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INTRODUCTION: Multiple myeloma (MM) is generally an incurable hematological malignancy with heterogeneous overall survival rates ranging from a few months to more than 10 years. Survival is especially poor for patients who developed disease that is refractory to immunomodulatory drugs and proteasome inhibitors. Areas covered: This review will discuss the importance of CD38-targeting antibodies for the treatment of MM patients to improve their outcome. Expert commentary: Intense immuno-oncological laboratory research has resulted in the development of functionally active monoclonal antibodies against cell surface markers present on MM cells. In this respect, CD38-targeting antibodies such as daratumumab, MOR202, and isatuximab, have high single agent activity in heavily pretreated MM patients by virtue of their pleiotropic mechanisms of action including Fc-dependent effector mechanisms and immunomodulatory activities. Importantly, CD38-targeting antibodies are well tolerated, with infusion reactions as most frequent adverse event. Altogether, this makes them attractive combination partners with other anti-MM agents. Daratumumab is already approved as monotherapy and in combination with lenalidomide-dexamethasone as well as bortezomib-dexamethasone in pretreated MM patients. Furthermore, results from studies evaluating CD38-targeting antibodies in newly diagnosed MM patients are also promising, indicating that CD38-targeting antibodies will be broadly used in MM, resulting in further improvements in survival.

Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction. Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed. Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab–CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138. Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance. The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498–511. ©2017 AACR.

Daratumumab is a CD38-targeted human monoclonal antibody with direct anti-myeloma cell mechanisms of action. Flow cytometry in relapsed and/or refractory multiple myeloma (RRMM) patients treated with daratumumab revealed cytotoxic T-cell expansion and reduction of immune-suppressive populations, suggesting immune modulation as an additional mechanism of action. Here, we performed an in-depth analysis of the effects of daratumumab on immune-cell subpopulations using high-dimensional mass cytometry. Whole-blood and bone-marrow baseline and on-treatment samples from RRMM patients who participated in daratumumab monotherapy studies (SIRIUS and GEN501) were evaluated with high-throughput immunophenotyping. In daratumumab-treated patients, the intensity of CD38 marker expression decreased on many immune cells in SIRIUS whole-blood samples. Natural killer (NK) cells were depleted with daratumumab, with remaining NK cells showing increased CD69 and CD127, decreased CD45RA, and trends for increased CD25, CD27, and CD137 and decreased granzyme B. Immune-suppressive population depletion paralleled previous findings, and a newly observed reduction in CD38+ basophils was seen in patients who received monotherapy. After 2 months of daratumumab, the T-cell population in whole-blood samples from responders shifted to a CD8 prevalence with higher granzyme B positivity (P = 0.017), suggesting increased killing capacity and supporting monotherapy-induced CD8+ T-cell activation. High-throughput cytometry immune profiling confirms and builds upon previous flow cytometry data, including comparable CD38 marker intensity on plasma cells, NK cells, monocytes, and B/T cells. Interestingly, a shift toward cytolytic granzyme B+ T cells was also observed and supports adaptive responses in patients that may contribute to depth of response. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

The prognosis of multiple myeloma (MM) patients who become refractory to lenalidomide and bortezomib is very poor, indicating the need for new therapeutic strategies for these patients. Next to the development of new drugs, the strategy of combining agents with synergistic activity may also result in clinical benefit for patients with advanced myeloma. We have previously shown in a retrospective analysis that lenalidomide combined with continuous low-dose cyclophosphamide and prednisone (REP) had remarkable activity in heavily pretreated, lenalidomide-refractory MM patients. To evaluate this combination prospectively, we initiated a phase 1/2 study to determine the optimal dose and to assess its efficacy and safety in lenalidomide-refractory MM patients. The maximum tolerated dose (MTD) was defined as 25 mg lenalidomide (days 1-21/28 days), combined with continuous cyclophosphamide (50 mg/d) and prednisone (20 mg/d). At the MTD (n = 67 patients), the overall response rate was 67%, and at least minimal response was achieved in 83% of the patients. Median progression-free survival and overall survival were 12.1 and 29.0 months, respectively. Similar results were achieved in the subset of patients with lenalidomide- and bortezomib-refractory disease as well as in patients with high-risk cytogenetic abnormalities, defined as t(4;14), t(14;16), del(17p), and/or ampl(1q) as assessed by fluorescence in situ hybridization. Neutropenia (22%) and thrombocytopenia (22%) were the most common grade 3-4 hematologic adverse events. Infections (21%) were the most common grade 3-5 nonhematologic adverse events. In conclusion, the addition of continuous low-dose oral cyclophosphamide to lenalidomide and prednisone offers a new therapeutic perspective for multidrug refractory MM patients. This trial was registered at www.clinicaltrials.gov as #NCT01352338.

Daratumumab, a human CD38 immunoglobulin G1 kappa (IgG1κ) monoclonal antibody, has activity as monotherapy in multiple myeloma (MM). This phase 1/2 study investigated daratumumab plus lenalidomide/dexamethasone in refractory and relapsed/refractory MM. Part 1 (dose escalation) evaluated 4 daratumumab doses plus lenalidomide (25mg/day orally on days 1-21 of each cycle) and dexamethasone (40 mg/week). Part 2 (dose expansion) evaluated daratumumab at the recommended phase 2 dose (RP2D) plus lenalidomide/dexamethasone. Safety, efficacy, pharmacokinetics, immunogenicity, and accelerated daratumumab infusions were studied. In part 1 (13 patients), no dose-limiting toxicities were observed, and 16 mg/kg was selected as the R2PD. In part 2 (32 patients), median time since diagnosis was 3.2 years,with amedian of 2 prior therapies (range, 1-3 prior therapies), including proteasome inhibitors (91%), alkylating agents (91%), autologous stem cell transplantation (78%), thalidomide (44%), and lenalidomide (34%); 22% of patients were refractory to the last line of therapy. Grade 3 to 4 adverse events (≥5%) included neutropenia, thrombocytopenia, and anemia. In part 2, infusion-related reactions (IRRs) occurred in 18 patients (56%); most were grade ≤2 (grade 3, 6.3%). IRRs predominantly occurred during first infusions and were more common during accelerated infusions. In part 2 (median follow-up of 15.6 months), overall response rate was 81%, with 8 stringent complete responses (25%), 3 complete responses (9%), and 9 very good partial responses (28%). Eighteen-month progression-free and overall survival rates were 72% (95% confidence interval, 51.7-85.0) and 90% (95% confidence interval, 73.1-96.8), respectively. Daratumumab plus lenalidomide/dexamethasone resulted in rapid, deep, durable responses. The combination was well tolerated and consistent with the safety profiles observed with lenalidomide/dexamethasone or daratumumabmonotherapy. This trial was registered atwww.clinicaltrials.gov as #NCT01615029.

