Daratumumab, a Novel Therapeutic Human CD38 Monoclonal Antibody, Induces Killing of Refractory Patient-Derived Multiple Myeloma Cells, Growing in a Novel Humanized Mouse MM Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 940-940
Author(s):  
Willy A Noort ◽  
Richard W.J. Groen ◽  
Reinier Raymakers ◽  
Linda Aalders ◽  
Frans M Hofhuis ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Research Funding ◽  
Humanized Mouse ◽  
Myeloma Cells ◽  
Tumor Load ◽  
Type 3

Abstract 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 <200 cmp/cm2) or late (day 50 and 72, 2 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.

Defining Primary Marrow Microenvironment-Induced Synthetic Lethality and Resistance for 2,684 Approved Drugs Across Molecularly Distinct Forms of Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 503-503 ◽  
Author(s):  
Megan Murnane ◽  
Eugen Dhimolea ◽  
Ruojing Li ◽  
Megan A. Bariteau ◽  
Diamond D. Wheeler ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Research Funding ◽  
Synthetic Lethality ◽  
Off Label ◽  
Drug Classes ◽  
Approved Drugs ◽  
Type 3

Abstract Multiple Myeloma (MM) is a prototypical neoplasm for the study of tumor-microenvironment interactions and influences on drug response. These interactions within the bone marrow (BM) alter the signaling state of MM cells and their relative dependence on pharmacological targets. Conversely, many efforts to identify and validate drug targets in MM are conducted outside of this context. This raises the possibility that systematic re-evaluation of the current pharmacopeia may identify drugs with previously unappreciated capacity for targeting MM cells within the marrow environment. To this end, we utilized the compartment-specific bioluminescence platform (CS-BLI) to characterize the activity of 2,684 FDA-approved drugs from The Johns Hopkins Drug Library (JHDL) in three distinct MM subtypes, in the presence or absence of patient-derived CD138-negative bone marrow stromal cells (BMSCs). Anti-MM activity was evaluated at 100 nM concentrations over 72 h in MM1S (t(14;16), KRASG12A, TRAF3LOF), L363 (t(20;22), NRASQ61H, p53S261T), and OPM2 (t(4;14), FGFRK560E, p53R175H) lines. These lines demonstrate phenotypes of strong, medium, and low BMSC-induced growth enhancement, respectively. Active drugs were placed into 4 categories: type 1 - having potent anti-MM activity independent of BMSC interactions (no stromal effect), type 2 - having anti-MM activity only in the presence of BMSCs (stroma-dependent "synthetic lethality"), type 3 - having anti-MM activity that is decreased in the context of BMSCs (stroma-dependent resistance), and type 4 - otherwise inactive agents that demonstrate pro-MM activity in context of BMSCs (stroma-dependent stimulants). In this study, for MM1S, L363, and OPM2, respectively, we identified 103, 118, and 108 type 1 drugs, 217, 105, and 76 type 2 drugs, 128, 75, and 16 type 3 drugs, and 124, 33, and 38 type 4 drugs. For each category of drug phenotype, we assessed overlap across the three MM cell lines. We observed high degree of overlap for type 1 drugs (67 drugs active in all three models), while more diversity between lines was evident across the 3 lines for type 2-4 drugs, whose activity is altered by interaction with BMSCs (Figure 1). Specifically, focusing on agents demonstrating BMSC-associated stimulation, adrenergic drugs consistently stimulated MM growth in context of BMSCs, while glucocorticoids consistently grouped as type 3 agents (demonstrating BMSC-associated resistance). Interestingly, carfilzomib was also subject to BMSC-associated resistance. Despite differences in drugs demonstrating stroma-induced lethality across the MM cell lines, salicylates were commonly represented in this category. In addition to the salicylates, tofacitinib, a Janus kinase (JAK) inhibitor, demonstrated a strong capacity to elicit a stroma-dependent synthetic lethal phenotype and ruxolitinib, another inhibitor in the same class, showed a similar, yet distinct pattern of stroma-mediated sensitization. In conjunction with our screen, we performed an RNA-seq analysis to assess differential gene expression between MM in monoculture vs. in co-culture with BMSCs. Expression analysis revealed 4.0 fold increase in JAK3 expression induced by co-culture with primary BMSCs, as well as induction of a STAT3 transcription factor fingerprint by ChIP-seq enrichment analysis. A detailed dose-response analysis of tofacitinib revealed no anti-MM activity against MM cells in isolation at physiological concentrations, but showed typical sigmoidal log-dose response dynamics in the presence of stroma and a dynamic range that completely abrogated the growth advantage attributable to stromal stimulation. This phenomenon of BMSC-dependent pharmacology identifies tofacitinib as an intriguing candidate for repurposing as an agent demonstrating stroma-induced synthetic lethality against MM. Further evaluation of this agent in combination with other anti-MM agents, like bortezomib, is also warranted. Taken together, this study demonstrates specific anti-MM activity for a wide array of clinically relevant drugs and drug classes in the context of BM microenvironment interactions and provides context for further validation and potential suitability for repurposing to treat MM within the medullary compartment. Figure 1. Figure 1. Disclosures Aftab: Cleave Biosciences, Inc.: Research Funding; Omniox, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Onyx Pharmaceuticals, Inc.: Research Funding. Off Label Use: The use of tofacitinib citrate and ruxolitinib will be discussed in preclinical contexts for treatment of multiple myeloma. Other approved drugs and drug classes will be generally presented in similar off-label context..

