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Cell-penetrating peptides (CPPs) are peptides able to promote uptake of various cargos, including proteins and plasmids. Advances in recent years imply the uptake to be endocytic, where the current hurdle for efficient intracellular delivery is material being retained in the endosomes. In this study we wanted to compare the ability of various established CPPs to deliver siRNA and induce gene silencing of luciferase, with a novel designed penetratin analog having endosomolytic properties, using a noncovalent strategy. In principal, the penetratin analog EB1 will, upon protonation in the early-late endosomes, be able to form an amphipathic alpha helix resulting in permeabilization of the endosomal membrane. We demonstrate that even though all CPPs evaluated in this study can form complexes with siRNA, there is not a direct relationship between the complex formation ability and delivery efficacy. More important, although all CPPs significantly promote siRNA uptake, in some cases no gene silencing effect can be observed unless endosomal escape is induced. We find the designed endosomolytic peptide EB1 to be far more effective both in forming complexes and transporting biologically active siRNA than its parent peptide penetratin. We believe that developing CPPs with increased endosomolytical properties is a necessary step toward achieving biological effects at low concentrations for future in vivo applications.

IntroductionMultiple myeloma (MM) is a malignant neoplasm characterized by clonal proliferation of plasma cells in the bone marrow (BM). This disease accounts for approximately 2% of all cancer deaths and nearly 20% of deaths caused by hematologic malignancies. 1 Although allogeneic stem-cell transplantation 2-5 occasionally cures these patients, and drugs such as thalidomide, lenalidomide, and bortezomib have improved outcomes, 6 high-dose chemotherapy followed by autologous stem-cell transplantation (ASCT) 7 still appears to be the best treatment for patients up to 70 years of age. However, the great majority of patients with MM are incurable due to the persistence of minimal residual disease. 8,9 Thus, novel modalities complementing or improving current treatment options are needed.Natural killer (NK) cells are cytotoxic lymphocytes that lyse certain tumor-and virus-infected cells without any prior stimulation or immunization. 10 The cytotoxic activity of NK cells is tightly controlled by a balance between activating and inhibitory signals from receptors on the cell surface. A main group of receptors that inhibit NK-cell activation is the inhibitory killer immunoglobulinlike receptors (KIRs). Upon recognition of self-MHC class I molecules on target cells, these receptors deliver an inhibitory signal that stops the activating signaling cascade, sparing cells with normal MHC class I expression from NK cell-mediated lysis. Activating receptors include the natural cytotoxicity receptors (NCRs) and NKG2D, all of which push the balance toward cytolytic action through engagement with separate ligands on the target cell surface. 11,12 This role of NK cells indicates their possible use for adoptive immunotherapeutic strategies, in particular against malignancies that express low levels of MHC class I molecules. 13 The aim of this study was to investigate whether, and under what conditions, NK cells from patients with MM can be expanded numerically using good manufacturing practice (GMP)-compliant components. Furthermore, we aimed to investigate if NK cells expanded ex vivo could target autologous MM cells and thus prove to be favorable candidates for immunotherapeutic approaches against MM. Methods Patients and acquisition of patient materialPeripheral blood and BM samples from 7 newly diagnosed patients at different stages of MM were included in this study. The patients were admitted to the Department of Hematology, Karolinska University Hospital Huddinge, Stockholm, Sweden. The study was approved by the south Stockholm research ethics committee. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Patients' characteristics at the time of blood and BM sampling are given in Table 1.Peripheral blood mononuclear cells (PBMCs) as well as BM mononuclear cells (BMMCs) were isolated by gradient centrifugation, using Lymphoprep (Axis-Shield, Oslo, Norway). PBMCs and BMMCs were washed twice with phosphate-buffered saline (PBS; Gibco, Grand Island, NY), and cell viability was assessed...

Abstract. Sutlu T, Alici E (Karolinska Institutet, Stockholm, Sweden). Natural killer cell-based immunotherapy in cancer: current insights and future prospects (Review). J Intern Med 2009; 266: 154-181.As our understanding of the molecular mechanisms governing natural killer (NK) cell activity increases, their potential in cancer immunotherapy is growing increasingly prominent. This review analyses the currently available preclinical and clinical data regarding NK cell-based immunotherapeutic approaches in cancer starting from a historical background and an overview of molecular mechanisms taking part in NK cell responses. The status of NK cells in cancer patients, currently investigated clinical applications such as in vivo modulation of NK cell activity, ex vivo purification ⁄ expansion and adoptive transfer as well as future possibilities such as genetic modifications are discussed in detail.

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The chimeric state after allogeneic hematopoietic stem cell transplantation provides a platform for adoptive immunotherapy using donor-derived immune cells. The major risk with donor lymphocyte infusions (DLIs) is the development of graft-versus-host disease (GvHD). Development of new DLI products with antitumor reactivity and reduced GvHD risk represents a challenging task in cancer immunotherapy. Although natural killer (NK) and NK-like T cells are promising owing to their antitumor activity, their low concentrations in peripheral blood mononuclear cells reduces their utility in DLIs. We have recently developed a system that allows expansion of clinical-grade NK and NK-like T cells in large numbers. In this study, the safety of donor-derived long-term ex vivo-expanded human NK and NK-like T cells given as DLIs was investigated as immunotherapy for cancer in five patients following allogeneic stem cell infusion. Infusion of the cells was safe whether administered alone or with IL-2 subcutaneously. No signs of acute GvHD were observed. One patient with hepatocellular carcinoma showed markedly decreased serum alpha-fetoprotein levels following cell infusions. These findings suggest that the use of ex vivo-expanded NK and NK-like T cells is safe and appears an attractive approach for further clinical evaluation in cancer patients.

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