Introduction The inclusion of "novel" agents including proteasome inhibitors or IMiD-derivatives in the treatment of multiple myeloma significantly improves patient survival. Results of several study groups suggest incorporating at least one "novel agent" in first-line treatment before and after high-dose chemotherapy (HDT) followed by autologous stem cell transplantation. Here we address four main questions: First, what determines (excellent) long-term survival for different treatment regimen? Second, can we show benefit of novel agents for all patients and molecular subentities, including low risk? Third, can the prognostic impact of molecular entities be explained by different association with response, proliferation, and renal impairment? Fourth, does it matter regarding long-term survival by which agents, i.e. "old" vs. "new", a response was reached? Patients and Methods Patients were included in the prospective phase III HOVON-65/GMMG-HD4-trial (German part, n=354) randomizing VAD-induction, autologous tandem-transplantation and thalidomide-maintenance vs. PAD-induction, tandem-transplantation and bortezomib-maintenance. Plasma cells after CD138-purification were subjected to interphase fluorescence in-situ hybridization and gene expression profiling using Affymetrix U133 2.0 DNA-microarrays. Median follow-up (time to censoring) was 93 months. Results Low proliferation, revised-ISS I and cyto-ISS I delineate excellent long-term survival (70-75% after eight years, both arms). Molecular entities are associated with proliferation-rate, i.e. higher (del17p13, del8p21, del13q14, 1q21+, t(4;14)) or lower proliferation (hyperdiploidy), and response: bad response/survival in case of del17p, bad response/no survival impact (t(11;14)), and good response/bad survival (1q21+, t(4;14), and del13q), depending on the treatment regimen. Thus, it does not hold true that good response = good survival if patients are substratified according to their molecular background. Renal insufficiency is associated with 1q21+, del17p13, and t(4;14). For patients with ≥1 of the chromosomal aberrations del17p13, t(4;14), 1q21+ (i.e. cytogenetic high risk, 27.5% of patients) or renal insufficiency (10.6%), risk is abrogated; in absence of these risk features, no benefit could be shown. Patients reaching a near complete remission or better (≥nCR) with VAD-based regimen, HDT followed by thalidomide maintenance show significantly better survival compared to those reaching ≥nCR after bortezomib-based induction/HDT followed by bortezomib maintenance treatment. Conclusions Taken together, adversely prognostic molecular entities are associated with proliferation but can show association with better or adverse remission. Bortezomib-based upfront treatment abrogates chromosomal high-risk aberrations and renal insufficiency; however, no long-term survival-benefit is evident for those without these risk factors or low proliferation, i.e. the majority of patients. Responses achieved by different regimen are not equal in transmission in long-term survival. Responses (≥nCR) are not equivalent regarding their biological and prognostic role in patients with different molecular background and different treatment regimen. Disclosures Seckinger: Celgene: Research Funding; Sanofi: Research Funding; EngMab: Research Funding. Salwender:Novartis: Honoraria, Other: travel suppport, Research Funding; Amgen: Honoraria, Other: travel suppport, Research Funding; Bristol-Myers Squibb: Honoraria, Other: travel suppport, Research Funding; Celgene: Honoraria, Other: travel suppport, Research Funding; Takeda: Honoraria; Janssen: Honoraria, Other: travel support, Research Funding. Scheid:Celgene: Honoraria; Janssen: Honoraria. Knauf:Janssen: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria; Gilead Sciences: Consultancy; Roche: Consultancy; Amgen: Consultancy, Honoraria; Mundipharma: Consultancy. Duehrsen:AbbVie: Consultancy, Honoraria; Amgen: Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Consultancy, Honoraria. Dürig:Roche: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria; Celgene: Honoraria. Schmidt-Wolf:Janssen: Research Funding; Novartis: Research Funding. Haenel:Novartis: Honoraria; Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria. Raab:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Sonneveld:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Blau:Celgene: Other: Advisory board, Research Funding; Janssen: Other: Advisory board, Research Funding; Amgen: Other: Advisory board; Takeda: Other: Advisory board; Novartis: Other: Advisory boards; BMS: Other: Advisory board. Hillengass:Takeda: Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Celgene: Consultancy, Honoraria, Other: Advisory Board, Research Funding; amgen: Consultancy, Honoraria, Other: Advisory Board; Sanofi: Research Funding; Janssen: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board. Weisel:Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, Celgene, Janssen, and Sanofi: Research Funding; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Chugai: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Adaptive Biotechnology: Consultancy; ArtTempi: Honoraria. Hose:Celgene: Honoraria, Research Funding; EngMab: Research Funding; Sanofi: Research Funding.

Allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusion are effective treatment modalities for various hematological malignancies. Their therapeutic effect, the graft-versus-tumor (GvT) effect, is based mainly on an alloimmune response of donor T cells directed at tumor cells, in which differences in the expression of minor histocompatibility Ags (mHags) on the cells of the patient and donor have a crucial role. However, these differences are also responsible for induction of sometimes detrimental GvHD. As relapse and development of GvHD pose major threats for a large proportion of allotransplanted patients, additional therapeutic strategies are required. To augment the GvT response without increasing the risk of GvHD, specific mHag-directed immunotherapeutic strategies have been developed. Over the past years, much effort has been put into the identification of therapeutically relevant mHags to enable these strategies for a substantial proportion of patients. Currently, the concept of mHag-directed immunotherapy is tested in clinical trials on feasibility, safety and efficacy. In this review, we will summarize the recent developments in mHag identification and the clinical data on mHag-specific immune responses and mHag-directed therapies in patients with hematological malignancies. Finally, we will outline the current challenges and future prospectives in the field.

This multicenter phase 2 study of the European Myeloma Network investigated the combination of carfilzomib, thalidomide, and dexamethasone (KTd) as induction/consolidation therapy for transplant-eligible patients with previously untreated multiple myeloma (N = 91). During KTd induction therapy, patients received 4 cycles of carfilzomib 20/27 mg/m(2) (n = 50), 20/36 mg/m(2)(n = 20), 20/45 mg/m(2)(n = 21), or 20/56 mg/m(2)(n = 20) on days 1, 2, 8, 9, 15, and 16 of a 28-day cycle; thalidomide 200 mg on days 1 to 28; and dexamethasone 20 mg on days 1, 2, 8, 9, 15, and 16. After autologous stem cell transplantation, patients proceeded to KTd consolidation therapy, where the target doses of carfilzomib were 27 mg/m(2), 36 mg/m(2), 45 mg/m(2), or 56 mg/m(2), respectively, and thalidomide 50 mg. Common grade 3/4 adverse events included respiratory (15%), gastrointestinal (12%), and skin disorders (10%); polyneuropathy was infrequent (1%). Complete response rates after induction and consolidation treatment were 25% and 63%, respectively; rates of very good partial response or better after induction and consolidation were 68% and 89%, respectively. At a median follow-up of 23 months, the 36-month progression-free survival rate was 72%. The KTd induction and consolidation regimens were active, safe, and well tolerated. This study was registered at http://www.trialregister.n1 as #NTR2422.

Objective The aim of this study was to understand the influence of disease and patient characteristics on exposure to daratumumab, an immunoglobulin Gκ (IgGκ) monoclonal antibody, and clinical outcomes in relapsed or refractory multiple myeloma (MM). Patients and Methods Baseline myeloma type, albumin levels, renal/hepatic function, age, sex, race, weight, Eastern Cooperative Oncology Group (ECOG) status, refractory status, and number of prior therapies were evaluated using data from two clinical studies—GEN501 (N = 104) and SIRIUS (N = 124). Results Daratumumab clearance was approximately 110% higher in IgG myeloma patients than non-IgG myeloma patients, leading to significantly lower exposure in IgG myeloma patients based on maximum trough serum concentrations (p

Daratumumab, a human CD38 imunoglobulin G 1κ monoclonal antibody, has demonstrated clinical activity and a manageable safety profile in monotherapy and combination therapy clinical trials in relapsed and/or refractory multiple myeloma. CD38 is expressed at high levels on myeloma cells and, to a lesser extent, on immune effector cells, including natural killer (NK) cells, which are important for daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Here, the pharmacodynamic effects of daratumumab monotherapy on NK cells, and the effect of NK cell dynamics on daratumumab efficacy and safety, were assessed. Daratumumab, like other CD38 antibodies, reduced NK-cell counts in peripheral blood mononuclear cells (PBMCs) of healthy donors in vitro. Data on NK-cell counts, clinical efficacy, and adverse events were pooled from two single-agent daratumumab studies, GEN501 and SIRIUS. In daratumumab-treated myeloma patients, total and activated NK-cell counts reduced rapidly in peripheral blood after the first dose, remained low over the course of treatment, and recovered after treatment ended. There was a clear maximum effect relationship between daratumumab dose and maximum reduction in NK cells. Similar reductions were observed in bone marrow. PBMCs from daratumumab-treated patients induced lysis by ADCC of CD38+ tumor cells in vitro, suggesting that the remaining NK cells retained cytotoxic functionality. There was no relationship between NK-cell count reduction and the efficacy or safety profile of daratumumab. Furthermore, although NK cell numbers are reduced after daratumumab treatment, they are not completely depleted and may still contribute to ADCC, clinical efficacy, and infection control.