scholarly journals Differential Activation of CD8+Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma

1998 ◽  
Vol 6 (3-4) ◽  
pp. 331-342 ◽  
Author(s):  
Christoph Specht ◽  
Hans-Gerd Pauels ◽  
Christian Becker ◽  
Eckehart Kölsch
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Tumor Cells ◽  
T Cell Subsets ◽  
Early Stages ◽  
Specific Tolerance

The involvement of counteractiveCD8+T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model.CD8+Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cellsin vivoandin vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinducedCD8+T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γas a suppressive factor. Whereas most longterm cultivated CD8+ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primaryin vitroTc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10.CD8+Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γor IL-12. In contrast, ADJ-PC- 5-specificCD8+Tc cells from immunized mice are IFN-γproducing Tc1 cells. Since the primaryin vitroTc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γthese Tc1 cells behave similar to the suppressiveCD8+T cells that are induced during early stages of ADJ-PC-5 tumorigenesis.

scholarly journals Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3017-3025 ◽  
Author(s):  
VS Goldmacher ◽  
LA Bourret ◽  
BA Levine ◽  
RA Rasmussen ◽  
M Pourshadi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Tumor Cell Line ◽  
Covalent Attachment ◽  
Myeloma Cells ◽  
Colony Forming Units

Abstract We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.

Anti-Tumor Activity of IPI-504, a Novel Hsp90 Inhibitor in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4922-4922 ◽  
Author(s):  
Vito J. Palombella ◽  
Emmanuel Normant ◽  
Janid Ali ◽  
John Barrett ◽  
Michael Foley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Aqueous Solubility ◽  
Hsp90 Inhibitor ◽  
Tumor Xenograft ◽  
Active Metabolites ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.

Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 601-601
Author(s):  
Rodger E. Tiedemann ◽  
Yuan Xiao Zhu ◽  
Jessica Schmidt ◽  
Hongwei Yin ◽  
Quick Que ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Epithelial Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Lymphoid Tissues ◽  
Myeloma Cells ◽  
Stat3 Phosphorylation ◽  
Human Multiple Myeloma

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1844-1844 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle Kuhne ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Mouse Models ◽  
Research Funding ◽  
Therapeutic Agents ◽  
Dependent Manner ◽  
Apoptotic Pathways ◽  
Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 277-277 ◽  
Author(s):  
Inger S. Nijhof ◽  
Willy A. Noort ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Joost M. Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lysis ◽  
In Vitro Assays ◽  
Human Antibody ◽  
Myeloma Cells ◽  
Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

scholarly journals Wish-Qol Study - Assessment of Health-Related Quality of Life (HRQoL) and Health-Economic Aspects in Patients with Von Willebrand Disease (VWD) in France: Results of the Women Cohort

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1395-1395
Author(s):  
Annie Borel-Derlon ◽  
Jenny Goudemand ◽  
Dominique Desprez ◽  
Fabienne Volot ◽  
Yves Gruel ◽  
...  
Keyword(s):  
Interim Analysis ◽  
Research Funding ◽  
Von Willebrand Disease ◽  
Female Group ◽  
Bleeding Score ◽  
Type 3 ◽  
Von Willebrand

