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Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM, we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients, we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However, we discovered, next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients), as well as CDC (56 patients). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients.

Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantationHuman mesenchymal stem cells (MSCs) are multipotent stem cells, able to differentiate into multiple mesenchymal lineages. [1][2][3] Previously, we have shown that human fetal lung-derived MSCs enhance the engraftment of human umbilical cord blood (UCB)-derived CD34 ϩ hematopoietic cells in nonobese diabetic-severe combined immunodeficiency mice. 1 Here we show that secondtrimester amniotic fluid is an abundant source of fetal MSCs that exhibit a phenotype and multilineage differentiation potential similar to that of postnatal bone marrow (BM)-derived MSCs. We suggest that amniotic fluid is an attractive source of MSCs for cotransplantation in conjunction with UCB-derived hematopoietic stem cells.Amniotic fluid was collected transcervically from 6 secondtrimester legal terminations of pregnancy (mean gestational age, 19 weeks [range, 17-22 weeks]) according to a protocol approved by the medical ethical review board of our hospital. Amniotic fluid samples, without visible contamination with blood, were centrifuged for 10 minutes at 1283 rpm. Pellets were resuspended and cultured as described previously. 1 Adherent cells were detached with trypsin/EDTA (ethylenediaminetetraacetic acid) and phenotypically characterized by flow cytometry using fluorescein isothiocyanate-or phycoerythrin-conjugated antibodies. The adipogenic and osteogenic differentiation capacity of culture-expanded MSCs was determined as previously reported. 1 To confirm the fetal origin of cultured cells, a molecular HLA typing was performed on DNA obtained from expanded MSCs, and fetal and maternal blood cells by polymerase chain reaction/sequence-specific oligonucleotide using a reverse dot blot method. 4 MSCs were cultured from all 6 consecutive samples of second-trimester amniotic fluid. A quantity of 2 mL amniotic fluid was sufficient to culture these cells. The expansion potential of amniotic fluid-derived MSCs exceeded that of BM-derived MSCs. As a result, we were able to expand amniotic fluid MSCs to about 180 ϫ 10 6 cells within 4 weeks (3 passages). The phenotype of the culture-expanded amniotic fluid-derived cells was similar to that reported for MSCs derived from secondtrimester fetal tissues and adult BM 1,2 (Table 1). Amniotic fluidderived MSCs showed multilineage differentiation potential into fibroblasts, adipocytes, and osteocytes. Molecular HLA typing of fetal and maternal cells confirmed that the cultured cells were of fetal origin, without detectable contamination of maternal cells (Figure 1).Following allogeneic transplantation, most studies indicate that MSCs remain of host origin, 5 possibly as a result of the low frequency of these cells in stem cell grafts. The frequency of MSCs in UCB is particularly low, and most laboratories have been unable to grow MSCs from UCB. 6,7 Supplementing stem cell grafts with MSCs to promote engraftment has been proposed. Studies in mice and sheep show that engraftment can be promoted by the addition of t...

Patients suffering from heart failure as a result of myocardial infarction are in need of heart transplantation. Unfortunately the number of donor hearts is very low and therefore new therapies are subject of investigation. Cell transplantation therapy upon myocardial infarction is a very promising strategy to replace the dead myocardium with viable cardiomyocytes, smooth muscle cells and endothelial cells, thereby reducing scarring and improving cardiac performance. Despite promising results, resulting in reduced infarct size and improved cardiac function on short term, only a few cells survive the ischemic milieu and are retained in the heart, thereby minimizing long-term effects. Although new capillaries and cardiomyocytes are formed around the infarcted area, only a small percentage of the transplanted cells can be detected months after myocardial infarction. This suggests the stimulation of an endogenous regenerative capacity of the heart upon cell transplantation, resulting from release of growth factor, cytokine and other paracrine molecules by the progenitor cells – the so-called paracrine hypothesis. Here, we focus on a relative new component of paracrine signalling, i.e. exosomes. We are interested in the release and function of exosomes derived from cardiac progenitor cells and studied their effects on the migratory capacity of endothelial cells.

A doptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38 + fractions of CD34 + hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38 + malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma

Purpose: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide-and bortezomib-refractory patients.Experimental Design: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide-and bortezomib-refractory multiple myeloma patients.Results: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide-and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumabmediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide-and bortezomib-refractory patient.Conclusions: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide-and bortezomib-refractory patients.

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model IntroductionIn the BM, specialized microenvironments such as hematopoietic niches regulate hematopoiesis. Within these niches, hematopoietic stem cells (HSCs) are present in a complex network consisting of mesenchymal stromal cells (MSCs), osteoblasts, osteoclasts, endothelial cells, and adipocytes embedded in an extracellular matrix. The bidirectional interactions with the hematopoietic niche are essential for HSC maintenance and function. [1][2][3][4] The BM niche is also thought to serve as a sanctuary site for leukemic stem cells (LSCs), which, in addition to their immortalizing genetic events, highly depend on interaction with the microenvironment to survive and proliferate. 5,6 Although the majority of leukemias initially respond to therapeutic intervention, relapse rates are high. [7][8][9] There is increasing evidence that the tumor niche plays a crucial role in the survival and drug resistance of LSCs. Interactions with the niche provide signals protecting the LSCs from apoptosis and eventually leading to the selection and outgrowth of a resistant cell. 10-13 Therefore, it is apparent that the hematopoietic niche plays an important role in hematopoietic development and in chemotherapy resistance of BM-localized leukemic and solid tumors.Although our understanding of how the BM niche regulates HSC self-renewal and confers therapy resistance has advanced greatly over the past years, most of this knowledge is based on genetic loss-of-function or gain-of-function murine models. 1,2,10,11,14 However, these murine models do not simulate human physiology and much of the constituents of the human hematopoietic niche remain largely unclear. [14][15][16] This emphasizes the need for more suitable models that recapitulate the human BM microenvironment and, very importantly, facilitate the engraftment and outgrowth of normal HSCs and patient-derived tumor cells within these protected sites.In the present study, we describe a unique humanized model that implements a novel scaffold-based technology for generating a human bone environment in RAG 2 Ϫ/Ϫ ...

Although mesenchymal stromal cells (MSCs) have been applied clinically to treat cardiac diseases, it is unclear how and to which extent transplanted MSCs exert their beneficial effects. To address these questions, pre-clinical MSC administrations are needed for which pigs appear to be the species of choice. This requires the use of porcine cells to prevent immune rejection. However, it is currently unknown to what extent porcine MSCs (pMSCs) resemble human MSCs (hMSCs). Aim of this study was to compare MSC from porcine bone marrow (BM) with human cells for phenotype, multi-lineage differentiation potential, immune-modulatory capacity and the effect on cardiac function after transplantation in a mouse model of myocardial infarction. Flow cytometric analysis revealed that pMSC expressed surface antigens also found on hMSC, including CD90, MSCA-1 (TNAP/W8B2 antigen), CD44, CD29 and SLA class I. Clonogenic outgrowth was significantly enriched following selection of CD271+ cells from BM of human and pig (129 ± 29 and 1961 ± 485 fold, respectively). hMSC and pMSC differentiated comparably into the adipogenic, osteogenic or chondrogenic lineages, although pMSC formed fat much faster than hMSC. Immuno-modulation, an important feature of hMSC, was clearly demonstrated for pMSC when co-cultured with porcine peripheral blood cells stimulated with PMA and pIL-2. Finally, pMSC transplantation after myocardial infarction attenuated adverse remodelling to a similar extent as hMSC when compared to control saline injection. These findings demonstrate that pMSCs have comparable characteristics and functionality with hMSCs, making reliable extrapolation of pre-clinical pMSC studies into a clinical setting very well possible.