Background: Treatment of multiple myeloma has evolved considerably in the past few years with availability of several news drugs as well as increasing use of multidrug combinations. These changes have no doubt led to the improved survival seen among patients with MM. We have previously shown that outcomes of patients intolerant or refractory to one of the IMiDs and bortezomib had a poor outcome. Since that time, other drugs of the same class as well as new classes of drugs have been introduced for the treatment of MM. We designed this retrospective study to estimate the outcomes in patients with relapsed myeloma, who have become refractory to the current generation IMiDs and proteasome inhibitors. Patients and Methods: Patients with relapsed multiple myeloma who have received at least 3 prior lines of therapy, is refractory to both an IMiD (lenalidomide or pomalidomide) AND a proteasome inhibitor (bortezomib or carfilzomib), and has been exposed to an alkylating agent were identified from multiple centers. The time patients met the above criteria was defined as T0, and details of all treatment regimens before and after T0 were collected using electronic CRFs. The study was approved by the IRB at the respective centers. Results: 543 patients were enrolled in this study; median age was 62 years (31-87) and 61% were males. Patients were enrolled from centers in North America (n=181), Europe (n=318), and Asia Pacific (n=44). Patients were diagnosed between 2006 and 2014, the median duration between diagnosis of myeloma and study entry (T0) was 3.1 years (0.3 to 9). The median (95% CI) estimated follow up from diagnosis and from T0 were 61 (57, 66) months and 13 (11, 15) months respectively. The median number of lines of therapy prior to T0 was 4 (3-13), 48% had a prior transplant. The median OS from T0 for the entire cohort was 13 (11, 15) months. For these 462 patients, the median number of recorded regimens was 2 (1-9). The overall response and the depth of response to each line of treatment following T0 are as shown in the table. The median (95% CI) PFS and OS from T0 was 5 (4, 6), and 15.2 (13, 17), respectively. The overall survival for the 81 patients with no treatment post T0 was only 2.1 months. In a multivariate analysis, duration from diagnosis to T0, ISS stage III and number of lines of therapy were all associated with inferior PFS, as well as OS, and in addition, serum creatinine>2 mg/dL at T0 also predicted inferior OS. Conclusions: The study provides the expected outcome following development of myeloma that is refractory to a PI and an IMiD. The outcomes of these patients appear to be better than we had seen historically in patients refractory/ intolerant to bortezomib and IMiDs, highlighting the increased treatment options available for these patients. However, there is decreasing response rate to sequential regimens highlighting the development of drug resistance. The data provides a bench mark for comparison of new therapies that are being evaluated in this disease. Table Table. Disclosures Dimopoulos: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kastritis:Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Terpos:BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria. Hillengass:Sanofi: Research Funding; Novartis: Research Funding; Amgen: Consultancy, Honoraria; BMS: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria. Leleu:TEVA: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; LeoPharma: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Pierre Fabre: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria. Oriol:Janssen: Honoraria, Other: Expert board committee; Amgen: Honoraria, Other: Expert board committee. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Mateos:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vij:Shire: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy; Karyopharma: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy, Research Funding. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. van de Donk:Amgen: Research Funding; Janssen: Research Funding; BMS: Research Funding; Celgene: Research Funding. Mark:Onyx: Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ludwig:Amgen: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Janssen: Speakers Bureau. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Usmani:Array: Research Funding; Britsol-Myers Squibb: Consultancy, Research Funding; Skyline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau. Dytfeld:Janssen Poland: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Moreau:Novartis: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau. Lee:Amgen: Membership on an entity's Board of Directors or advisory committees. Shustik:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. de la Rubia:Celgene: Consultancy; Bristol Myers: Consultancy; Amgen,: Consultancy; Janssen: Consultancy. Durie:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy.

Background: Novel agents such as IMIDs and proteasome inhibitors have substantially changed the therapeutic landscape in the first line treatment of multiple myeloma (MM). Better response rates and prolonged progression-free survival have lead to an improvement in overall survival (OS) with median values well beyond 5 years. Therefore to assess whether first line therapy strategies have an impact on the prognosis for patients with MM, long-term results of clinical trials with follow up covering >10 years are necessary. Methods: The HOVON-65/GMMG-HD4 study is a prospective randomized trial testing bortezomib+adriamycin+dexamethasone (PAD) for 3 cycles as induction prior to high-dose chemotherapy (HDT) and autologous stem cell transplantation compared to vincristine+adriamycin+dexamethasone (VAD) in the control arm. After one (HOVON) or two (GMMG) HDT maintenance was given for 2 years consisting of bortezomib every 2 weeks in the PAD arm and thalidomide 50 mg daily in the VAD arm. The study results were initially reported in 2012 (1) and with a median follow up of 91 months in 2018 (2). In this analysis we present OS results after a median follow up of 137 months. All hazard ratios (HR) are given with 95% confidence intervals (CI). Results: Overall survival at 12 years was 32% (CI 27-37%) in the VAD arm versus 36% (CI 31-41%) in the PAD arm without significant difference in the univariate Cox model (HR 0.87, CI 0.73 - 1.03, p=0. 11 or in multivariate Cox model including ISS stage and treatment arm (HR 0.87, CI 0.73 - 1.04, p=0.12; the primary analysis) as specified in the study protocol. When other factors including age, sex, ISS stage, WHO performance status, Immunoglobulin-type, Durie and Salmon-stage, LDH, del 13q, study group and renal impairment (RI, defined as serum creatinine ≥ 2 mg/dl) were added to the Cox model, treatment in the PAD arm was a significant factor for improved OS (HR 0.84, CI 0.7 - 1.0, p=0.048). Of the remaining factors age (HR 1.02, CI 1.01 - 1.03, p=0.002), female sex (HR 0.83, CI 0.69 - 0.99, p=0.044), ISS stage (HR 1.19, CI 1.04 - 1.35, p=0.01), WHO performance status (HR 1.32, CI 1.17 - 1.48, pULN (HR 1.44, CI 1.14 - 1.82, p=0.002), del 13q (HR 1.42, CI 1.17 - 1.73, p Discussion: Long-term results of the HOVON-65/GMMG-HD4 trial show that one third of patients receiving HDT with either thalidomide-based or bortezomib-based maintenance are still alive at 12 years. In contrast to earlier analyses with shorter follow up (1,2) the use of bortezomib in the induction and maintenance treatment provided a significant OS benefit when adjusting for other risk in a multivariate Cox model, although not in the primary analysis. A particular OS benefit was found in patients with RI receiving bortezomib before and after HDT and this could completely abolish the negative prognostic impact of RI. Similarly bortezomib used in combination with tandem-HDT improved OS in patients with del17p so that more than a third of these patients with high-risk MM survived more than 10 years. Our results underline that despite the growing options for treatment at relapse the choice of an optimal first-line therapy is of critical prognostic importance, in particular for patients with high-risk myeloma. References: 1) Sonneveld et al., J Clin Oncol, 2012; 30:2946-2955 2) Goldschmidt et al., Leukemia 2018; 32: 383-390 Figure 1 Disclosures Scheid: Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Bertsch:Celgene: Other: travel support; Sanofi: Other: travel support. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Weisel:Celgene: Consultancy, Honoraria, Research Funding; Juno: Consultancy; Bristol-Myers Squibb: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; GSK: Honoraria; Adaptive Biotech: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Kersten:Gilead: Honoraria; Kite Pharma: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Miltenyi: Honoraria; Roche: Honoraria, Research Funding; Takeda Oncology: Research Funding; Celgene: Honoraria, Research Funding. Mai:Mundipharma: Other: travel support; Takeda: Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support; Celgene: Consultancy, Honoraria, Other: travel support. Hillengass:Amgen: Consultancy, Honoraria; Janssen: Honoraria. Stilgenbauer:AbbVie, AstraZeneca, Celgene, Gilead Sciences, Inc., GSK, Hoffmann La-Roche, Janssen, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Broyl:Celgene, amgen, Janssen,Takeda: Honoraria. Bos:Kiadis Pharma: Other: Shareholder (of Cytosen, acquired by Kiadis); Celgene: Research Funding. Dührsen:Amgen: Consultancy, Honoraria, Research Funding; CPT: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Gilead: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Honoraria; Celgene: Research Funding. Salwender:Celgene: Honoraria, Other: Travel or accommodations; Sanofi: Honoraria, Other: Travel or accommodations; Takeda: Honoraria, Other: Travel or accommodations; Bristol-Myers Squibb: Honoraria, Other: Travel or accommodations; AbbVie: Honoraria; Amgen: Honoraria, Other: Travel or accommodations; Janssen Cilag: Honoraria, Other: Travel or accommodations. Goldschmidt:Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Amgen: Consultancy, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; John-Hopkins University: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Janssen: Consultancy, Research Funding. Sonneveld:Amgen: Honoraria, Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; SkylineDx: Research Funding. OffLabel Disclosure: bortezomib maintenance thalidomide maintenance

