ALBERT

Description: Previously, we have reported that our human bone marrow (BM)-like scaffold xenograft model allows the engraftment and outgrowth of normal and malignant hematopoiesis (e.g. multiple myeloma (MM), acute myelocytic/lymphocytic leukemia (AML/ALL) and MDS (Groen et al. Blood 2012; Gutierrez et al. JCI 2014 and data not shown). Whereas the presence of osteoblasts and bone of human origin mimics a human BM-like niche more closely than the murine BM in standard xenotransplant models (e.g. NOD-SCID/NSG mice), still some essential components of the human BM niche, i.e. human blood vessels, are missing. To this end, in addition to human mesenchymal stromal cells we now incorporated cord blood-derived endothelial progenitor cells (CB-EPCs) in the hybrid scaffold production process, to create a multi-tissue compartment that "maximally humanizes" the BM-like niche of our scaffolds. Towards successful implementation of a human vascular system we compared: i) scaffold material composition (biphasic calcium phosphate (BCP) vs. tricalcium phosphate (TCP)); ii) scaffold shape (particles vs. tubes); iii) different types of matrigel for CB-EPC embedding. Histological analysis of the humanized scaffolds, eight weeks after implantation in mice, showed a large number of functional human blood vessels, as indicated by hCD31+ staining and the presence of erythrocytes within. Comparison of the composition and the shapes of the scaffolds indicated superiority of TCP and tube-shaped scaffolds in supporting the formation of vessels. Further analysis of scaffolds for CD44, CD146, LEPR and nestin-positive cells, revealed the presence of other stromal niche cells besides human osteoblasts and endothelial cells. Irradiation of mice carrying these humanized implants did not have a significant deleterious effect on the established human vessels, allowing their further functional evaluation in xenotransplantation. Additionally, mice carrying tubes with and without human CB-EPC derived vessels (on either flank) were subsequently inoculated with adult BM-derived CD34-positive cells by intracardiac injection. Upon analysis 12 weeks later, all tubes showed multi-lineage hematopoietic outgrowth. Interestingly, CB-EPC embedment resulted in increased numbers of CD45+ (2-fold), CD13+ (4-fold) and CD7+ (2-fold), while CD19+ cell numbers were equal. In contrast, in mouse BM almost only CD19+ cells could be detected. Moreover, we observed that the use of CB-EPCs in our scaffolds provides faster kinetics of in vivo engraftment and growth of both patient-derived MM or AML cells. With the addition of both human CB-EPCs and human BM stromal cells, our scaffold systems now simulate both human endosteal and vascular niches of the BM, thereby more closely recapitulating the human hematopoietic niche. Disclosures Yuan: Xpand Biotechnology BV: Employment. de Bruijn:Xpand Biotechnology BV: Employment. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding. Martens:Johnson & Johnson: Research Funding. Groen:Johnson & Johnson: Research Funding.

