and target-specific ADCC against controls.

Although all HER2.CAR t-haNK polyclonal cultures and cell clones propagated well in the absence of exogenous IL-2, the expression of er-IL2 and the amount of erIL-2 released in the medium were also tested . Exemplary results are shown in Figure 12, which depicts a small release of erIL-2 into the culture medium. Notably, as shown in Figure 12, extracellular erIL-2 release was significantly lower than that of the closely related haNK003 cell line. Such reduced release may further advantageously reduce potential systemic effects otherwise attributable to IL-2 (eg, vascular permeability, systemic vascular leakage, etc.).

To test the in vivo efficacy of HER2.CAR t-haNK cells, NSG mice (NOD.Cg-Prkdc scid ^{Il2rg tml Wjl} /SzJ) were subcutaneously implanted with MDA-MB-453 tumor cells and the tumor volume was measured. When tumors reached an average of approximately 120 mm, ^HER2.CAR t-haNK cells were administered intratumorally or intravenously twice weekly during the study period. Following administration of HER2.CARt-haNK cells, tumor volume was sustainably reduced compared to vehicle control treatment, as demonstrated by the exemplary results in FIG. 13 . It will be appreciated that intratumoral administration requires significantly fewer HER2.CAR t-haNK cells than systemic intravenous administration for the same reduction in tumor volume.

Similarly, the in vivo efficacy of HER2.CAR t-haNK cells was tested in NSG mice subcutaneously implanted with BT-474 tumor cells, and tumor volume was measured over time. When tumors reached an average of approximately 120 mm, ^HER2.CAR t-haNK cells were administered intratumorally or intravenously twice weekly during the study period. After administration of HER2.CAR t-haNK cells, the increase in tumor volume was significantly reduced over the time period tested, as shown in the exemplary results in FIG. 14 . Again, it should be appreciated that intratumoral administration requires significantly fewer HER2.CAR t-haNK cells than systemic intravenous administration for the same reduction in tumor growth.

Example 2: EGFR.CAR t-haNK cells

To generate EGFR.CAR t-haNK cells, a recombinant DNA molecule was assembled as schematically depicted in FIG. CD16 ^158V ), and this in turn is followed by an IRES sequence element upstream of the sequence encoding erIL-2. Unless otherwise stated, EGFR.CAR has a structure as exemplarily shown in FIG. 1 (however, has an Fcε signal sequence) and has a nucleic acid sequence of SEQ ID NO:33. Of course, it should be noted that other suitable sequences for generating EGFR.CAR may have at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of SEQ ID NO: 33 sequence identity. Primary transcripts of the tricistronic nucleic acid then lead to the formation of EGFR.CAR, CD16 ^158V and erIL-2 as recombinant polypeptides. Figure 16 schematically depicts linearized recombinant nucleic acid for transfection of NK-92 cells.

EGFR.CAR and CD16 expression on the surface of NK-92 cells was determined by flow cytometry using biotinylated anti-scFv antibody and APC-labeled streptavidin and fluorescently-labeled anti-CD16 antibody. Exemplary results for EGFR.CAR and CD16 expression (polyclonal) are shown in Figure 17 along with aNK (CD16 not expressed) and haNK (CD16 expressed) controls. As can be easily seen in Figure 17, the polyclonal cell cultures had significant and strong expression for both EGFR.CAR and CD16 ^158V . In this context, it should again be noted that EGFR.CAR was expressed at significantly higher levels, with HER2.CAR having an FcεRIγ signaling moiety compared to the other signaling moieties (data not shown).

The native cytotoxicity of polyclonal cell cultures of EGFR.CAR t-haNK cells was determined and FIG. 18 depicts an exemplary set of results. Here, native cytotoxicity against K562 cells did exceed that of aNK control cells. Similarly, polyclonal cell cultures of EGFR.CAR t-haNK were assayed for CAR-mediated cytotoxicity against HCT116 target cells and against A549 cells, both of which express EGFR. As can be seen from the graph in Figure 19, CAR-mediated cytotoxicity was significant and specific. In a further experiment, the inventors tested the ADCC of EGFR.CAR t-haNK polyclonal cells against SUP-B15 ^EGFR-/CD20+ cells using rituximab as target-specific antibody and Herceptin as control antibody . As can be seen from Figure 20, all EGFR.CAR t-haNK cells again had significant, potent and target-specific ADCC relative to controls.

