Chimeric Antigen Receptor Natural Killer Cells Research Articles

GMP-compliant, automated process for generation of CAR NK cells in a closed system for clinical use

Background & Aim Major advances have been made in harnessing natural killer (NK) cells in cancer immunotherapy in recent years. Regulated by their germ-line encoded activating and inhibitory receptors, NK cells can recognize and eliminate tumor cells rapidly without prior sensitization. Clinical evidence has shown that donor-derived NK cells have low risk in inducing graft-versus-host-disease (GvHD) as well as a reduced risk of life-threatening cytokine storms during therapy. Both properties make NK cells ideal for allogeneic application. To enhance the anti-tumor specificity and efficiency, NK cells can be further modified by chimeric antigen receptors (CARs). Promising clinical outcome of CAR-engineered NK cells has been recently reported for patients with relapsed or refractory B cell malignancies. Methods, Results & Conclusion In this study purified NK cells were cultivated with a feeder cell-free protocol including cytokines in NK MACS GMP Medium. For generation of CAR NK cells we used a Baboon endogenous virus (BaEV)-pseudotyped lentiviral vector system to efficiently transduce NK cells with various CARs and routinely obtained transduction efficiencies between 30-70%. To facilitate the clinical application of CAR NK cells, we implemented this protocol into a highly efficient, automated process to generate CAR NK cells under good manufacturing practice (GMP)-compliant conditions in a closed system by using CliniMACS Prodigy device. The process covers the complete procedure of NK cell manufacturing, including separation, activation, gene modification, and expansion/cultivation. High purity of NK cells (mean 94%) could be achieved by CD3 depletion followed by subsequent CD56 enrichment, resulting in a mean log depletion of T cells of 4.3. Inhouse evaluation runs with leukapheresis products (n=11) achieved a mean transduction efficiency of 52.0% CAR+ cells, resulting in 5.2 – 17.9 x 108 (mean 7.17 x 108) CAR+ NK cells after 14 days of culture. In summary, we developed a novel process for automated NK cell purification, transduction, and cultivation in a closed GMP-compatible system. The high level of automation enables standardized, consistent, and operator independent genetic engineering of NK cells for therapeutic applications.

Abstract A61: Human leukocyte antigen G as a novel target for switch-based chimeric antigen receptor natural killer cell therapy of solid cancers

Abstract Human leukocyte antigen G (HLA-G) is a molecule within the tumor microenvironment (TME) that modulates the innate and adaptive immune systems by interacting with inhibitory receptors on the surface of immune cells and thus functions as an immune checkpoint. It could be potentially expressed by all tumor types but is not expressed by either healthy tissues surrounding the tumor cells or vital normal tissues. Chimeric antigen receptors (CARs) for adoptive cell therapy (ACT) have been successful in clinical trials against hematologic cancers; however, challenges have been encountered when applying this approach to the treatment of solid tumors. These obstacles are mainly due to the lack of a ubiquitous tumor-associated antigen (TAA) across different tumor types without “on-target/off-tumor” reactivity and the immunosuppressive nonphysical TME. To address these issues, we developed a novel switch HLA-G CAR carrying an inducible Caspase9 (iC9) suicide gene system that binds to HLA-G1~G7 isoforms and expressed this CAR in natural killer (NK) cells. We tested these HLA-G CAR-NK cells in a variety of adult cancer models and discovered that they mediate significant tumor cytolysis in triple-negative breast cancer (TNBC), glioblastoma (GBM), pancreatic (PA) cancer, and ovarian (OV) cancer in vitro and in TNBC and GBM xenograft models in vivo. Coculturing HLA-G CAR-NK cells with vital normal cell lines did not cause cell damage. We further discovered that surface-exposed HLA-G is chemoinducible, which in turn increases tumor sensitivity to both HLA-G CAR effector-mediated antitumor responses and tumor-infiltrative NK cells. The underlying mechanism of tumor and HLA-G CAR NK cell interaction may be through upregulation of membranous HLA-G via demethylation of the TAP-1 promoter or enhanced activity of the TAP1/signal peptide peptidase (SPP) pathways. In conclusion, HLA-G CAR-NK cells could be an option for treating solid tumors of different cell types, and a regimen comprising chemotherapy followed by HLA-G CAR immunotherapy may improve responses compared to those achieved with CAR-T cell immunotherapy alone. Citation Format: Chia-Ing Jan, Shi-Wei Huang, Peter Canoll, Yu-Chuan Lin, Hsin-Man Lu, Shao-Chih Chio, Der-Yang Cho. Human leukocyte antigen G as a novel target for switch-based chimeric antigen receptor natural killer cell therapy of solid cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A61.