The tumor microenvironment of multiple myeloma (MM) is known to play a critical role in disease pathogenesis, MM cell survival, and drug resistance. In addition, we have previously demonstrated the impact of the microenvironment also on immunotherapies. We discovered that bone marrow mesenchymal stromal cells (BMMSCs) protected MM cells from HLA restricted CD4 and CD8 cytotoxic T cells and NK cell-mediated daratumumab dependent cytotoxicity through a cell-adhesion mediated immune resistance (CAM-IR). This CAM-IR corresponded with the upregulation of anti-apoptotic proteins Survivin and Mcl-1 in MM cells and could be modulated by small inhibitors of these molecules. We now extended our studies to investigate the impact of the microenvironment on novel immunotherapeutic approaches such as chimeric antigen receptor-transduced T cells (CAR T cells) and death receptor mediated antibody treatments, which can induce direct apoptosis in MM cells. We also investigated the possibility to modulate CAM-IR with small molecule inhibitors of Survivin, XIAP, and Mcl-1. To this end, we tested a panel of MM reactive CAR T cells directed against CD38, BCMA, and CD138, with different target affinities, for their potential to kill MM cells in the absence or presence of BMMSCs. We observed no effect of BMMSCs on the cytotoxic capacity of BCMA- and CD38-targeting CAR T cells with high affinity for the target and that were capable of inducing high levels of MM cell lysis at very low effector to target ratios. In contrast, BMMSCs effectively protected MM cells against killing by BCMA- and CD38-targeting CAR T cells with relative low affinity, or CD138-targeting CAR T cells that were less powerful in their lytic activity. Taken together, we discovered a significant inverse correlation between the lytic capacity of the CAR T cells and the extent of BMMSC-mediated protection. Additionally, we found that BMMSCs protected MM cells from apoptosis induction by death receptor 5 (DR5; or TRAIL receptor 2) antibodies. In all cases of BMMSC-mediated protection against CAR T cells as well as DR5 antibodies or daratumumab, the protection could be abrogated by an inhibitor of Survivin, XIAP, and Mcl-1. This indicates that BMMSC-mediated protection against CAR T cell or antibody-mediated lysis is indeed associated with an upregulation of anti-apoptotic proteins, similarly to the well-described cell adhesion mediated drug resistance. In conclusion, our results confirm the potential negative impact of the tumor microenvironment in the development of an adaptive resistance of MM cells against immunotherapies. Our data further suggest that this microenvironmental shielding of MM cells can be overcome either by increasing the avidity of immune killer cells or through combination of immunotherapy with inhibitors of anti-apoptotic mediators. Figure 1 Disclosures Li: Canget Bio Tekpharma LLC: Membership on an entity's Board of Directors or advisory committees. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Themeli:Covagen: Consultancy. Van De Donk:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Mutis:Aduro: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding; Onkimmune: Research Funding; Janssen Pharmaceuticals: Research Funding; Novartis: Research Funding.

MHC-bound peptides from aberrant proteins may be a specific immunotherapeutic target on cancer cells. Because of difficulties in identifying such antigens, viral or model antigens have so far been used to study their biological relevance. We here identify a naturally existing human T-cell epitope derived from a truncated protein. The antigenic peptide is derived from the gene TTK only through an alternative transcript containing a premature termination codon that may target the transcript for nonsense-mediated decay (NMD). This antigen is recognized by HLA-A*02:01–restricted CD8+ T cells derived from an allotransplanted leukemia patient. Functional analyses showed that these T cells failed to recognize several HLA-matched primary leukemic cells that expressed the alternative TTK transcript. Conventional antigen processing and presentation were not affected, suggesting that leukemic cells modify the generation of antigens processed from aberrant proteins. This natural TTK epitope provides insights in the source of transcripts producing antigenic epitopes in healthy and leukemic cells. Our data underscore potential pitfalls of targeting NMD-derived or other unconventionally generated epitopes as immunotherapeutic approach.

The introduction of autologous stem cell transplantation as well as novel agents such as proteasome inhibitors (bortezomib) and immunomodulatory drugs (IMiDs; thalidomide and lenalidomide) have significantly improved long-term outcome of multiple myeloma patients. However, patients with high-risk disease at diagnosis had less benefit from these new strategies. In addition, myeloma patients with lenalidomide and bortezomib double-refractory disease have a very poor survival.Several next generation novel agents are active in patients with double-refractory disease including carfilzomib and pomalidomide. Various monoclonal antibodies are also promising in the setting of relapsed/refractory disease, including daratumumab, elotuzumab and lorvotuzumab mertansine. This editorial will focus on the most promising next generation novel agents for the treatment of multiple myeloma.Incorporation of these new novel agents in frontline therapies will lead to more effective and less toxic combination therapies. Furthermore, new diagnostic techniques such as gene-expression profiling and next-generation sequencing will hopefully result in more personalized treatments for molecularly-defined subgroups.

The anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow-localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).