After dilution propagation, the selected EGFR.CAR t-haNK cell clones were tested for natural cytotoxicity against 562 cells. Notably, all selected EGFR.CAR t-haNK cell clones had essentially the same native cytotoxicity comparable to the aNK control, as can be seen from FIG. 21 . The selected EGFR.CAR t-haNK cell clones were then tested for CAR-mediated cytotoxicity against HCT116 tumor cells, and exemplary results are shown in the graph of FIG. 22 . Again, all EGFR.CAR t-haNK cell clones had very significant CAR-mediated cytotoxicity against HCT116 tumor cells, approaching 90% specific lysis. Similar results were obtained using A549 tumor cells as target cells, as shown in FIG. 23 .

In a further experiment, the inventors measured the selected EGFR.CAR t using SUP-B15 ^EGFR-/CD20+ cells as target cells, rituximab as target-specific antibody and Herceptin as control antibody. - ADCC of haNK cell clones. As already observed with polyclonal cell cultures and as can be seen from Figure 24, selected EGFR.CAR t-haNK cell clones exhibited potent ADCC in the presence of cognate antibodies.

Although all EGFR.CAR t-haNK polyclonal cultures and cell clones propagated well in the absence of exogenous IL-2, the expression of er-IL2 and the amount of erIL-2 released in the medium were also tested . Exemplary results are shown in Figure 25, which depicts that for some clones, less erIL-2 was released into the medium, while other clones had comparable erIL-2 release to the control. As noted above, reduced erIL-2 release may further advantageously reduce potential systemic effects otherwise attributable to IL-2 (eg, vascular permeability, systemic vascular leakage, etc.).

Example 3: Tetracistronic structure

Although the above examples were performed with tricistronic constructs, it should be understood that the same constructs can also be implemented in tetracistronic constructs to express IL-12, TGF-beta traps or homing receptors, Thereby reducing immunosuppression in the tumor microenvironment and enriching the NK cells thus modified in the tumor microenvironment. An exemplary tetracistronic construct is schematically shown in Figure 26, wherein the nucleic acid sequence encoding the TGF-beta trap is upstream of the tricistronic construct described above. As can be seen from Figure 26, the P2A sequence is located between the TGF-β trap and the CAR to ensure synergistic expression while producing different proteins. Expression of TGF-beta traps was then tested in an ELISA to confirm that the tetracistronic constructs indeed produced functional TGF-beta traps, and Figure 27 depicts exemplary results relative to controls. Notably, expression levels of TGF-β wells were significant in all recombinant clones with the tetracistronic construct.

Similarly, tetracistronic constructs were prepared using secreted IL-12 single-chain heterodimers, as schematically depicted in FIG. 28 . Again, the P2A sequence was placed between the IL-12 single chain heterodimer sequence and the CAR sequence. Figure 29 shows an exemplary example of a single-stranded heterodimer with alternating orders of p35 and p40 subunits, both separated by a linker sequence. As can be seen from the expression levels tested, both single chain heterodimers were expressed equally well in the cells tested relative to the control. In another example, a tetracistronic construct was made using CCR7 as a homing receptor, and an exemplary tetracistronic arrangement is depicted in FIG. 30 .

Of course, it should be recognized that for all nucleic acid sequences provided herein, the corresponding encoded proteins are also expressly contemplated herein. Likewise, for all amino acid sequences, the corresponding nucleic acid sequences are contemplated herein (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 following claims, 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", "a" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

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

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 range and include all values between the endpoints of the range. All ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can readily be considered sufficiently descriptive and the same range can be broken down into at least equal halves, two-thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be easily broken down into a lower third, a middle third, an upper third, and so on. As will also be understood by those skilled in the art, all language such as "at most," "at least," etc., includes the recited numerical value and refers to a range that can then be subdivided into sub-ranges as noted above. Finally, as understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2 or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4 or 5 cells, etc.