Use of chimeric antigen receptor NK-92 cells to target mesothelin in ovarian cancer

Mesothelin (MSLN) has been reported to be overexpressed in ovarian cancer and may be an ideal target for immunotherapy. Recent studies have suggested that natural killer (NK) cells may be better chimeric antigen receptor (CAR) drivers because of their favorable innate characteristics, such as directly recognizing and killing tumor cells, resulting in a graft-versus-tumor effect but irresponsible for graft-versus-host disease (GVHD). The therapeutic effects of CAR-engineered NK cells targeting MSLN in ovarian cancer have not been evaluated. In this study, MSLN- and CD19-targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed. Both MSLN- and CD19-CAR molecules were highly expressed on the surface of NK-92 cells following lentiviral gene transduction. MSLN-CAR NK cells specifically killed MSLN-positive ovarian cancer cells (OVCAR-3 and SK-OV-3), rather than MSLN-negative cells (SK-HEP-1), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretion was detected in MSLN-CAR NK cells cocultured with OVCAR-3 and SK-OV-3. Furthermore, MSLN-CAR NK cells effectively eliminated ovarian cancer cells in both subcutaneous and intraperitoneal tumor models; these cells also significantly prolonged the survival of intraperitoneally tumor-bearing mice. These results demonstrate that MSLN-CAR NK cells have robust specific antitumor activity, both in vitro and in vivo, suggesting that mesothelin could be a potential target for CAR NK cells and could be applied in the treatment of ovarian cancer.

To PARP or not to PARP?-Toward sensitizing acute myeloid leukemia stem cells to immunotherapy.

A new study reveals that leukemia stem cells (LSCs) in acute myeloid leukemia downregulate natural killer cell-activating receptor ligands to evade immune surveillance via the transcriptional co-factor PARP1. The inhibition of PARP1 sensitizes LSCs to immunotherapy, highlighting its potential as a therapeutic target.

Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1–2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log −3.6 after CD3 depletion and log −3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2–8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector–target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.

Genetically engineered CAR NK cells display selective cytotoxicity against FLT3-positive B-ALL and inhibit in vivo leukemia growth.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising effector cell type for adoptive cancer immunotherapy. Both, genetically modified donor-derived NK cells as well as continuously expanding NK-92 cells are currently under clinical development. To enhance their therapeutic utility for the treatment of pre-B-cell acute lymphoblastic leukemia (B-ALL), we engineered NK-92 cells by lentiviral gene transfer to express a FMS-like tyrosine kinase 3 (FLT3)-specific CAR that contains a composite CD28-CD3ζ signaling domain. FLT3 has primarily been described as a therapeutic target for acute myeloid leukemia, but overexpression of FLT3 has also been reported in B-ALL. Exposure of FLT3-positive targets to CAR NK-92 cells resulted in conjugate formation between NK and leukemia cells, NK-cell degranulation and selective cytotoxicity toward established B-ALL cell lines and primary blasts that were resistant to parental NK-92. In a SEM B-ALL xenograft model in NOD-SCID IL2R γnull mice, treatment with CAR NK-92 but not parental NK-92 cells markedly inhibited disease progression, demonstrating high antileukemic activity in vivo. As FLT3 is known to be also expressed on precursor cells, we assessed the feasibility of incorporating an inducible caspase-9 (iCasp9) suicide switch to enhance safety of our approach. Upon addition of the chemical dimerizer AP20187 to NK-92 cells coexpressing the FLT3-specific CAR and iCasp9, rapid iCasp9 activation was observed, precluding further CAR-mediated cytotoxicity. Our data demonstrate that B-ALL can be effectively targeted by FLT3-specific CAR NK cells which may complement CD19-directed immunotherapies, particularly in cases of inherent or acquired resistance to the latter.