The efficacy and favorable safety profile of daratumumab monotherapy in multiple myeloma (MM) was previously reported. Here, we present an updated pooled analysis of 148 patients treated with daratumumab 16 mg/kg. Data were combined from part 2 of a first-in-human phase 1/2 study of patientswhorelapsed after or were refractory to ‡2 prior therapies and a phase 2 study of patients previously treated with ‡3 prior lines of therapy (including a proteasome inhibitor [PI] and an immunomodulatory drug [IMiD]) or were double refractory. Among the pooled population, patients received a median of 5 prior lines of therapy (range, 2 to 14 prior lines of therapy), and 86.5% were double refractory to a PI and an IMiD. Overall response rate was 31.1%, including 13 very good partial responses, 4 complete responses, and 3 stringent complete responses. The median duration of response was 7.6 months (95% confidence interval [CI], 5.6 to not evaluable [NE]). The median progression-free survival (PFS) and overall survival (OS) were 4.0 months (95% CI, 2.8-5.6 months) and 20.1 months (95% CI, 16.6 months to NE), respectively. When stratified by responders vs stable disease/minimal response vs progressive disease/NE, median PFS was 15.0 months (95% CI, 7.4 months to NE) vs 3.0 months(95% CI, 2.8-3.7months) vs 0.9months (95% CI, 0.9-1.0months), respectively, andmedianOSwasNE(95% CI,NEto NE) vs 18.5 months (95% CI, 15.1-22.4 months) vs 3.7 months (95% CI, 1.7-7.6 months), respectively. No new safety signals were identified. In this pooled data set, daratumumab 16 mg/kg monotherapy demonstrated rapid, deep, and durable responses, with a clinical benefit that extended to patients with stable disease or better. (Blood. 2016;128(1):37-44)

CD38 is a multifunctional cell surface protein that has receptor as well as enzyme functions. The protein is generally expressed at low levels on various hematological and solid tissues, while plasma cells express particularly high levels of CD38. The protein is also expressed in a subset of hematological tumors, and shows especially broad and high expression levels in plasma cell tumors such as multiple myeloma (MM). Together, this triggered the development of various therapeutic CD38 antibodies, including daratumumab, isatuximab, and MOR202. Daratumumab binds a unique CD38 epitope and showed strong anti-tumor activity in preclinical models. The antibody engages diverse mechanisms of action, including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, modulation of enzymatic activity, and immunomodulatory activity. CD38-targeting antibodies have a favorable toxicity profile in patients, and early clinical data show a marked activity in MM, while studies in other hematological malignancies are ongoing. Daratumumab has single agent activity and a limited toxicity profile, allowing favorable combination therapies with existing as well as emerging therapies, which are currently evaluated in the clinic. Finally, CD38 antibodies may have a role in the treatment of diseases beyond hematological malignancies, including solid tumors and antibody-mediated autoimmune diseases. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Monoclonal antibodies (mAbs) are a recent addition to multiple myeloma (MM) therapies and a number of mAbs directed at myeloma cell surface molecules are in development. Daratumumab is a CD38 mAb that has demonstrated substantial activity and good tolerability in four phase I, phase I/II and phase II studies as monotherapy, as well as in combination with current standard treatments in MM. The positive results obtained in the relapsed/refractory setting in patients with advanced-stage disease and in a small number of patients with newly diagnosed disease provide the rationale for the investigation of the agent in a number of ongoing phase III trials. mAbs are generally better tolerated than conventional chemotherapy; however, their use requires other special considerations. Such factors include those common to all mAbs, namely infusion-related reactions, but also factors that are observed with mAbs used in myeloma, such as interference with response assessment, or factors that are related to CD38 mAbs such as daratumumab, for instance blood typing interference. Our review provides an overview of the results from the daratumumab clinical trials conducted to date, as well as practical management considerations for the use of daratumumab based on our experience with the agent.

Primary plasma cell leukemia (pPCL) is a rare and aggressive plasma cell proliferative disorder with a very poor prognosis and with distinct biologic, clinical, and laboratory features. Compared with multiple myeloma, pPCL presents more often with extramedullary involvement, anemia, thrombocytopenia, hypercalcemia, elevated serum β2-microglobulin and lactate dehydrogenase levels, as well as impaired renal function. Many of the genetic aberrations observed in newly diagnosed pPCL are typically found in advanced multiple myeloma. These cytogenetic abnormalities and mutations lead to increased proliferation, enhanced inhibition of apoptosis, escape from immune surveillance, and independence from the BM microenvironment, with changes in expression of adhesion molecules or chemokine receptors. The outcome of pPCL has improved with the introduction of autologous stem cell transplantation and combination approaches with novel agents, including bortezomib and immunomodulatory drugs, such as lenalidomide. In this review, we provide an overview of currently available therapeutic options with recommendations of how these treatment modalities can best be used to improve outcome for plasma cell leukemia 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 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.

Most patients with newly diagnosed multiple myeloma (MM) are aged > 65 years with 30% aged > 75 years. Many elderly patients are also vulnerable because of comorbidities that complicate the management of MM. The prevalence of MM is expected to rise over time because of an aging population. Most elderly patients with MM are ineligible for autologous transplantation, and the standard treatment has, until recently, been melphalan plus prednisone. The introduction of novel agents, such as thalidomide, bortezomib, and lenalidomide, has improved outcomes; however, elderly patients with MM are more susceptible to side effects and are often unable to tolerate full drug doses. For these patients, lower-dose-intensity regimens improve the safety profile and thus optimize treatment outcome. Further research into the best treatment strategies for vulnerable elderly patients is urgently needed. Appropriate screening for vulnerability and an assessment of cardiac, pulmonary, renal, hepatic, and neurologic functions, as well as age > 75 years, at the start of therapy allows treatment strategies to be individualized and drug doses to be tailored to improve tolerability and optimize efficacy. Similarly, occurrence of serious nonhematologic adverse events during treatment should be carefully taken into account to adjust doses and optimize outcomes.

To improve the outcome of allogeneic stem cell transplantation (allo-SCT) in multiple myeloma as part of first-line treatment, we prospectively investigated the feasibility and efficacy of lenalidomide maintenance. Patients started maintenance 1 to 6 months after nonmyeloablative allo-SCT. Lenalidomide was dosed 10 mg on days 1 to 21 of a 28-day schedule for a total of 24 cycles. Peripheral blood samples were taken to evaluate immune modulating effects. Thirty-five eligible patients were enrolled, and 30 started with lenalidomide. After 2 cycles, 14 patients (47%) had to stop treatment, mainly because of the development of acute graft versus host disease (GVHD). In total, 13 patients (43%) stopped treatment because of development of GVHD, 5 patients (17%) because of other adverse events, and 5 patients (17%) because of progression. Responses improved in 37% of patients, and the estimated 1-year progression-free survival from start of maintenance was 69% (90% confidence interval, 53%-81%). Lenalidomide increased the frequency of human leukocyte antigen-DR+ T cells and regulatory T cells, without correlation with clinical parameters. In conclusion, lenalidomide maintenance 10 mg daily after nonmyeloablative allo-SCT with unmanipulated graft in multiple myeloma patients is not feasible, mainly because of the induction of acute GVHD. This trial was registered at www.trialregister.nl as #NTR1645.