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

"Optional" or "optionally" means that the subsequently described event or circumstance can or cannot 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 compositions and methods include the listed elements, but do not exclude other elements. "Consisting essentially of" when used to define compositions and methods shall mean excluding other elements of any significance to the combination. For example, a composition consisting essentially of elements defined herein will not exclude other elements that do not materially affect the basic and novel characteristics of the claimed invention. "Consisting of" means excluding more than trace amounts of other ingredients and the enumerated substantive process steps. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

As used herein, "immunotherapy" refers to NK-92 cells using modified or unmodified, naturally occurring or modified NK cells or T cells, whether used alone or in combination, and Capable of inducing cytotoxicity upon contact with target cells.

As used herein, "natural killer (NK) cells" are cells of the immune system that kill target cells without stimulation by a specific antigen, and are not limited according to major histocompatibility complex (MHC) class. Target cells can be tumor cells or virus-carrying cells. 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 distinct from the NK-92 cell line. Endogenous NK cells are generally a heterogeneous population of cells in which NK cells have been enriched. Endogenous NK cells can be used for autologous or allogeneic therapy of patients.

The term "NK-92" refers to natural ^killer cells derived from a highly potent unique cell line described by Gong et al. ). Immortalized NK cell lines were originally obtained from patients with non-Hodgkin's lymphoma. Unless otherwise stated, the term "NK-92 ^™ " refers to the original NK-92 cell line as well as NK-92 cell lines that have been modified (eg, by introducing foreign genes). NK-92 ^™ cells and exemplary and non-limiting modifications thereof are described in U.S. Patent Nos. 7,618,817, 8,034,332, 8,313,943, 9,181,322, 9,150,636 and published U.S. Application No. 10/008,955, all of which are incorporated herein by reference in their entirety, And including wild-type NK-92 ^™ , NK-92 ^™ -CD16, NK-92 ^™ -CD16-γ, NK-92 ^™ -CD16-ζ, NK-92 ^™ -CD16(F176V), NK-92 ^™ MI and NK-92 ^™ CI. NK-92 cells are known to those of ordinary skill in the art and such cells are readily available from Nant Guist.

细胞”)。在一些实施例中，CD16+NK-92^TM细胞在细胞表面包含高亲和力CD16受体。术语“taNK”是指源自Gong等人(1994)所述的高度有效的独特细胞系的未经修饰的自然杀伤细胞，其权利归南特圭斯特公司所有，其经修饰以表达嵌合抗原受体(下文为“CAR修饰的NK-92^TM细胞”或“

细胞”)。术语“t-haNK”是指源自Gong等人(1994)所述的高度有效的独特细胞系的未经修饰的自然杀伤细胞，其权利归南特圭斯特公司所有，其经修饰以在细胞表面表达CD16并表达嵌合抗原受体(下文为“CAR修饰的CD16+NK-92^TM细胞”或“t-haNK^TM细胞”)。在一些实施例中，t-haNK^TM细胞在细胞表面表达高亲和力CD16受体。The term " ^aNK " refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al. ). The term "haNK" refers to unmodified natural killer cells derived from a highly potent unique cell line described by Gong et al. Expressing CD16 (hereinafter "CD16+NK-92 ^TM cells" or "

cells"). In some embodiments, CD16+NK-92 ^™ cells comprise high affinity CD16 receptors on the cell surface. The term "taNK" refers to a highly potent unique cell line derived from Gong et al. (1994) The unmodified natural killer cells of the Nant Guist company, whose rights are owned by Nant Guist, have been modified to express chimeric antigen receptors (hereinafter referred to as "CAR-modified NK-92 ^TM cells" 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. Modified to express CD16 on the cell surface and to express a chimeric antigen receptor (hereinafter "CAR-modified CD16+ NK-92 ^TM cells" 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 (such as IL-2 or IL-12), and/or suicide genes. In some embodiments, the modified NK-92 cells comprise vectors encoding transgenes, such as Fc receptors, CARs, cytokines (such as IL-2 or IL-12), and/or suicide genes. In one embodiment, the modified NK-92 cells express at least one transgenic protein.