Therapeutic Targeting of Follicular Helper T Cells with Chimeric Antigen Receptor-Expressing Natural Killer Cells

Follicular helper T cells (TFH) are critical for vaccine and infection elicitation of long-lived humoral immunity, but exaggerated TFH responses can promote autoimmunity and other pathologies. Unfortunately, no clinical interventions are currently available for selective depletion of TFH cells to alleviate these disease conditions. We engineered a novel chimeric antigen receptor (CAR) that facilitates specific targeting of cells highly expressing human programmed cell death protein 1 (PD-1), a cardinal feature of TFH cells. CAR-expressing natural killer (NK) cells robustly degranulate in response to PD-1, resulting in discriminatory elimination of human tonsil TFH cells while sparing other T and B-cell subsets. Given that CAR NK cells are emerging as a safe and effective alternative to CAR T cells in cancer immunotherapy, our results suggest a new, clinically-translatable application of CAR NK cells for selective depletion of pathogenic TFH cells in specific disease states.

Abstract B044: Engineering CAR NK cells for antigen-dependent autocrine expansion

Abstract Given promising results with chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies attention has turned to whether CAR natural killer (NK) cells might have utility in cancer immunotherapy. NK cells are known to have potent cytolytic function and the ability to secrete interferons and cytokines, including GM-CSF and interferon-γ (IFNγ) upon stimulation. In pre-clinical models CAR NK cells have been shown to have efficacy and possibly some advantages over CAR T-cells. Several biotechs and pharmas are currently exploring this approach, but a major limitation of NK cells is their inability to produce autocrine growth factors to promote their own expansion and activation. In the context of CAR NK cell therapy, a major limitation is the inability of the infused CAR NK cells to expand in the absence of cytokine signals to initiate their proliferation and enhance their effector functions. IL-12 and IL-15 are essential for the clonal expansion of NK cells; however, recombinant IL-12 and IL-15 are quite toxic when administered systemically. Additionally, engineering NK cells to constitutively secrete IL-12 or IL-15 is not an attractive strategy as this may also result in off-target toxicity. Our goal is to engineer chimeric receptors with the intracellular signaling domains of IL-12 or IL-15 receptors fused to the extracellular domains of cytokine receptors. Consequently, self-secreted cytokines by activated NK cells or recognition of tumor cells will provide autocrine growth signal, permit NK clonal expansion and enhance NK effector functions. Citation Format: Avishai Shemesh, Kole T. Roybal, Lewis L. Lanier. Engineering CAR NK cells for antigen-dependent autocrine expansion [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B044.

Novel Human NK Cell Line Carrying CAR Targeting EGFRvIII Induces Antitumor Effects in Glioblastoma Cells.

Natural killer (NK) cells are considered potential antitumor effector cells. The aim of this study was to establish a novel type of a chimeric antigen receptor (CAR) NK cell line (CAR-KHYG-1) specific for epidermal growth factor receptor variant III (EGFRvIII)-expressing tumors and investigate the anti-tumor activity of EGFRvIII-specific-CAR-KHYG-1 (EvCAR-KHYG-1). EvCAR-KHYG-1 was established by self-inactivated lentiviral-based transduction of the EvCAR gene and magnetic bead-based purification of EvCAR-expressing NK cells. The anti-tumor effects of EvCAR-KHYG-1 were evaluated using growth inhibition and apoptosis detection assays in glioblastoma (GBM) cell lines (EGFRvIII-expressing and non-expressing U87MG). The findings demonstrated that EvCAR-KHYG-1 inhibited GBM cell-growth via apoptosis in an EGFRvIII-expressing specific manner. This is the first study to establish a CAR NK cell line based on the human NK cell line KHYG-1. Therapy with EvCAR-KHYG-1 may be an effective treatment option for GBM patients.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3-) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO™10, CellGro®, TexMACS™, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

The therapeutic potential of natural killer cells in treating systemic lupus erythematosus.

Abstract Systemic lupus erythematosus (SLE) is a chronic disease defined by rampant autoantibody production and multi-organ pathology. Standard treatments for SLE are broadly immunosuppressive and often ineffective, as ~38% of patients develop lupus nephritis (LN) that can result in kidney failure and death. Current therapeutics may lack efficacy because they do not target the specific immune processes underlying SLE. For example, lupus autoantibodies can arise from aberrant germinal centers (GCs) driven by dysregulated follicular helper T cells (TFH). We have shown that natural killer (NK) cells suppress activated TFH during immunization, resulting in blunted GCs and antibody production. We hypothesize that an effective new strategy to treat SLE would involve harnessing of this NK cell-mediated humoral regulation. To assess whether therapeutic elevation of NK cell numbers can ameliorate disease, we used IL-2/αIL-2 complexes to expand endogenous populations of NK cells in the bm12 cGVHD mouse model of SLE-like disease. We also evaluated ex vivo activation, expansion, and infusion of NK cells as an immunotherapeutic means to ameliorate disease in mouse models of SLE. Finally, we are engineering human NK cells expressing a programmed death-ligand 1 (PD-L1)-based chimeric antigen receptor (CAR) that will facilitate cytolytic elimination of TFH and B cells that highly express programmed cell death protein 1 (PD-1). We plan to evaluate the capacity of these CAR NK cells to selectively kill PD-1+ targets and to reduce autoantibody-mediated LN in humanized mouse models of SLE. These CAR NK cells represent an innovative means to curtail autoantibody production, particularly during disease flares, and thereby promote disease remission in SLE.