Description: Abstract 1834 During progression of Multiple Myeloma (MM), the disease spreads to multiple sites in the bone marrow (BM) and towards terminal stages also to extra-medullary sites. If spreading of MM cells via the circulation could be prevented, it would reduce subsequent sites of MM growth and prevent additional bone lesions. To study this hypothesis an animal model that truly mimics human disease is essential. Recently, we described a novel human-mouse hybrid model of MM, based on the generation of a human bone microenvironment (BME) in RAG2−/−gc−/− mice by combining a ceramic scaffold with culture-expanded mesenchymal stromal cells (MSCs). This BME acts as a hematopoietic niche and supports outgrowth of patient-derived MM cells (pMM) (Groen et al, Blood 2012). By marking pMM cells with the luciferase gene and using bioluminescent imaging (BLI), we were able to monitor pMM outgrowth in time in humanized scaffolds and visualize effects of treatment. pMM cells, injected in scaffolds located at one side of the mice showed local outgrowth to MM tumors but were also found to migrate to non-injected scaffolds at the contralateral side. pMM cells circulated in a low numbers in mouse blood and were found to colonize mouse BM. These combined phenomena provided us with the ideal MM model to study therapeutic agent(s) not only targeting the pMM tumors, but also to study targeting of circulating pMM cells and thus inhibit spreading to secondary locations, i.e. to humanized scaffolds located contra-lateral or to mouse BM. One such novel agent is daratumumab (DARA), which is in clinical development for MM. DARA is a human CD38 antibody with broad-spectrum killing activity. DARA exerts its effects via complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC/ADCP). In clinical studies DARA showed marked reductions in paraprotein and BM plasma cells and adverse events were manageable. To study whether DARA could prevent spreading of disease to other sites, mice carrying humanized scaffolds, in which luciferase marked pMM cells that were refractory to chemotherapy were growing, were treated with DARA at early (day 34, 50 and 72) or late (day 50 and 72) stage disease. Parallel groups of mice were treated with melphalan, bortezomib or dexamethason. Growth of scaffold-injected pMM cells and response to DARA- or chemo -therapy, was monitored with BLI. Animals were treated early (small tumors) or late (large tumors) during disease progression with different dose levels of DARA, i.e. 1× 50 μg, or 2 or 3 × 200 μg/mouse. The 50 μg dose of late stage disease, strongly reduced the number of circulating CD138+ MM cells in blood and, interestingly, also in BM. Treatment at late stage with melphalan, bortezomib or dexamethasone resulted in only a marginal effect on the outgrowth of pMM grafts in scaffolds. 200 μg DARA at early stage (day 35, 50, 72) resulted in a strong anti-pMM effect with complete elimination of the CD38+ fraction from the scaffolds, as analyzed by immunohistochemistry and FACS. Late treatment (day 50,72) with 200 μg DARA also resulted in a strong anti-pMM effect, but the surviving MM tumors had a mixed phenotype, consisting of 4 subpopulations, being CD38/CD138 positive and negative cells. In the blood a low percentage of circulating tumor cells (CD38+/CD138+) was observed before treatment (day 34: 0.06%). This stayed low in the early treatment group; on day 70, (∼40-fold lower) as compared to controls. On day 90 it reached 4%, but consisted only of CD38− cells. In the late treatment group the number of circulating tumor cells was ∼3–6 fold lower at day 70, as compared to controls. As DARA plasma levels were very low in late treatment groups, suggesting target-mediated clearance of DARA by the pMM tumors. This also suggests that DARA dosing may be increased to optimize treatment. In summary, whilst conventional drug treatment did not effectively inhibit growth of scaffold-grafted multiple-drug refractory pMM tumors, low dose DARA was already able to reduce pMM plasma cells in the circulation and to reduce spreading to other medullary sites, e.g. mouse BM. At a high dose level, treatment at an early time point induced elimination of CD38+ cells from blood, with only CD38− cells surviving in scaffolds. Treatment with high dose DARA at a late time point temporarily reduced tumor growth but the optimal dose levels in this animal model did not seem to be reached. This requires further investigation. Disclosures: Groen: Genmab: Research Funding. Raymakers:Novartis: Consultancy. Parren:genmab: Employment. Lokhorst:Celgene: Honoraria. Mutis:genmab: Research Funding. Martens:Genmab: Research Funding.