As used herein, "non-irradiated NK-92 cells" are NK-92 cells that have not been irradiated. Irradiation renders cells unable to grow and proliferate. It is envisioned that NK-92 cells will be irradiated at the therapeutic facility or at other points prior to patient treatment, as the time between irradiation and infusion should not exceed four hours to maintain optimal viability. Alternatively, NK-92 cell proliferation can be prevented by another mechanism.

As used herein, "inactivation" of NK-92 cells renders them unable to grow. Inactivation may also be related to the death of NK-92 cells. It is envisioned that after ex vivo samples of NK-92 cells that have effectively cleared cells associated with pathology in therapeutic applications, or that remain in a mammal long enough to effectively kill many or all target cells in vivo, may Inactivate. As a non-limiting example, inactivation can be induced by administering an inactivating agent to which NK-92 cells are sensitive.

As used herein, the terms "cytotoxic" and "cytolytic" are intended to be synonymous when used to describe the activity of effector cells, such as NK-92 cells. In general, cytotoxic activity involves killing target cells by any of a variety of biological, biochemical or biophysical mechanisms. Cytolysis refers more specifically to the activity of effectors that cleave the plasma membrane of a target cell, thereby destroying its physical integrity. This results in the killing of 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" of a cell/population of cells is intended to include any type of manipulation that would result in the death of the cell/population of cells.

The term "Fc receptor" refers to a protein found on the surface of certain cells (eg, 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). FcRs are classified according to the type of antibody they recognize. For example, Fc-γ receptors (FCγRs) bind IgG class antibodies. FCγRIII-A is a low-affinity Fc receptor that binds to IgG antibodies and activates ADCC. FCγRIII-A is normally found on NK cells. NK-92 cells do not express FCγRIII-A. The Fc-ε receptor (FcεR) binds to the Fc region of an IgE antibody.

As used herein, the term "chimeric antigen receptor" (CAR) refers to an extracellular antigen binding domain fused to an intracellular signaling domain. CAR can be expressed in T cells or NK cells to increase cytotoxicity. Typically, the extracellular antigen binding domain is a scFv specific for an antigen found on the cell of interest. Based on the specificity of the scFv domain, CAR-expressing NK-92 cells were targeted to cells expressing certain antigens on the cell surface. The scFv domain can be engineered to recognize any antigen, including tumor-specific and virus-specific antigens. For example, the CD19CAR recognizes CD19, a cell surface marker expressed by certain cancers.

The term "tumor-specific antigen" as used herein refers to an antigen that is present on cancer cells or neoplastic cells but cannot be detected on normal cells derived from the same tissue or lineage as cancer cells. As used herein, a tumor-specific antigen also refers to a tumor-associated antigen, ie, an antigen that is expressed at higher levels on cancer cells 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 cannot be detected 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 to refer to a polymeric form of nucleotides of any length, ie deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, Ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. Nucleotide structural modifications, if present, can be made before or after polynucleotide assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated 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 that are known or predicted to make up the double-stranded form.

A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); (U) in place of thymine. Thus, the term "polynucleotide sequence" is an alphabetic designation for a polynucleotide molecule.

"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing 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 the sequence identity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing positions in each sequence that 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 identical at that position. Homologous nucleotide sequences include sequences encoding natural 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 containing conservative amino acid substitutions and polypeptides having the same binding and/or activity. In some embodiments, homologous amino acid sequences have 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 sequences are at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or greater identical to the sequences 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 by the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Gene Computer Group, University Research Park, Madison) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math. [Advanced Applied Mathematics], 1981, 2, 482-489). Algorithms suitable for determining percent sequence identity include the BLAST and BLAST 2.0 algorithms described in Altschul et al. (Nuc. Acids Res. [Nucleic Acids Research] 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990). Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (see web site at ncbi.nlm.nih.gov). The 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 wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program defaults to using a wordlength of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989) the comparison (B) is 50, the expected value (E) is 10, M=5, N=-4.