A role for multiple chimeric antigen receptor-expressing leukocytes in antigen-specific responses to cancer.

While adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells can induce remission of some tumors, the role of other CAR-modified leukocytes is not well characterized. In this study, we characterize the function of leukocytes including natural killer (NK) cells, macrophages and CAR T cells from transgenic mice expressing a CAR under the control of the pan-hematopoietic promoter, vav, and determine the ability of these mice to respond to ERB expressing tumors. We demonstrate the anti-tumor functions of leukocytes, including antigen specific cytotoxicity and cytokine secretion. The adoptive transfer of CAR T cells provided a greater survival advantage in the E0771ERB tumor model than their wildtype (WT) counterparts. In addition, CAR NK cells and CAR T cells also mediated increased survival in the RMAERB tumor model. When challenged with Her2 expressing tumors, F38 mice were shown to mount an effective immune response, resulting in tumor rejection and long-term survival. This was shown to be predominantly dependent on both CD8+ T cells and NK cells. However, macrophages and CD4+ T cells were also shown to contribute to this response. Overall, this study highlights the use of the vav-CAR mouse model as a unique tool to determine the anti-tumor function of various immune subsets, either alone or when acting alongside CAR T cells in adoptive immunotherapy.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival

ABSTRACTEpidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

ErbB2/HER2-Specific NK Cells for Targeted Therapy of Glioblastoma.

Glioblastoma (GBM) is the most common and malignant intracranial tumor in adults and currently incurable. To specifically target natural killer (NK) cell activity to GBM, we employed NK-92/5.28.z cells that are continuously expanding human NK cells expressing an ErbB2-specific chimeric antigen receptor (CAR). ErbB2 expression in 56 primary tumors, four primary cell cultures, and seven established cell lines was assessed by immunohistochemistry and flow cytometry. Cell killing activity of NK-92/5.28.z cells was analyzed in in vitro cytotoxicity assays. In vivo antitumor activity was evaluated in NOD-SCID IL2Rγ(null) (NSG) mice carrying orthotopic human GBM xenografts (6 to 11 mice per group) and C57BL/6 mice carrying subcutaneous and orthotopic ErbB2-expressing murine GBM tumors (5 to 8 mice per group). Statistical tests were two-sided. We found elevated ErbB2 protein expression in 41% of primary GBM samples and in the majority of GBM cell lines investigated. In in vitro assays, NK-92/5.28.z in contrast to untargeted NK-92 cells lysed all ErbB2-positive established and primary GBM cells analyzed. Potent in vivo antitumor activity of NK-92/5.28.z was observed in orthotopic GBM xenograft models in NSG mice, leading to a marked extension of symptom-free survival upon repeated stereotactic injection of CAR NK cells into the tumor area (median survival of 200.5 days upon treatment with NK-92/5.28.z vs 73 days upon treatment with parental NK-92 cells, P < .001). In immunocompetent mice, local therapy with NK-92/5.28.z cells resulted in cures of transplanted syngeneic GBM in four of five mice carrying subcutaneous tumors and five of eight mice carrying intracranial tumors, induction of endogenous antitumor immunity, and long-term protection against tumor rechallenge at distant sites. Our data demonstrate the potential of ErbB2-specific NK-92/5.28.z cells for adoptive immunotherapy of glioblastoma, justifying evaluation of this approach for the treatment of ErbB2-positive GBM in clinical studies.