Introduction: Conflicting results from two recently reported randomized studies comparing double vs single autotransplantation (ASCT) for newly diagnosed multiple myeloma (MM) patients (pts) [(Cavo M et al, Blood 2017;130(1); Stadtmauer EA et al, Blood 2016;128(22)] are likely to reflect differences in the design of the trials. To address this controversial issue, we performed a long-term follow-up analysis of pt-level data from three phase 3 trials of bortezomib-thalidomide-dexamethasone (VTD) (Cavo M et al, Lancet 376; 2075-85, 2010; Rosinol L et al, Blood 120; 1589-96, 2012) or bortezomib-doxorubicin-dexamethasone (PAD) (Sonneveld P et al, J Clin Oncol 30; 2946-55, 2012) as induction therapy before ASCT, followed by post-ASCT bortezomib-based consolidation and/or maintenance treatment. According to study design, patients were assigned to receive either a single or double ASCT (ASCT-1 or ASCT-2), thus allowing a comparison between these treatments. Methods: The intent-to-treat population included 909 pts who were randomized to either VTD or PAD arms of the studies and for whom ASCT-1 (n=501) or ASCT-2 (n=408) were planned at study entry. Median age was 58 yrs in both groups; the rate of ISS stage III was 20% and 17%, respectively, while 18% and 23% of pts in ASCT-1 and ASCT-2 groups were positive for t(4;14) and/or del(17p) (cut-off levels ≥10% and ≥20%, respectively) by FISH analysis. Results: With a median follow up of 117 mos (IQR 91-126), assignment to ASCT-2 resulted in superior PFS (median: 47 vs 38 mos; HR 0.76, 95%CI=0.65-0.89, p=0.0008) and OS (estimated 10-yr probability: 58% vs 47%; HR 0.69, CI 0.56-0.84, p=0.0002) in comparison with ASCT-1 (Figure 1). PFS benefit with ASCT-2 was retained across prespecified subgroups, including pts with both standard-risk (median: 53 vs 43 mos; HR 0.74, CI 0.61-0.91, p=0.005) and high-risk cytogenetics (cyto) (median: 36 vs 20 mos; HR 0.67, CI 0.46-0.97, p=0.032). The 10-yr OS rates were 72% with ASCT-2 vs 60% with ASCT-1 (HR 0.68, CI 0.52-0.88, p=0.004) for pts with standard-risk and 51% vs 34% (HR 0.54, CI 0.36-0.83, p=0.004) for those with high-risk cyto. In a multivariate Cox regression analysis, independent predictors for prolonged PFS included ASCT-2 (HR 0.81, CI 0.66-0.99, p=0.048), platelet (PLT) count >150.000/mmc (HR 0.74, CI 0.54-0.99, p=0.049), ISS stage I+II (HR 0.62, CI 0.48-0.80, p HR estimates of the leading, not including therapy, predictors of outcomes (ISS stage II+III, high-risk cyto and failure to achieve best CR) were used to build a score index that stratified patients into 3 subgroups at low-risk (20%, none of the 3 adverse variables), intermediate-risk (42%, 1 adverse variable) and high-risk (38%, 2 or 3 adverse variables). Median PFS for these subgroups was 87, 53 and 27 mos (p Conclusions: Results of this pooled analysis of phase 3 studies incorporating bortezomib-based triplets into ASCT confirmed the superiority of ASCT-2 over ASCT-1 in terms of extended PFS and OS. The subgroup of pts at high-risk mostly benefited from ASCT-2, in particular those who had advanced ISS stage, adverse cyto and failed to achieve CR. Disclosures Cavo: GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Adaptive Biotechnology: Consultancy; Sanofi: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; ArtTempi: Honoraria; Janssen: Consultancy, Honoraria, Research Funding. Rosinol:Janssen, Celgene, Amgen, Takeda: Honoraria. Zweegman:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corp.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Salwender:Novartis: Honoraria, Other: travel suppport, Research Funding; Amgen: Honoraria, Other: travel suppport, Research Funding; Takeda: Honoraria; Bristol-Myers Squibb: Honoraria, Other: travel suppport, Research Funding; Janssen: Honoraria, Other: travel support, Research Funding; Celgene: Honoraria, Other: travel suppport, Research Funding. Lahuerta:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Petrucci:Bristol-Myers Squibb: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; Amgen: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board; Janssen-Cilag: Honoraria, Other: Advisory Board. Oriol:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Weisel:Amgen, Celgene, Janssen, and Sanofi: Research Funding; Amgen, BMS, Celgene, Janssen, and Takeda: Honoraria; Amgen, BMS, Celgene, Janssen, Juno, Sanofi, and Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rios:Amgen, Celgene, Janssen, and Takeda: Consultancy. Patriarca:Celgene: Other: Advisory Role; Travel, accommodations, expenses; Medac: Other: Travel, accommodations, expenses; Jazz: Other: Travel, accommodations, expenses; Janssen: Other: Advisory role; MSD Italy: Other: Advisory Role. Mateos:GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Galli:Sigma-Tau: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Bristol-Myers Squibb: Honoraria. San-Miguel:Novartis: Honoraria; Sanofi: Honoraria; Roche: Honoraria; BMS: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Boccadoro:Novartis: Honoraria, Research Funding; AbbVie: Honoraria; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Blade:Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria. Sonneveld:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Background: In our efforts to develop novel effective treatment regimens for multiple myeloma we evaluated the potential benefits of combining the immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis. Design and Methods To explore the effect of lenalidomide combined with daratumumab, we first carried out standard antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+ multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from patients were used as target cells. We also tested the effect of lenalidomide on daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear cells of multiple myeloma patients. Finally, we determined the daratumumab-dependent cell-mediated cytotoxicity using peripheral blood mononuclear cells of multiple myeloma patients receiving lenalidomide treatment. Results Daratumumab-dependent cell-mediated cytotoxicity of purified primary multiple myeloma cells, as well as of the UM-9 cell line, was significantly augmented by lenalidomide pre-treatment of the effector cells derived from peripheral blood mononuclear cells from healthy individuals. More importantly, we demonstrated a clear synergy between lenalidomide and daratumumab- induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of multiple myeloma patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural environment of malignant cells. Finally, daratumumab-dependent cellmediated cytotoxicity was significantly up-regulated in peripheral blood mononuclear cells derived from 3 multiple myeloma patients during lenalidomide treatment. Conclusions Our results indicate that powerful and complementary effects may be achieved by combining lenalidomide and daratumumab in the clinical management of multiple myeloma

Purpose: The essential role of CD4+ T cells as helpers of anticancer immunity is indisputable. Little is known, however, about their capacity to serve as effector cells in cancer treatment. Therefore, we explored the efficacy of immunotherapy with sole CD4+ cytotoxic human T cells directed at a hematopoietic-restricted minor histocompatibility antigen (mHag). Experimental Design: In macrophage-depleted Rag2−/−γc−/− mice, which were also devoid of T, B, and natural killer cells, mHag-specific native T cells or tetanus toxoid (TT)-specific T cells transduced with the mHag-specific T-cell receptor (TCR) were injected to treat full-blown mHag+ human multiple myeloma tumors. Results: mHag-specific antitumor responses were achieved after injection of native or mHag-TCR-transduced T cells. Although the therapy completely eradicated the primary tumors in the bone marrow, it failed to control extramedullary relapses, even after repeated T-cell injections. Detailed analyses ruled out mHag or MHC downregulation as mechanisms of extramedullary tumor escape. Impaired T-cell survival in vivo or defective homing to the tumor site were also ruled out as mechanisms behind extramedullary relapses, because injections of TT-loaded antigen presenting cells could facilitate homing of long-term surviving T cells to s.c. tumor sites. Moreover, intratumoral treatment of extramedullary tumors with 3AB11 was also ineffective. Conclusions: Taken together, these results for the first time show the feasibility of immunotherapy of primary bone marrow tumors with sole CD4+ human T cells directed to a tumor-associated mHag. Extramedullary relapses, probably due to microenvironment-dependent inhibitory mechanisms, remain a challenging issue towards effective cellular immunotherapy of hematologic malignancies. Clin Cancer Res; 16(22); 5481–8. ©2010 AACR.