Abstract Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder with a prevalence of 1% in the general population. VWD results from a deficiency in or a dysfunction of von Willebrand factor which is a protein that is necessary for normal platelet adhesion and protection of factor VIII from proteolysis in the circulation. Nevertheless, prevalence of the most symptomatic forms such as bleeds requiring replacement treatment and /or hospitalization is about 0.01%. Although VWD affects both genders, there is a higher proportion in females than in males.VWD seems to be more symptomatic in women because of their reproductive life. Women with VWD have an increased bleeding risk in numerous situations including anemia, menorrhagia, bleeding during pregnancy, postpartum hemorrhage and impairments in their quality of life (QoL).The prevalence of menorrhagia in women with VWD is 74-92%. According to the Francecoag Network, the referral-based prevalence of moderate-to-severe VWD patients is about 1,750 cases in France. Aim: Since the disease and its treatment can affect every-day life of patients and their families, a French HRQoL Study (WiSH-QoL) exploring this impact started 22 months ago. Methods: This non-interventional 5-year study evaluates patients HRQoL and costs of care in France. At least 350 patients will be followed for 24 months in minimum 30 centers. HRQoL is assessed with the generic SF-36 and the disease-specific VWD-QoL questionnaires. Bleeding severity was measured using the Tosetto Bleeding Score (BS). Results: Since October 2014, 245 patients have been included. We present here the first interim analysis with a focus on the female group. At the first interim analysis, data from 140 patients were documented: 91 adults with a median age of 40.0 years [18.3-78.0] and 49 children with a median age of 10.1 years [2.9-17.5]. VWD Types were already identified for 122 (87%) of these patients: 33 with VWD type 1 (27%) including 5 type 1 Vicenza; 76 type 2 (62%) and 13 type 3 (11%). The median Tosetto bleeding score reported for 124 patients (males and females) was +7 ranging from -1 to +28. From the 95 female patients, 70 were aged ≥18 years, 21 were adolescents between 8-17 years and 4 were girls below 4 years of age. Median age was 29.4 (range 4.3-78.0) years. A total of 25 women had type 1 VWD (31%), 49 had type 2 VWD (60%), and 7 had type 3 VWD (9%), for 14 patients VWD type is undetermined. The median Tosetto bleeding score of the female group was +8 ranging from -1 to +28. Out of 95 patients, 45 patients (47.4%) have received a concomitant treatment due to menorrhagia, such as iron therapy, oral contraceptive, levonorgestrel intrauterin system: 5/21 patients in the group between 8 and 17 years and 40/70 in the group ≥18 years. Out of the 60 women of childbearing potential defined as age between 15-50 years, 6 women were pregnant at time of inclusion. A total of 46 patients, aged 18 years or more have had obstetrical history prior to study inclusion. The mean number of childbirth was more than 2 i.e 2.39 range (1-8) per woman, 75% of these deliveries were natural delivery and 25% were caesarean section. Out of 108 deliveries, 28 (26%) were experienced with post-partum hemorrhages. Conclusions: With the results of the WiSH-QoL study, the first prospective study of von Willebrand disease conducted in France, especially the VWD-specific evaluation of HRQoL and treatment satisfaction a deeper insight will be gathered into the patients' daily life, their perception of well-being and their specific health care needs. With the additional domain 'pregnancy' included in the French version of the VWD-QoL questionnaire for female adult patients, it will possible to better understand how women may be affected by VWD during childbearing years. Disclosures Borel-Derlon: LFB: Other: Reference expert and national coordinator for VWD; Octapharma: Research Funding; NovoNordisk: Other: Expert for scientific committee; Shire - Baxalta: Research Funding. Chatelanaz:LFB Biomedicaments: Employment. Doriat-Robin:LFB Biomedicaments: Employment. von Mackensen:SOBI: Research Funding; Shire: Research Funding.

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals Semiautomatic VWF Multimer Densitometric Analysis: Validation of the Clinical Accuracy and Clinical Implications in Von Willebrand Disease

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Ferdows Atiq ◽  
Johan Boender ◽  
Marjon H. Cnossen ◽  
Johanna G van der Bom ◽  
Karin Fijnvandraat ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Research Funding ◽  
Von Willebrand Disease ◽  
Densitometric Analysis ◽  
Travel Grant ◽  
Bleeding Score ◽  
Type 3 ◽  
Von Willebrand

