Halvard Bonig

Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab

Blockade of CD49d-mediated lymphocyte trafficking has been used therapeutically for certain autoimmune diseases, such as multiple sclerosis (MS). In addition to negative effects on the trafficking of mature lymphocytes to sites of inflammation, CD49d blockade in mice and monkeys rapidly mobilizes hematopoietic stem/progenitor cells (HSPCs) capable of short- and long-term engraftment. Here we aimed to ascertain the effects of treatment with antifunctional anti-CD49d antibody in humans (MS patients receiving infusions of the CD49d-blocking antibody natalizumab) on levels of circulating HSPCs after a single dose of antibody or after long-term treatment. On average, 6-fold elevated levels of circulating CD34+ cells and colony-forming unit-culture (CFU-C) were achieved within 1 day of the first dose of natalizumab, and similar levels were continuously maintained under monthly natalizumab infusions. The blood of natalizumab-treated subjects also contained SCID-repopulating cells. The fate of these circulating HSPCs and their clinical relevance for MS patients remains to be determined.

Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

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 CD3ζ 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. γ-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.

Hematopoietic stem cell mobilization: updated conceptual renditions

Despite its specific clinical relevance, the field of hematopoietic stem cell mobilization has received broad attention, owing mainly to the belief that pharmacologic stem cell mobilization might provide clues as to how stem cells are retained in their natural environment, the bone marrow ‘niche’. Inherent to this knowledge is also the desire to optimally engineer stem cells to interact with their target niche (such as after transplantation), or to lure malignant stem cells out of their protective niches (in order to kill them), and in general to decipher the niche’s structural components and its organization. Whereas, with the exception of the recent addition of CXCR4 antagonists to the armamentarium for mobilization of patients refractory to granulocyte colony-stimulating factor alone, clinical stem cell mobilization has not changed significantly over the last decade or so, much effort has been made trying to explain the complex mechanism(s) by which hematopoietic stem and progenitor cells leave the marrow. This brief review will report some of the more recent advances about mobilization, with an attempt to reconcile some of the seemingly inconsistent data in mobilization and to interject some commonalities among different mobilization regimes.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS The strategy for quality control testing depends on the products cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Epigenetic Regulation of Endothelial Lineage Committed Genes in Pro-Angiogenic Hematopoietic and Endothelial Progenitor Cells

Rationale: Proangiogenic hematopoietic and endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, but the mechanisms regulating differentiation to the endothelial lineage are unclear. Objective: To elucidate the epigenetic control of endothelial gene expression in proangiogenic cells and EPCs. Methods and Results: Here we demonstrate that the endothelial nitric oxide synthase (eNOS) promoter is epigenetically silenced in proangiogenic cells (early EPCs), CD34+ cells, and mesoangioblasts by DNA methylation and prominent repressive histone H3K27me3 marks. In order to reverse epigenetic silencing to facilitate endothelial commitment, we used 3-deazaneplanocin A, which inhibits the histone methyltransferase enhancer of zest homolog 2 and, thereby, reduces H3K27me3. 3-Deazaneplanocin A was not sufficient to increase eNOS expression, but the combination of 3-deazaneplanocin A and the histone deacetylase inhibitor Trichostatin A augmented eNOS expression, indicating that the concomitant inhibition of silencing histone modification and enhancement of activating histone modification facilitates eNOS expression. In ischemic tissue, hypoxia plays a role in recruiting progenitor cells. Therefore, we examined the effect of hypoxia on epigenetic modifications. Hypoxia modulated the balance of repressive to active histone marks and increased eNOS mRNA expression. The reduction of repressive H3K27me3 was associated with an increase of the histone demethylase Jmjd3. Silencing of Jmjd3 induced apoptosis and senescence in proangiogenic cells and inhibited hypoxia-mediated up-regulation of eNOS expression in mesoangioblasts. Conclusions: These findings provide evidence that histone modifications epigenetically control the eNOS promoter in proangiogenic cells.

Allogeneic donor peripheral blood "stem cell" apheresis: prospective comparison of two apheresis systems.

