[Show abstract][Hide abstract] ABSTRACT: Malignant brain tumors have a dismal prognosis, with residual after surgery necessitating adjuvant chemoradiotherapy. We previously demonstrated that targeted Natural Killer (NK-92) cells could be delivered to the brain using a combination of MRI-guided focused ultrasound and Definity microbubbles. Once in the CNS, they can track to malignant tissues without inflicting collateral damage. The HER2 receptor is expressed by epithelial tumours including both breast and glioblastoma; breast tumors with HER2-amplification have a higher risk of CNS metastasis, and poorer prognosis.
We investigated whether multiple combined treatments of targeted NK-92 cells and focused ultrasound with microbubbles could slow tumour growth and improve survival in an orthotopic HER2-amplified rodent brain tumour model using a human breast cancer line as a prototype.
Early daily treatments with targeted NK-92 cells and ultrasound improved survival and decreased tumour volumes compared with bi-weekly treatments, or either treatment alone. The intensive treatment paradigm resulted in cure in 50% of subjects.
Many tumour proteins could be exploited for targeted therapy with the NK-92 cell line, and combined with the mounting safety evidence for transcranial ultrasound, this may soon provide a non-invasive and highly targeted treatment option for patients with brain tumours.
The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques 05/2015; 42(S1):S11. DOI:10.1017/cjn.2015.76 · 1.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The disialoganglioside GD2 is a well-established target antigen for passive immunotherapy in neuroblastoma (NB). Despite the recent success of passive immunotherapy with the anti-GD2 antibody ch14.18 and cytokines, treatment of high-risk NB remains challenging. We expanded the approach of GD2-specific, antibody-based immunotherapy to an application of a GD2-specific natural killer (NK) cell line, NK-92-scFv(ch14.18)-zeta. NK-92-scFv(ch14.18)-zeta is genetically engineered to express a GD2-specific chimeric antigen receptor generated from ch14.18. Here, we show that chimeric receptor expression enables NK-92-scFv(ch14.18)-zeta to effectively lyse GD2(+) NB cells also including partially or multidrug-resistant lines. Our data suggest that recognition of GD2 by the chimeric receptor is the primary mechanism involved in NK-92-scFv(ch14.18)-zeta-mediated lysis and is independent of activating NK cell receptor/ligand interactions. Furthermore, we demonstrate that NK-92-scFv(ch14.18)-zeta is able to mediate a significant anti-tumor response in vivo in a drug-resistant GD2(+) NB xenograft mouse model. NK-92-scFv(ch14.18)-zeta is an NB-specific NK cell line that has potential for future clinical development due to its high stability and activity toward GD2(+) NB cell lines.
Cancer Immunology and Immunotherapy 02/2015; 64(5). DOI:10.1007/s00262-015-1669-5 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.
Frontiers in Pharmacology 02/2015; 6:21. DOI:10.3389/fphar.2015.00021 · 3.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3? signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. ?-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.Molecular Therapy (2014); doi:10.1038/mt.2014.219.
[Show abstract][Hide abstract] ABSTRACT: While EGFRvIII appears a logical target for immunotherapy, only a subpopulation of tumor cells express EGFRvIII and immune escape has been demonstrated. ErbB2 is overexpressed in a substantial proportion of glioblastomas and has been successfully utilized in immunotherapies. Natural killer (NK) cells are the first line of defense against viral infections and malignant cells. The continuously growing cytotoxic cell line NK-92 holds promise for cancer immunotherapy. Safety of infusion of high doses of NK-92 was established in previous phase I clinical trials utilizing irradiated cells to prevent permanent engraftment.