Description: TH and MW contributed equally to this work. Background Multiple myeloma is caused by an accumulation of malignant plasma cells in the bone marrow. Myeloma is characterized by an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily. BMP-signaling is important for both pre- and postnatal bone formation. Additionally, several BMPs induce growth arrest and apoptosis in myeloma cells. Thus, increasing BMP-signaling in myeloma patients may reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma. Methods Calcium phosphate scaffolds with human mesenchymal stromal cells (MSCs) were implanted in RAG2-/-GC-/- mice and the MSCs were left to differentiate in vivo for 8 weeks to create a humanized bone microenvironment. Then, adeno-associated virus (AAV), AAV8-BMP4, which has tropism for liver cells and expresses murine Bmp4 under the control of the liver specific human α1-antitrypsin (hAAT1) promoter, were administered by tail-vein injection. Empty viral vectors, AAV8-CTRL, were used for the control group. After 2 weeks, when BMP4 was detectable in circulation, we injected fluorescently labelled KJON myeloma cells in 3 out of 4 scaffolds in each mouse. The KJON cells are hyperdiploid, have a relatively slow growth rate and rely on interleukin (IL)-6 supplementation in the absence of a supporting microenvironment, thus resembling primary human myeloma cells. Tumor growth was examined by weekly imaging until end-point, 6 weeks after tumor cell injection. Results At end-point, serum levels of BMP4 in AAV8-BMP4 mice were in the range of 50-200 ng/mL, but not detectable in AAV8-CTRL mice. Strikingly, tumor growth as quantified by imaging was significantly reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice (p

Description: 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 resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

Description: Key Points Humanized niche xenograft mouse models were generated that enabled engraftment of patients’ leukemia cells covering all risk groups. Self-renewal was better maintained in the humanized niches as determined by serial transplantation and genome-wide transcriptome studies.

Description: Abstract 940 The evolution of multiple myeloma (MM) is a multi-step process during which mature B cells acquire genetic mutations in multiple genes, which typically takes place in the bone marrow (BM) microenvironment. This, together with the difficulty to culture MM in vitro or to grow MM in vivo in animal models has been the main reason during past decades for poor progress in preclinical research with patient-derived myeloma (pMM) cells. Recently, we developed a unique human-mouse hybrid model that allows engraftment and outgrowth of pMM cells by implementing a technology that is based on first generating a human bone environment in immune deficient mice (Groen et al. 2012) and that is subsequently capable of supporting growth of injected pMM cells. The model offers the opportunity (1) to study the pathobiology of myeloma, and (2) to evaluate, preclinically, new therapeutics for MM treatment, including antibody testing against pMM cells, obtained from patients who acquired resistance to conventional and novel drugs. Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. Daratumumab induces effective killing of MM tumor cells via complement dependent cytolysis (CDC), ADCC (antibody dependent cellular cytolysis) and ADCP (antibody-dependent phagocytosis). DARA represents a novel promising treatment for MM and other hematological malignancies and is currently tested in Phase I/II clinical trials. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. In the current study, we asked whether DARA was able to inhibit growth of refractory tumor cells in our human-mouse hybrid model. To this end, immune-deficient RAG2−/−gc−/−-mice were implanted subcutaneously with biphasic calcium phosphate (BCP) particles (2–3 mm) loaded with culture expanded human mesenchymal stromal cells (MSCs). Eight weeks later, the humanized scaffolds in mice (n=45) were injected with 0.5–5×106 pMM cells obtained from different refractory, MM patients. The pMM cells were gene-marked with a GFP-luciferase lentiviral construct for imaging of viable tumor cells. Bioluminescent imaging (BLI) was used to follow myeloma outgrowth in time and to visualize the effect of treatment. The pMM cells were obtained from patients at diagnosis (type 1); at end stage disease, after a history of MPT (melphalan, prednisone, thalidomide, type 2); or from a patient refractory to chemotherapy with bortezomib (BORT), adriamycine and dexamethasone (DEX) (type 3). Mice carrying the pMM cells received similar treatment as the patients or were treated with DARA in a dose range of 1x 50 μg (low dose (LD)) or 2 to 3x 200 μg/mouse (high dose (HD)). BLI showed that the type 1 pMM-bearing mice responded well to all treatments, including DARA; type 2-bearing pMM mice showed no reduction in tumor growth after chemotherapy, but DARA treatment (LD) resulted in an almost complete elimination of circulating MM cells, as assessed with a CD138 antibody, in blood and BM. In a second experiment, type 2-pMM bearing mice were treated with a high DARA dose early (day 34, 50 and 72, 3 times HD, tumor size/BLI signal 8000 cpm/cm2). A significant reduction of overall tumor load, as measured with BLI was observed, which interestingly did not differ between the high and low tumor load group. A reduction of circulating tumor cells (CD138+) was observed for both conditions, which was most obvious in the early treated mice and in agreement with the observations in the first experiment. Type 3 (resistant) pMM-bearing mice showed only a minor response to DEX and BORT, but were highly sensitive to melphalan. When DEX- and BORT-treated mice were treated with a single injection of DARA, this resulted in a complete remission in 3 out of 4 mice and a reduction of the tumor load by 50% in the fourth BORT-treated mouse. In conclusion, our results demonstrate that DARA is effective against multiple myeloma cells derived from therapy- naïve or -refractory patients grafted in a humanized mouse model. In addition, this humanized MM model can be used to study the potential and mechanism of action of DARA in vivo. This novel MM model might be used to predict responsiveness of myeloma patients to particular treatments. Disclosures: Groen: Genmab BV: Research Funding. Raymakers:Novartis: Consultancy. Lammerts van Bueren:Genmab BV: Employment. Parren:genmab: Employment. Mutis:genmab: Research Funding. Martens:Genmab BV: Research Funding.