Modulation of Natural Killer Cell Activity in the Setting of Oncolytic Virotherapy and with a Chimeric Antigen Receptor

Natural killer (NK) cells are innate lymphocytes that can rapidly eradicate tumor cells, especially those lacking MHC Class I molecules. NK cells can also rapidly eradicate herpes virus-infected cells. We designed an oncolytic herpes virus (oHSV) to selectively infect, replicate within, and lyse glioblastoma (GBM), a devastating brain tumor with a median survival of only 15 months following diagnosis. We have shown that the rapid influx of NK cells limits oHSV efficacy in GBM as they impede oHSV replication and spread [Alvarez-Breckenridge et al., Nat Med, 2012, 18(12):1827-34]. In the current study, we developed NK cell-based novel GBM therapies by decreasing the brain influx of NK cells to enhance the efficacy of oHSV, while arming NK cells in the brain with a chimeric antigen receptor (CAR) that targets both the wild-type EGFR and its mutant form EGFRvIII, two GBM tumor-associated antigens. We then investigated the synergistic effects between EGFR-CAR NK cells and oHSV.Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine of NK cells [Yu et al, Immunity, 2006, 24(5):575-90]. We first determined if oHSV efficacy for treatment of GBM would be augmented by inhibiting anti-oHSV activity of NK cells with TGF-β pre-treatment. In vitro, NK cells pre-treated with TGF-β displayed less cytolytic capacity against oHSV-infected GBM cell lines and patient-derived GBM stem-like cells. In viral replication assays, co-culturing oHSV-infected GBM cells with NK cells pre-treated with TGF-β significantly increased virus titers. In an immunocompetent syngeneic GBM mouse model,administration of TGF-β to GBM-bearing mice prior to oHSV injection significantly inhibited intracranial infiltration and activation of NK cells (P < 0.05). In orthotopic human GBM xenograft mouse models and in syngeneic GBM mouse models, TGF-β treatment in vivo prior to oHSV therapy resulted in inhibition of NK cell infiltration, suppression of tumor growth and significantly prolonged survival of GBM-bearing mice (P < 0.05). Furthermore, depletion of NK cells incompletely blocked the positive effects of in vivo treatment of GBM with TGF-β on survival, suggesting that TGF-β may also directly act on other innate immune cells such as macrophages/microglia. These data demonstrate a single dose of TGF-β prior to oHSV administration enhances anti-tumor efficacy for GBM at least in part through the transient inhibition of the innate immune responses to oHSV infection.We next investigated whether NK cell activity could be enhanced to more directly target brain tumors while sparing eradication of oHSV. We therefore infected both human NK-92 cells and primary human NK cells to express the second generation CAR targeting both EGFR and EGFRvIII that we designed. Further, we asked if the treatment with EGFR-CAR NK cells plus oHSV could create a therapeutic synergy for the treatment to brain tumors. In vitro, compared with mock-transduced CAR-NK-cells, EGFR-CAR NK cells exhibited significantly higher cytotoxicity and IFN-γ production when co-cultured with tumor cells, for both NK-92 and primary NK cells (P < 0.01). Further, significantly higher cytolytic activity against tumor cells was obtained when CAR NK cells were combined with oHSV-1 infection of tumor cells, compared to either of the monotherapies alone (P < 0.05). In mice, to avoid oHSV clearance by the EGFR-CAR NK cells following the inoculation of the mouse with tumor cells, we administered these two agents sequentially; administering EGFR-CAR NK cells directly into the tumor first as a single injection of 2 × 106 cells, followed by intracranial infection with 2 × 105 plaque-forming units oHSV five days later, presumably after EGFR-CAR NK survival has diminished. Compared to vehicle controls, intracranial administration of either EGFR-CAR NK cells or oHSV blunted tumor growth. However, the combination of EGFR-CAR NK cells followed by oHSV infection resulted in significantly more efficient killing of tumor cells (P < 0.05) and significantly longer survival for tumor-bearing mice when compared to either monotherapy alone.Collectively, our studies demonstrate that in animal tumor models, we can combine novel NK cell and oHSV therapies to significantly improve survival. DisclosuresNo relevant conflicts of interest to declare.

