[Show abstract][Hide abstract] ABSTRACT: Epidermal 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 wildtype 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.
[Show abstract][Hide abstract] ABSTRACT: Natural killer (NK) cells are increasingly considered as immunotherapeutic agents in particular in the fight against cancers. NK cell therapies are potentially broadly applicable and, different from their T cell counterparts, do not cause graft-versus-host disease. Efficacy and clinical in vitro or in vivo expansion of primary NK cells will however always remain variable due to individual differences of donors or patients. Long-term storage of clinical NK cell lots to allow repeated clinical applications remains an additional challenge. In contrast, the established and well-characterized cell line NK-92 can be easily and reproducibly expanded from a good manufacturing practice (GMP)-compliant cryopreserved master cell bank. Moreover, no cost-intensive cell purification methods are required. To date, NK-92 has been intensively studied. The cells displayed superior cytotoxicity against a number of tumor types tested, which was confirmed in preclinical mouse studies. Subsequent clinical testing demonstrated safety of NK-92 infusions even at high doses. Despite the phase I nature of the trials conducted so far, some efficacy was noted, particularly against lung tumors. Furthermore, to overcome tumor resistance and for specific targeting, NK-92 has been engineered to express a number of different chimeric antigen receptors (CARs), including targeting, for example, CD19 or CD20 (anti-B cell malignancies), CD38 (anti-myeloma) or human epidermal growth factor receptor 2 (HER2; ErbB2; anti-epithelial cancers). The concept of an NK cell line as an allogeneic cell therapeutic produced 'off-the-shelf' on demand holds great promise for the development of effective treatments.
No preview · Article · Nov 2015 · Cancer Immunology and Immunotherapy
[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.
Preview · Article · May 2015 · The Canadian journal of neurological sciences. Le journal canadien des sciences neurologiques
[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.
Full-text · Article · Feb 2015 · Cancer Immunology and Immunotherapy
[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.
Preview · Article · Feb 2015 · Frontiers in Pharmacology
[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.
Full-text · Article · Nov 2014 · Molecular Therapy
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: 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. METHODS: To provide NK-92 cells with pre-determined tumor-cell specificity, we generated a lentiviral
second generation chimeric antigen receptor (CAR) construct (5.28.z) employing the ErbB2 (HER2)-specific scFv(FRP5) antibody
fragment for target cell recognition, and human CD28-CD3 ζ as a composite signaling moiety. An ErbB2-specific single cell
clone (NK-92/5.28.z) was isolated, which showed high and selective cytotoxicity towards ErbB2-expressing tumor cells of various
origins in vitro. We evaluated the cytotoxicity of NK-92/5.28.z cells against a panel of glioblastoma cell lines and primary
glioblastoma cultures with different levels of Erb2 expression in vitro and in vivo. RESULTS: 13 out of a total of 15 established
glioblastoma cell lines and 5 out of 5 primary glioblastoma cell lines were specifically lysed by NK-92/5.28.z cells, although
to varying degrees. ErbB2 expression was necessary for cytotoxicity of NK-92/5.28.z cells towards glioblastoma cells, and
ectopic overexpression of ErbB2 enhanced their cytotoxicity. In subcutaneous glioblastoma xenograft models, injections of
NK-92/5.28.z induced prolonged regression in NOD-scid/gamma (NSG) mice with grafts of LN-319 cells and retarded the growth
of LNT-229 grafts. In an orthotopic intracranial model utilizing LN-319 cells, tumor size in mice intratumorally injected
with NK-92/5.28.z cells weekly was substantially smaller compared to controls as assessed by MR imaging. Injections of NK-92/5.28.z
cells also conferred an impressive advantage in symptom free survival in two independent experiments (median 73 days in controls
versus 140 days in NK-92/5.28.z treated animals in experiment A, p < 0,05, and median 77 days in controls versus 163 days
in NK-92/5.28.z treated animals in experiment B, p < 0,01). In preparation of a phase I trial, GMP-compliant protocols for
expansion of the NK-92/5.28.z line were established. CONCLUSIONS: Adoptive immunotherapy with application of ErbB2-specific
NK-92/5.28.z cells may be a promising new immunotherapy approach for ErB2 positive glioblastoma. A phase I trial for glioblastoma
patients with local injections of NK-92/5.28.z cells is in preparation.
[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.
[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.
No preview · Article · Jun 2014 · International Journal of Cancer
[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.