Contains fulltext : 88517.pdf (Publisher’s version ) (Closed access) To identify molecularly defined subgroups in multiple myeloma, gene expression profiling was performed on purified CD138(+) plasma cells of 320 newly diagnosed myeloma patients included in the Dutch-Belgian/German HOVON-65/GMMG-HD4 trial. Hierarchical clustering identified 10 subgroups; 6 corresponded to clusters described in the University of Arkansas for Medical Science (UAMS) classification, CD-1 (n = 13, 4.1%), CD-2 (n = 34, 1.6%), MF (n = 32, 1.0%), MS (n = 33, 1.3%), proliferation-associated genes (n = 15, 4.7%), and hyperdiploid (n = 77, 24.1%). Moreover, the UAMS low percentage of bone disease cluster was identified as a subcluster of the MF cluster (n = 15, 4.7%). One subgroup (n = 39, 12.2%) showed a myeloid signature. Three novel subgroups were defined, including a subgroup of 37 patients (11.6%) characterized by high expression of genes involved in the nuclear factor kappa light-chain-enhancer of activated B cells pathway, which include TNFAIP3 and CD40. Another subgroup of 22 patients (6.9%) was characterized by distinct overexpression of cancer testis antigens without overexpression of proliferation genes. The third novel cluster of 9 patients (2.8%) showed up-regulation of protein tyrosine phosphatases PRL-3 and PTPRZ1 as well as SOCS3. To conclude, in addition to 7 clusters described in the UAMS classification, we identified 3 novel subsets of multiple myeloma that may represent unique diagnostic entities.

Contains fulltext : 87501.pdf (Publisher’s version ) (Closed access) The phase 3 trial HOVON-50 was designed to evaluate the effect of thalidomide during induction treatment and as maintenance in patients with multiple myeloma who were transplant candidates. A total of 556 patients was randomly assigned to arm A: 3 cycles of vincristine, adriamycin, and dexamethasone, or to arm B: thalidomide 200 mg orally, days 1 to 28 plus adriamycin and dexamethasone. After induction therapy and stem cell mobilization, patients were to receive high-dose melphalan, 200 mg/m(2), followed by maintenance with alpha-interferon (arm A) or thalidomide 50 mg daily (arm B). Thalidomide significantly improved overall response rate as well as quality of the response before and after high dose melphalan. Best overall response rate on protocol was 88% and 79% (P = .005), at least very good partial remission 66% and 54% (P = .005), and complete remission 31% and 23% (P = .04), respectively, in favor of the thalidomide arm. Thalidomide also significantly improved event-free survival from median 22 months to 34 months (P < .001), and prolonged progression free from median 25 months to 34 months (P < .001). Median survival was longer in the thalidomide arm, 73 versus 60 months; however, this difference was not significant (P = .77). Patients randomized to thalidomide had strongly reduced survival after relapse. This trial was registered on www.controlled-trials.com as ISRCTN06413384.

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Background: Venous thromboembolism (VTE) occurs frequently in multiple myeloma patients, especially during induction treatment with thalidomide in combination with anthracyclines and/or dexamethasone. Several coagulation abnormalities have been described in untreated myeloma patients, but these have not been prospectively evaluated during and after treatment. Patients and methods: We performed a prospective study in 138 multiple myeloma patients in whom coagulation factor levels were evaluated longitudinally before, during induction and after intensification. Patients were randomized to induction treatment consisting of adriamycin and dexamethason, in combination with either vincristin (VAD), thalidomide (TAD), or bortezomib (PAD) followed by high-dose melphalan (HDM) and autologous stem cell transplant (ASCT). Results: Factor VHLC (FVIII:C) and von Willebrand factor (VWF) were significantly elevated before treatment (median FVIII:C 2.26U/ml, VWF:Ag 1.95 U/ml). Irrespective of the type of induction regimen, these variables increased strongly during induction therapy (FVIII:C 2.55 U/ml and VWF:Ag 2.96 U/ml). Fibrinogen also showed a significant increase after induction therapy (3.5 g/l pre-treatment and 4.0 g/l after treatment, respectively, P < 0.001). This was significantly higher in TAD than VAD treated patients. Three to six month after ASCT levels of VWF and FVIII:C had decreased to values lower than observed before treatment (1.71 and 1.67 U/ml respectively). There was no correlation between the increased levels at start and the response of multiple myeloma to treatment. High levels of VWF, fibrinogen and FVIII:C before start of treatment were significantly associated with mortality. Fourteen patients (10%) developed a venous thrombotic event (VTE). The coagulation factor abnormalities before and during treatment were not associated with the development of VTE. Conclusion: During induction treatment several changes in coagulation factor levels are observed, which may result in a prothrombotic state. Larger studies are required to establish whether the changes in these coagulation factors during induction treatment contribute to the increased risk of venous thromboembolism in multiple myeloma patients. (c) 2007 Elsevier Ltd. All rights reserved.

Contains fulltext : 70575.pdf (Publisher’s version ) (Open Access) Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)-matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4(+) T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19(L)-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19(L)-specific CD4(+) T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8(+) mHag-specific T cells. They also lysed CD19(L)-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19(L)-derived HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.

The occurrence and nature of cytogenetic aberrations in polyneuropathy associated with IgM monoclonal gammopathy was determined. Therefore, interphase fluorescence in situ hybridization (FISH) was applied in 22 patients with polyneuropathy associated with IgM monoclonal gammopathy, multiplex ligation-dependent probe amplification (MLPA) assay in 18 of these patients and genome-wide-array-based comparative genomic hybridization (CGH) in eight of these 18 patients. Four patients had 10-20% and one patient had 30% B cells with IgH rearrangements; one patient had additional loss of 14qter; one patient had amplification of 6p and loss of 6q. Cytogenetic aberrations may be found in one third of the patients with neuropathy associated with IgM monoclonal gammopathy and are mainly associated with indolent Waldenstrom's Macroglobulinemia.

Purpose: Donor T cells directed to hematopoietic minor histocompatibility antigens (mHag) are appealing tools for adoptive immunotherapy of hematological malignancies after allogeneic stem cell transplantation (allo-SCT). Toward the development of a convenient strategy for ex vivo generation of human leukocyte antigen (HLA) class II–restricted mHag-specific T cells, we evaluated the feasibility of rebuilding mHag-specific T cell functions in donor-derived recall antigen-specific T cells via T cell receptor (TCR) transfer.Experimental Design: TCR α- and β-chains of an HLA-DPB1*0401–restricted T-cell clone recognizing a multiple myeloma-associated mHag were retrovirally transferred into a tetanus toxoid (TT)–specific clone derived from the original stem cell donor. TCR double-transduced cells were compared with the parent mHag- and TT-specific clones for antigen specificity, cytokine secretion, and cytotoxic activity and were analyzed for their in vitro expansion capacity in a TT- or mHag-specific fashion.Results: mHag-TCR–transduced TT-specific cells displayed both TT and mHag specificity. Similar to the parent cells, they secreted Th-1 cytokines and exerted significant cytotoxic activity against TT-pulsed or mHag+ target cells, including multiple myeloma cells. A 4-week expansion of TCR-transduced cells via the TT-specific TCR had no negative influence on the mHag-specific cytotoxic activity and resulted in 10- to 100-fold better cell yields as compared with mHag-specific expansion.Conclusions: HLA class II–restricted, mHag-specific effector functions can be successfully reconstructed in donor-derived TT-specific T cells via TCR transfer. Effective expansion of these T cells via TT-specific TCRs illustrate the suitability of this strategy for ex vivo expansion and possibly for in vivo TT-specific reboosting of HLA class II–restricted immunotherapeutic T cells.

*Laboratory for Thrombosis and Haemostasis, Department of Clinical Chemistry and Haematology, University Medical Center Utrecht and theInstitute for Biomembranes, Utrecht; Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, Universityof Groningen, Groningen; and Department of Hematology, University Medical Center Utrecht, Utrecht, the NetherlandsTo cite this article: Bouma B, Maas C, Hazenberg BPC, Lokhorst HM, Gebbink MFBG. Increased plasmin-a2-antiplasmin levels indicate activationof the fibrinolytic system in systemic amyloidoses. J Thromb Haemost 2007; 5: 1139–42.See also Hammarstro¨m P. The bloody path of amyloids and prions. This issue, pp 1136–8.