BACKGROUND: Granulocyte–colony‐stimulating factor–mobilized peripheral blood stem cells, collected by white blood cell apheresis, are used for more than 80% of allogeneic and most autologous hematopoietic stem cell transplantations. Optimal donor and recipient outcomes require maximized stem cell collection efficiency and minimized non–target cell contamination. Therefore, improved apheresis technology is desirable. The safety and feasibility of apheresis collections with the novel, electronics‐assisted apheresis system Spectra Optia v.5.0 (CaridianBCT) were recently demonstrated. An unpublished optimization trial had furthermore determined that different settings than manufacturer‐installed default might result in improved apheresis yields.

Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy

Bone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210(+)] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.

Concurrent Blockade of α4-Integrin and CXCR4 in Hematopoietic Stem/Progenitor Cell Mobilization†‡

The important contributions of the α4 integrin VLA‐4 and the CXCR4/SDF‐1 axis in mobilization have been demonstrated and thereby, these pathways can be suggested as rational targets for clinical stem cell mobilization in the absence of cytokine use. α4‐blockade alone (in humans, macaques and mice), or genetic ablation of α4‐integrin in mice, provides reproducible, but modest mobilization. Similarly, CXCR4 blockade with small‐molecule antagonists mobilizes hematopoietic stem cells in all three species, but at least with the established single‐injection schedule, the mobilization efficiency is marginally sufficient for clinical purposes. Hypothesizing that the different molecular targets (α4‐integrin vs. CXCR4) might allow for additive mobilization effects, we therefore tested the efficacy of the combination of α4‐integrin blockade with anti‐functional antibodies and CXCR4 blockade with the small‐molecule inhibitor AMD3100 in macaques, or the combination of conditional α4‐integrin ablation and AMD3100 in mice. Mobilization was at least additive. While the prolonged effects of α4‐blocking antibodies may not be suitable for clinical mobilization, future availability of small‐molecule α4‐antagonists in combination with AMD3100 could provide an alternative to granulocyte colony‐stimulating factor. STEM CELLS 2009;27:836–837

Mobilized allogeneic peripheral stem/progenitor cell apheresis with Spectra Optia v.5·0, a novel, automatic interface‐controlled apheresis system: results from the first feasibility trial

Background and Objectives  G‐CSF mobilized peripheral blood stem/progenitor cells are frequently used for allogeneic transplantation. Available manual apheresis systems generate stem cell products of consistently high quality. Short‐comings include need for continuous interface monitoring/adjustment, interface instability in donors with inconsistent blood flow, high collection variability, high platelet loss, and failure to electronically document apheresis parameters.

Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly

Apoptosis resistance contributes to poor outcome in pediatric acute lymphoblastic leukemia (ALL). Here, we identify a novel synergistic combination of Smac mimetic BV6 and glucocorticoids (GCs) (ie, dexamethasone, prednisolone) to trigger apoptosis in ALL cells. BV6 and GCs similarly cooperate to induce apoptosis in patient-derived leukemia samples, underlining the clinical relevance. Importantly, BV6/dexamethasone cotreatment is significantly more effective than monotherapy to delay leukemia growth in a patient-derived xenograft model of pediatric ALL without causing additional side effects. In contrast, BV6 does not increase cytotoxicity of dexamethasone against nonmalignant peripheral blood lymphocytes, mesenchymal stromal cells, and CD34-positive hematopoietic cells. We identify a novel mechanism by showing that BV6 and dexamethasone cooperate to deplete cIAP1, cIAP2, and XIAP, thereby promoting assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing complex. This complex is critical and is required for BV6/dexamethasone-induced cell death, because RIP1 knockdown reduces caspase activation, reactive oxygen species production, and cell death. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, because blocking antibodies for TNFα, tumor necrosis factor-related apoptosis-inducing ligand, or CD95 ligand or knockdown of death receptors fail to rescue BV6/dexamethasone-induced cell death. This is the first report showing that BV6 sensitizes for GC-triggered cell death by promoting ripoptosome formation with important implications for apoptosis-targeted therapies of ALL.