Description: Abstract 3412 Interactions with the hematopoietic niche in the bone marrow (BM) microenvironment are essential for hematopoietic stem cell (HSC) self-renewal. In addition, in hematological malignancies this niche is considered to serve as a sanctuary site for leukemic stem cells during chemotherapy, and to contribute to disease relapse. Although many advances have been made in understanding how the niche regulates HSC self-renewal and confers therapy resistance, most of this knowledge is based on genetic loss- or gain-of-function murine models. Since these models do not recapitulate the human physiology, there is a need for models that more closely resemble the human niche. Here, we describe a unique humanized model, which implements a novel scaffold-based technology for generating a human bone environment in RAG2−/−gc−/−-mice. Inoculation of these mice with normal human CD34+ hematopoietic progenitor cells, isolated from umbilical cord blood, resulted in homing to the human bone environment and the generation of human hematopoietic cells of distinct lineages, but more importantly also the engraftment of CD34+ cells themselves. In a next series of experiments the supportive nature of the humanized niche was further investigated with patient-derived acute myeloid leukemia (pAML) and multiple myeloma (pMM) cells, two hematopoietic malignancies that are highly dependent on the BM microenvironment for survival and growth. Inoculation of the humanized mice with pAML cells, obtained from a poor-risk patient (M1; complex karyotype) or cells from a good risk AML patient (M4; inv(16)) revealed the ability of the reconstructed human bone environment to support outgrowth of the leukemia with the cells having a similar phenotype as those from the patient sample. Interestingly, engraftment of good risk AML samples, including inv(16), has been reported to be very difficult in the NOD/SCID-based AML xenotransplant model. The humanized model that we developed was further substantiated by the ability to support the outgrowth of pMM from 7 out of 7 patients. MM is a hematological malignancy that fails to grow in mouse tissues without extra support, e.g. fetal human bone chips. Moreover, the outgrowth of pMM in our humanized model is accompanied by an increase in osteoclast activity, indicating the presence of bone resorption, one of the most relevant clinical sequelae of MM. In addition, by gene-marking pMM cells with luciferase and using bioluminescent imaging, we were able to follow myeloma outgrowth in time. Treatment of pMM-bearing mice with identical drugs as given to the patients showed that the pMM cells growing in the humanized environment in the mice responded similar as the MM patients. Hence, our model allows, for the first time, to investigate essential interactions within the human BM microenvironment for the development of normal and malignant hematopoiesis and thus for therapy development. Disclosures: de Bruijn: Xpand Biotechnology BV: Employment. Weers:Genmab BV: Employment. Parren:Genmab BV: Employment.