In some embodiments, the nucleic acid sequence is codon-optimized for expression in a particular species, for example, a mouse sequence can be codon-optimized for expression in a human (the expression of the protein encoded by the codon-optimized nucleic acid sequence). Express). Accordingly, in some embodiments, a codon-optimized nucleic acid sequence is at least 60%, at least 65%, at least 70%, at least 80%, or at least 85% or greater identical to a nucleic acid sequence described herein. In some embodiments, the codon-optimized nucleic acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence described herein. identity.

The term "expression" refers to the production of a gene product (eg, protein). The term "transient" when referring to expression means that the polynucleotide is not incorporated into the genome of the cell. The term "stabilized" when referring to expression refers to the incorporation of a polynucleotide into the genome of a cell, or the maintenance of expression of a transgene using a positive selection marker (i.e., a foreign gene expressed by a cell that is beneficial under certain growth conditions) .

The term "cytokine or cytokines" refers to the general class of biological molecules 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 preferred embodiments, the cytokine is IL-2.

As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising a complete replicon such that the vector can replicate when placed in a permissive cell, eg, through a transformation process. A vector can replicate in one cell type, such as a bacterium, but has limited or no ability to replicate in another cell type, such as a mammalian cell. Vectors can be viral or non-viral. Exemplary non-viral vectors for the delivery of 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, containing DNA complexed with cationic polymers Condensed DNA, such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, in some cases contained in liposomes; and ternary complexes containing virus and polylysine-DNA the use of. In one embodiment, the vector is a viral vector, eg, 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 or outside of a cell. Targeting is typically achieved by a signal or targeting peptide, which is a stretch of amino acid residues in a polypeptide chain. These signal peptides can be located anywhere within the polypeptide sequence, but are usually N-terminal. Polypeptides can also be engineered to have a signal peptide at the C-terminus. Signal peptides can direct polypeptides for extracellular cleavage and localization to the plasma membrane, Golgi apparatus, endosomes, endoplasmic reticulum, and other cellular compartments. For example, a polypeptide with a specific amino acid sequence at its C-terminus (eg, KDEL) is retained in or transported back to the lumen of the ER.

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

As used herein, the term "transfection" refers to the insertion of nucleic acid into a cell. Transfection can be performed using any means that allows the nucleic acid to enter the cell. DNA and/or mRNA can be transfected into cells. Preferably, the transfected cells express the gene product (ie, protein) encoded by the nucleic acid.

The term "suicide gene" refers to a transgene that allows negative selection of cells expressing the transgene. A suicide gene is used as a safety system, allowing cells expressing the gene to be killed by introducing a selection agent. Multiple suicide gene systems have been identified, including herpes simplex virus thymidine kinase (TK) gene, cytosine deaminase gene, varicella-zoster virus thymidine kinase gene, nitroreductase gene, Escherichia coli gpt Genes and Escherichia coli (E. coli) Deo genes (see also, eg, Yazawa K, Fisher W E, Brunicardi F C: Current progress in suicide gene therapy for cancer. World J. Surg. Surgery Journal] 2002 Jul;26(7):783-9). In one embodiment, the suicide gene is the thymidine kinase (TK) gene. The TK gene can be a wild type or a mutant TK gene (eg, tk30, tk75, sr39tk). Cells expressing TK proteins can be killed using ganciclovir.

Claims (as amended under Article 19 of the Treaty)

1. A genetically modified NK cell transfected with one or more recombinant nucleic acids encoding and expressing the following substances

(i) Membrane-bound recombinant chimeric antigen receptor (CAR), which comprises an extracellular binding domain, a hinge domain, a transmembrane structure having SEQ ID NO: 41 in a single polypeptide chain domain and a signaling domain, wherein the extracellular binding domain specifically binds to EGFR superfamily receptors;

(ii) recombinant CD16;

(iii) autocrine growth stimulating cytokines; 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 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 FcεRIγ signaling domain or a CD3ζ signaling domain.