CS1-Specific Chimeric Antigen Receptor (CAR)-Engineered NK Cells and T Cells Enhance In Vitro and In Vivo Anti-Tumor Activity Against Human Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by the aberrant clonal expansion of plasma cells (PCs) within the bone marrow (BM). Despite the use of proteasome inhibitors and immune-modulating drugs, which have improved overall survival, MM remains an incurable malignancy for which novel therapeutic approaches are urgently needed. Immunotherapy that specifically targets antigens expressed by MM would be a promising approach to treat MM patients refractory to any current treatments. Chimeric antigen receptors (CARs) are engineered fusion proteins containing tumor antigen-recognition moieties and immune cell activation domains. CAR-expressing T cells have been demonstrated successful in the clinic to treat chronic lymphocytic leukemia (CLL) and acute lymphoid leukemia (ALL). However, the potential utility of antigen-specific CAR-engineered natural killer (NK) cells or T cells to target MM-expressed CS1 to treat MM has not been previously explored, and is the focus of our study.CS1 is a surface glycoprotein and represents an ideal target for the treatment of MM. CS1 is highly, and nearly ubiquitously, expressed on MM cells, while expression remains very low on NK cells, some T-cell subsets, and normal B cells, and also it is almost undetectable on myeloid cells. In addition, monoclonal antibody directed against CS1, elotuzumab, has already been proven safe in phase 1 and 2 clinical trials, and phase 3 trials are ongoing. Therefore, it should be safe to target CS1 for the treatment of MM.We successfully generated a specific CS1-CAR construct with a lentiviral vector backbone, sequentially containing a signal peptide (SP), a heavy chain variable region (VH), a linker, a light chain variable region (VL), a hinge, CD28 and CD3ζ. Flow cytometry analysis with an antibody against VH and VL regions indicated that CS1-CAR was successfully expressed on the surface of NK cells and T cells transduced with the CAR construct. In vitro, CS1-CAR NK cells and T cells displayed enhanced MM cytolysis (detected by Cr51 release assay) and augmented production of cytokines (determined by enzyme-linked immunosorbent assay, ELISA), such as IFN-g for NK and T cells and IL-2 for T cells, when co-cultured with CS1-expressing MM cell lines. These effects relied on CS1-dependent recognition of MM cells because CS1-CAR NK or T cells possessed higher activity when they were co-cultured with CS1-expressing cells, but remained much lower activity when they were co-cultured with CS1-negative cells. However, CS1-CAR NK or T cells launched significantly higher killing of MM cells and secrete more abundant cytokines when the CS1-negative MM cells ectopically overexpressed CS1 and were co-cultured with the CAR cells. Ex vivo, compared to NK or T cells transduced with the empty vector, NK or T cells transduced with CS1-CAR also showed significantly enhanced effector functions when responding to purified primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, when compared to untreated mice or mice treated with empty vector-tranduced NK or T cells, adoptive transfer of 5 × 106 NK or T cells expressing CS1-CAR once every five days efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged survival of mice bearing IM9 MM cells. Our efforts to translate these findings into clinical trials are ongoing.In summary, CS1 is a promising target for using CAR NK or T cells for MM treatment, and we have generated a CAR that recognizes CS1. We demonstrate that NK cells or T cells armed with this CS1-CAR can recognize and eradicate myeloma cells in vitro and in vivo. Autologous or allogeneic transplantation of these CS1-specific CAR NK cells or CAR T cells may be a promising strategy to treat MM. Disclosures:Caligiuri:Innate Pharma: Membership on an entity's Board of Directors or advisory committees. Hofmeister:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

8714 Trabedersen (AP 12009) in recurrent or refractory high-grade glioma patients: results of a phase IIb study and outlook

Pancreatic cancer (PC) is the third leading cause of cancer-related death with a 5-year survival rate of approximately 10%. It typically presents as a late-stage incurable cancer and chemotherapy provides modest benefit. Here, we demonstrate the feasibility, safety, and potency of a novel human natural killer (NK) cell-based immunotherapy to treat PC.The expression of prostate stem cell antigen (PSCA) was evaluated in primary PC at messenger RNA and protein levels. The processes of retroviral transduction, expansion, activation, and cryopreservation of primary human NK cells obtained from umbilical cord blood were optimized, allowing us to develop frozen, off-the-shelf, allogeneic PSCA chimeric antigen receptor (CAR) NK cells. The safety and efficacy of PSCA CAR NK cells also expressing soluble (s) interleukin 15 (PSCA CAR_s15 NK cells) were evaluated in vitro and in vivo.PSCA was elevated in primary human PC compared with the adjacent or other normal tissues. PSCA CAR_s15 NK cells displayed significant tumor-suppressive effects against PSCA(+) PC in vitro before and after 1 cycle of freeze-thaw. The viability of frozen PSCA CAR_s15 NK cells persisted more than 90 days in vivo after their last infusion and significantly prolonged the survival of mice engrafted with human PC.PSCA CAR_s15 NK cells showed therapeutic efficacy in human metastatic PC models without signs of systematic toxicity, providing a strong rationale to support clinical development.