Introduction Von Willebrand factor (VWF) multimer analysis is an essential tool in the diagnosis and classification of von Willebrand disease (VWD). Current visual VWF multimer analysis is observer dependent, time consuming and is inaccurate in detecting subtle changes in multimer patterns. Therefore, recent studies have investigated VWF multimer quantification using semiautomatic densitometric analysis. The accuracy of VWF multimer densitometric analysis in clinical practice needs further investigation before it can be widely used. The aim of the study was to validate the accuracy of VWF multimer densitometric analysis in clinical practice. Additionally, we aimed to identify patient characteristics associated with VWF multimer densitometry outcomes in type 1 and type 2 VWD patients, and we investigated whether subtle differences in VWF multimer pattern are associated with the bleeding phenotype of VWD patients. Methods We included patients from the nationwide Willebrand in the Netherlands (WiN) study. The inclusion criteria of the WiN study were a personal hemorrhagic diathesis or family history of VWD, and historically lowest VWF antigen (VWF:Ag), VWF activity (measured with the monoclonal antibody assay: VWF:Ab) or VWF collagen binding (VWF:CB) ≤0.30 IU/mL or FVIII activity (FVIII:C) ≤0.40 IU/mL in case of type 2N VWD. At inclusion in the WiN study, blood was drawn and patients filled in an extensive questionnaire containing a self-administered Tosetto bleeding score (BS). For multimer analysis, citrated blood samples were separated on 0.9% agarose gel and visualized by Western blotting. We used IMAGEJ for densitometric analysis. The five smallest bands on densitometric images were defined as small multimers, next five bands were defined as medium multimers and the remaining bands were defined as large multimers. Medium-large VWF multimer index was calculated by dividing the patient's multimer ratio (intensity of the medium and large multimers divided by the total intensity of all multimers) by the multimer ratio of a normal control in the same western blot. If no multimers could be detected, the multimer index was set as 0. Results We included 561 VWD patients: 328 type 1, 211 type 2 and 21 type 3 patients. The median age was 44 [IQR 29-58] and 351 patients (62.7%) were female (Table 1). Figure 1 illustrates typical densitometric outcomes of a type 1 VWD patient with normal VWF multimers (A) and a type 2A patient with reduced high-molecular-weight (HMW) VWF multimers (B). Medium-large VWF multimer index was 1.06 [0.99-1.12] in type 1 and 0.53 [0.29-0.89] in type 2 and 0.00 [0.00-0.00] in type 3 VWD. Medium-large VWF multimer index was in patients visually classified as normal, reduced and absent HMW VWF multimers, respectively 1.07 [1.02-1.12], 0.84 [0.71-0.91] and 0.31 [0.20-0.44] (p&lt;0.001, Figure 2A). With visual examination as gold standard, medium-large VWF multimer index had a very good accuracy in distinguishing normal VWF multimers from reduced HMW VWF multimers (AUC: 0.96 (0.94-0.98) p&lt;0.001, Figure 2B). It could also accurately distinguish reduced HMW VWF multimers from absence of HMW multimers, with an AUC of 0.95 (0.92-0.97, p&lt;0.001), and type 2A and 2B from type 2M and 2N (AUC: 0.96 (0.94-0.99), p&lt;0.001, Figure 2C and 2D). From VWF activity measurements, medium-large VWF multimer index was strongest correlated with VWF:CB (ρ=0.79, p&lt;0.001). From the ratio of the various functional VWF measurements (divided by VWF:Ag), the strongest correlation was again found for VWF:CB/VWF:Ag ratio (ρ=0.80, p&lt;0.001). In type 1 VWD, an increased clearance of VWF (defined as VWFpropeptide/VWF:Ag ratio ≥2.2) was independently associated with lower medium-large VWF multimer index (β=-0.10 (-0.14; -0.07), p&lt;0.001). Also, type 1 VWD patients with a VWF gene variant had relatively lower medium-large VWF multimer index compared to type 1 patients without a VWF variant, respectively 1.03 [0.95-1.10] vs 1.08 [1.04-1.12] (p&lt;0.001). In the total population, higher medium-large VWF multimer index was associated with a lower bleeding score: β=-4.6 (-7.2; -2.0), p=0.001, adjusted for age, sex, blood group and type of VWD. Conclusion Semiautomatic densitometric analysis of VWF multimers has an excellent accuracy in clinical practice, and may have an additional value in providing a better understanding of the clinical features such as the bleeding phenotype of VWD patients. Disclosures Atiq: CSL Behring: Research Funding; SOBI: Other: travel grant. Boender:SOBI: Current Employment; CSL Behring: Research Funding. Cnossen:Bayer: Research Funding; Novo Nordisk: Research Funding; Nordic Pharma: Research Funding; Sobi: Research Funding; Takeda: Research Funding; CSL behring: Research Funding; Pfizer: Research Funding; Shire: Research Funding; Baxter: Research Funding. van der Bom:Bayer: Speakers Bureau. Fijnvandraat:SOBI: Research Funding; NovoNordisk: Consultancy; Grifols: Consultancy; Takeda: Consultancy; Roche: Consultancy; CSL Behring: Research Funding; NovoNordisk: Research Funding. Van Galen:Bayer: Research Funding; Takeda: Speakers Bureau; CSL Behring: Research Funding. Laros-Van Gorkom:Baxter: Other: Educational grant; CSL Behring: Other: Educational grant. Meijer:Bayer: Research Funding; Sanquin: Research Funding; Pfizer: Research Funding; Bayer: Speakers Bureau; Sanquin: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; BMS: Speakers Bureau; Aspen: Speakers Bureau; Uniqure: Consultancy. Eikenboom:CSL Behring: Research Funding; Roche: Other: Teacher on educational activities. Leebeek:Roche: Other: DSMB member for a study; SOBI: Other: Travel grant; Novo Nordisk: Consultancy; Shire/Takeda: Consultancy; Uniqure: Consultancy; Shire/Takeda: Research Funding; CSL Behring: Research Funding.

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