5. Cancellation.

6. The genetically modified NK cell of claim 1, wherein the recombinant CD16 is a CD16 ^158V mutant.

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

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

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

10. The genetically modified NK cell of claim 1, wherein the TGF-beta well comprises a single-chain dimer of the TGF-beta receptor II ectodomain.

11. The genetically modified NK cell of claim 10, wherein the TGF-beta well is a secreted form of a single-chain dimer of the TGF-beta receptor II ectodomain.

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

13. The genetically modified NK cell as claimed in claim 12, wherein the homing receptor is selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, Group consisting of CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC and CXCL14 receptors.

14. A genetically modified NK cell comprising:

Recombinant nucleic acids encoding

(i) Membrane-bound recombinant chimeric antigen receptor (CAR), which comprises an extracellular binding domain, a hinge domain, a transmembrane structure having SEQ ID NO: 41 in a single polypeptide chain domain and a signaling domain, wherein the extracellular binding domain specifically binds to EGFR superfamily receptors;

(ii) recombinant CD16;

(iii) autocrine growth stimulating cytokines; and

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

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

16. The genetically modified NK cell of claim 14, wherein the NK cell is a NK-92 cell.

17. The genetically modified NK cell of any one of claims 14-16, wherein the extracellular binding domain comprises scFv.

18. The genetically modified NK cell of claim 14, wherein the signaling domain comprises an FcεRIγ signaling domain or a CD3ζ signaling domain.

19. Cancellation.

20. The genetically modified NK cell of claim 14, wherein the recombinant CD16 is a CD16 ^158V mutant.

21. The genetically modified NK cell of claim 14, wherein the autocrine growth stimulating cytokine is IL-2 or IL-15.

22. The genetically modified NK cell of claim 21, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence.

23. The genetically modified NK cell of claim 14, wherein the IL-12 is a single chain IL-12 heterodimer.

24. The genetically modified NK cell of claim 14, wherein the TGF-beta well comprises a single-chain dimer of the TGF-beta receptor II ectodomain.

25. The genetically modified NK cell of claim 24, wherein the TGF-beta well is a secreted form of a single-chain dimer of the TGF-beta receptor II ectodomain.

26. The genetically modified NK cell of claim 14, wherein the homing receptor is a cell adhesion molecule, selectin, integrin, C-C chemokine receptor or C-X-C chemokine receptor.

27. The genetically modified NK cell of claim 26, wherein the homing receptor is selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, Group consisting of CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC and CXCL14 receptors.

28. A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the genetically modified NK cells of claim 1 or claim 14, thereby treating the cancer.

29. The method of claim 28, 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 grafts, and tumor-targeting cytokines.

30. The method of claim 28, wherein the cancer is lung cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, or head and neck cancer.

31. The method of claim 28, wherein about 1 x ¹⁰⁸ to about ¹ x 1011 cells per ^m2 of patient body surface area are administered to the patient.

32. Use of the genetically modified NK cells of claim 1 or claim 14 in the treatment of cancer.

Claims (32)