Cancer Research 07/2014; 16 Suppl 3(8 Supplement):iii44. DOI:10.1093/neuonc/nou209.9 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) plays an important role in essential cellular processes such as proliferation, survival and migration. Aberrant activation of EGFR is frequently found in human cancers of various origins and has been implicated in cancer pathogenesis. The therapeutic antibody cetuximab (Erbitux) inhibits tumor growth by binding to the extracellular domain of EGFR, thereby preventing ligand binding and receptor activation. This activity is shared by the single chain antibody fragment scFv(225) that contains the same antigen binding domain. The unrelated EGFR-specific antibody fragment scFv(30) binds to the intracellular domain of the receptor and retains antigen binding upon expression as an intrabody in the reducing environment of the cytosol. Here, we used scFv(225) and scFv(30) domains to generate a novel type of bispecific transmembrane antibody termed 225.TM.30, that simultaneously targets intra- and extracellular EGFR epitopes. Bispecific 225.TM.30 and related membrane-anchored monospecific 225.TM and TM.30 proteins carrying extracellular scFv(225) or intracellular scFv(30) antibody fragments linked to a transmembrane domain were expressed in EGFR-overexpressing tumor cells using a doxycycline-inducible retroviral system. Induced expression of 225.TM.30 and 225.TM, but not TM.30 reduced EGFR surface levels and ligand-induced EGFR activation, while all three molecules markedly inhibited tumor cell growth. Co-localization of 225.TM with EGFR was predominantly found on the cell surface, while interaction with 225.TM.30 and TM.30 proteins resulted in the redistribution of EGFR to perinuclear compartments. Our data demonstrate functionality of this novel type of membrane-anchored intrabodies in tumor cells and suggest distinct modes of action of mono- and bispecific variants.
International Journal of Cancer 06/2014; 134(11). DOI:10.1002/ijc.28585 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background aims
Cytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential.
In this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells.
In vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro.
Our study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.
[Show abstract][Hide abstract] ABSTRACT: Multiple natural killer (NK) cell-based anticancer therapies are currently under development. Here, we compare the efficiency of genetically modified NK-92 cells expressing chimeric antigen receptors (CARs) at killing NK cell-resistant B-lymphoid leukemia cells to the antibody-dependent cell-mediated cytotoxicity (ADCC) of NK-92 cells expressing a high affinity variant of the IgG Fc receptor (FcγRIII). First, we compared in vitro the abilities of NK-92 cells expressing CD20-targeting CARs to kill primary chronic lymphocytic leukemia (CLL) cells derived from 9 patients with active, untreated disease to the cytotoxicity of NK-92 cells expressing FcγRIII combined with either of the anti-CD20 monoclonal antibodies (mAbs) rituximab or ofatumumab. We found that CAR-expressing NK-92 cells effectively kill NK cell-resistant primary CLL cells and that such a cytotoxic response is significantly stronger than that resulting from ADCC. For studying CAR-expressing NK cell-based immunotherapy in vivo, we established xenograft mouse models of residual leukemia using the human BCR-ABL1(+) cell lines SUP-B15 (CD19(+)CD20(-)) and TMD-5 (CD19(+)CD20(+)), two acute lymphoblastic leukemia (ALL) lines that are resistant to parental NK-92 cells. Intravenous injection of NK-92 cells expressing CD19-targeting CARs eliminated SUP-B15 cells, whereas they had no such effect on TMD-5 cells. However, the intrafemoral injection of NK-92 cells expressing CD19-targeting CAR resulted in the depletion of TMD-5 cells from the bone marrow environment. Comparative studies in which NK-92 cells expressing either CD19- or CD20-targeting CARs were directly injected into subcutaneous CD19(+)CD20(+) Daudi lymphoma xenografts revealed that CD20-targeting CAR is superior to its CD19-specific counterpart in controlling local tumor growth. In summary, we show here that CAR-expressing NK-92 cells can be functionally superior to ADCC (as mediated by anti-CD20 mAbs) in the elimination of primary CLL cells. Moreover, we provide data demonstrating that the systemic administration of CAR-expressing NK-92 cells can control lymphoblastic leukemia in immunocompromised mice. Our results also suggest that the direct injection of CAR-expressing NK-92 cells to neoplastic lesions could be an effective treatment modality against lymphoma.
[Show abstract][Hide abstract] ABSTRACT: Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer.
PLoS ONE 08/2013; 8(8):e72756. DOI:10.1371/journal.pone.0072756 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells hold great promise for adoptive cancer immunotherapy. The antitumor activity of NK cells can be enhanced by the transgene driven expression of chimeric antigen receptors that facilitate the selective recognition and killing of malignant cells. Recent data from our laboratory suggest that NK cells may similarly be "armed" against neoplastic cells by the expression of cancer-specific granzyme B-containing fusion proteins that are released as soluble factors upon NK-cell activation.
[Show abstract][Hide abstract] ABSTRACT: Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor-the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment.Molecular Therapy-Nucleic Acids (2013) 2, e105; doi:10.1038/mtna.2013.32; published online 9 July 2013.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.
PLoS ONE 04/2013; 8(4):e61267. DOI:10.1371/journal.pone.0061267 · 3.23 Impact Factor