1. A genetically modified NK cell expressing (i) a membrane-bound recombinant chimeric antigen receptor (CAR) comprising an extracellular binding domain, a hinge domain, a transmembrane domain and a signaling domain in a single polypeptide chain, Wherein the extracellular binding domain specifically binds to EGFR superfamily receptors; (ii) recombinant CD16; (iii) autocrine growth stimulating cytokines; 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 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 FcεRIγ signaling domain or a CD3ζ signaling domain. 5. The genetically modified NK cell of any one of the preceding claims, wherein the EGFR superfamily receptor is HER-2 or EGFR. 6. The genetically modified NK cell of any one of the preceding claims, wherein the recombinant CD16 is a CD16 ^158V mutant. 7. The genetically modified NK cell of any one of the preceding claims, wherein the autocrine growth stimulating cytokine is IL-2 or IL-15. 8. The genetically modified NK cell of claim 7, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence. 9. The genetically modified NK cell of any one of the preceding claims, wherein the IL-12 is a single chain IL-12 heterodimer. 10. The genetically modified NK cell of any one of the preceding claims, wherein the TGF-beta well comprises a single-chain dimer of the TGF-beta receptor II ectodomain. 11. The genetically modified NK cell of claim 10, wherein the TGF-beta well is a secreted form of a single-chain dimer of the TGF-beta receptor II ectodomain. 12. The genetically modified NK cell of any one of the preceding claims, wherein the homing receptor is a cell adhesion molecule, selectin, integrin, C-C chemokine receptor or C-X-C chemokine receptor body. 13. The genetically modified NK cell as claimed in claim 12, wherein the homing receptor is selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, Group consisting of CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC and CXCL14 receptors. 14. A genetically modified NK cell comprising: Recombinant nucleic acids encoding (i) a membrane-bound recombinant chimeric antigen receptor (CAR) comprising an extracellular binding domain, a hinge domain, a transmembrane domain and a signaling domain in a single polypeptide chain, Wherein the extracellular binding domain specifically binds to EGFR superfamily receptors; (ii) recombinant CD16; (iii) autocrine growth stimulating cytokines; and (iv) Optionally one of IL-12, TGF-beta trap or homing receptor. 15. The genetically modified NK cell of claim 14, wherein the recombinant nucleic acid is polycistronic RNA. 16. The genetically modified NK cell of claim 14, wherein the NK cell is a NK-92 cell. 17. The genetically modified NK cell of any one of claims 14-16, wherein the extracellular binding domain comprises scFv. 18. The genetically modified NK cell of any one of claims 14-17, wherein the signaling domain comprises an FcεRIγ signaling domain or a CD3ζ signaling domain. 19. The genetically modified NK cell of any one of claims 14-18, wherein the EGFR superfamily receptor is HER-2 or EGFR. 20. The genetically modified NK cell of any one of claims 14-19, wherein the recombinant CD16 is a CD16 ^158V mutant. 21. The genetically modified NK cell of any one of claims 14-20, wherein the autocrine growth stimulating cytokine is IL-2 or IL-15. 22. The genetically modified NK cell of claim 21, wherein the autocrine IL-2 or IL-15 further comprises an endoplasmic retention sequence. 23. The genetically modified NK cell of any one of claims 14-22, wherein the IL-12 is a single chain IL-12 heterodimer. 24. The genetically modified NK cell of any one of claims 14-23, wherein the TGF-beta well comprises a single-chain dimer of the TGF-beta receptor II extracellular domain. 25. The genetically modified NK cell of claim 24, wherein the TGF-beta well is a secreted form of a single-chain dimer of the TGF-beta receptor II ectodomain. 26. The genetically modified NK cell of any one of claims 14-25, wherein the homing receptor is a cell adhesion molecule, selectin, integrin, C-C chemokine receptor or C-X-C chemoattractant factor receptors. 27. The genetically modified NK cell of claim 26, wherein the homing receptor is selected from the group consisting of CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, Group consisting of CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1, CCXCKR, D6, DARC and CXCL14 receptors. 28. A method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of any of the genetically modified NK cells of claims 1-27, thereby treating the cancer . 29. The method of claim 28, 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 grafts, and tumor-targeting cytokines. 30. The method of claim 28 or 29, wherein the cancer is lung cancer, breast cancer, thyroid cancer, esophageal cancer, gastric cancer, gastroesophageal cancer or head and neck cancer. 31. The method of any one of claims 28-30, wherein about 1 x ¹⁰⁸ to about ¹ x 1011 cells/ ^m2 patient body surface area is administered to the patient. 32. Use of a genetically modified NK cell according to any one of claims 1-27 in the treatment of cancer.

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