Allo-SCT followed by DLIs can establish long-term remissions in multiple myeloma (MM) patients. In many patients, however, the immunotherapeutic graft-versus-tumor (GVT) effect is moderate and not sustained, implying that immune suppression is mediated, among other factors, by regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs). Towards a better understanding and, eventually, manipulation of the immune-regulatory mechanisms in transplanted MM patients, we retrospectively sought a correlation between DLI outcome and circulating CD14(+) MDSCs, CD14(-) MDSCs and Tregs in 53 MM patients before their first DLI. We found significantly elevated frequencies of highly suppressive CD14(+) MDSCs, CD14(-) MDSCs and Tregs in pre-DLI samples from patients. Higher frequencies of Tregs, but not of MDSCs, were significantly associated with non-responsiveness to DLI. Furthermore, a lower frequency of Tregs predicted the development of chronic GVHD, which, in turn, displayed a high association with GVT. Elevated Treg frequencies before DLI were also associated with significantly shorter PFS and OS. Hence, our data reinforce the idea of active suppression of antitumor responses by Tregs in MM patients and therefore suggest that targeting patient Tregs before DLI may improve outcome of DLI.

Patients with multiple myeloma have variable survival and require reliable prognostic and predictive scoring systems. Currently, clinical and biological risk markers are used independently. Here, International Staging System (ISS), fluorescence in situ hybridization (FISH) markers, and gene expression (GEP) classifiers were combined to identify novel risk classifications in a discovery/validation setting. We used the datasets of the Dutch-Belgium Hemato-Oncology Group and German-speaking Myeloma Multicenter Group (HO65/GMMG-HD4), University of Arkansas for Medical Sciences-TT2 (UAMS-TT2), UAMS-TT3, Medical Research Council-IX, Assessment of Proteasome Inhibition for Extending Remissions, and Intergroupe Francophone du Myelome (IFM-G) (total number of patients: 4750). Twenty risk markers were evaluated, including t(4;14) and deletion of 17p (FISH), EMC92, and UAMS70 (GEP classifiers), and ISS. The novel risk classifications demonstrated that ISS is a valuable partner to GEP classifiers and FISH. Ranking all novel and existing risk classifications showed that the EMC92-ISS combination is the strongest predictor for overall survival, resulting in a 4-group risk classification. The median survival was 24 months for the highest risk group, 47 and 61 months for the intermediate risk groups, and the median was not reached after 96 months for the lowest risk group. The EMC92-ISS classification is a novel prognostic tool, based on biological and clinical parameters, which is superior to current markers and offers a robust, clinically relevant 4-group model.

Despite recent treatment improvements, multiple myeloma remains an incurable disease. Since antibody-dependent cell-mediated cytotoxicity is an important effector mechanism of daratumumab, we explored the possibility of improving daratumumab-mediated cell-mediated cytotoxicity by blocking natural killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines, but it did significantly augment daratumumab-induced myeloma cell lysis. Also in an ex vivo setting, IPH2102 synergistically improved daratumumab-dependent lysis of primary myeloma cells in bone marrow mononuclear cells (n=21), especially in patients carrying the FcγRIIIa-158F allele or the FcγRIIa-131R allele, who bind IgG1 with lower affinity than patients carrying the FcγRIIIa-158V allele or the FcγRIIa-131H allele. Finally, a further synergistically improved myeloma cell lysis with the daratumumab-IPH2102 combination was observed by adding lenalidomide, which suggests that more effective treatment strategies can be designed for multiple myeloma by combining daratumumab with agents that independently modulate natural killer cell function.

Antineural antibody activity is the implicated pathogenic mechanism in polyneuropathy associated with monoclonal gammopathy. Recognition of antigen depends on immunoglobulin variable regions, encoded by V genes. We studied V(H)DJ(H) and V(L)J(L) gene use in monoclonal B cells by clonal analysis in 20 patients with polyneuropathy and IgM monoclonal gammopathy. V genes associated with bacterial responses appear over-represented and V(H)3-23 was preferentially used, without association with specific D, J(H) or V(L)J(L). V genes revealed somatic mutation and intraclonal variation was found in 9 of 20 patients. Polyneuropathy associated with monoclonal gammopathy may be caused by an immune response to bacterial antigens, which recruit somatically mutated autoreactive B cells.

HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.

Venous thromboembolism (VTE) is a major complication in patients with multiple myeloma (MM) during treatment with thalidomide combined with chemotherapy and/or dexamethasone. The pathophysiology is not clear. We performed a cross-sectional study in 20 MM patients who were treated with thalidomide for refractory/relapsed disease. Seven patients (35%) experienced an episode of VTE. Plasma samples were analyzed for known risk factors for VTE and compared with those from 30 MM patients without thalidomide treatment. The patients groups differed in their baseline characteristics in activity status only. Extremely high levels of factor VIII-coagulant activity (FVIII:C, mean 352%) and von Willebrand factor antigen (VWF-Ag, 374%) were found in all patients using thalidomide. All other prothrombotic risk factors were normal. In patients with VTE, VWF-Ag but not FVIII:C, levels were significantly higher as compared with patients without VTE. Patients without thalidomide treatment had significantly lower levels of both coagulation factors but the difference was only due to difference in activity status. High FVIII:C/VWF-Ag levels are found in patients with active MM and this is probably a reflection of increased bone marrow angiogenesis in MM. These prothrombogenic circumstances could contribute to the high incidence of VTE during treatment with thalidomide in combination with dexamethasone/chemotherapy.

Virus or tumor Ag–derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients.

Background: Daratumumab (DARA) (HuMax™-CD38), a human IgG1κ monoclonal antibody effectively mediates destruction of CD38-expressing malignant plasma cells. In the first-in-human dose-escalation study, 42% of heavily pretreated patients with relapsed, or relapsed, refractory (RR) multiple myeloma (MM) treated with DARA alone (≥4mg/kg) achieved partial response (PR) and 25% had minimal response (MR) (modified IMWG guidelines) (1). In preclinical studies, DARA + lenalidomide (LEN) enhanced killing of MM cells in vitro (2). We evaluated safety, pharmacokinetics (PK) and efficacy of DARA + LEN + low-dose dexamethasone (DEX) in patients with relapsed or RR MM. Methods: This ongoing phase I/II open-label multicenter study consisted of 2 parts: Part1 was dose-escalation study in which patients (≥ 18 years old) with life expectancy ≥3 months and ECOG status 0, 1 or 2 received DARA+LEN+DEX (DARA [2-16 mg/kg] per week [8 weeks], twice a month [16 weeks], then, once monthly until disease progression, unmanageable toxicity or 24 months in total; LEN [25 mg PO day 1 through 21 of 28-days cycles]; DEX [40 mg] once weekly). Part 2 was cohort expansion study which explored the testing of maximum tolerated DARA dose (MTD) (16 mg/kg) determined in part 1 along with LEN (25 mg mg PO day 1 through 21 of 28-days cycles) and DEX (40 mg) once weekly. Results: Data from 22 patients (13 patients [fully enrolled] from part 1 and 9 patients from part 2, [ongoing enrollment]) were presented at ASCO earlier this year (3). These results demonstrated that the most frequent (>30% patients) adverse events (AEs) were neutropenia and diarrhea; no dose limiting toxicities (DLTs) were reported. Infusion reactions (grade 1 and 2) were reported in 4 patients. 8 serious AEs were reported, all assessed as unrelated to DARA. MTD was not reached. DARA+LEN+DEX PK-profile was similar to DARA alone suggesting LEN and DEX do not affect the DARA PK-profile. Available preliminary efficacy data from 20 patients demonstrated marked decrease in M-protein in all patients; 15/20 patients achieved PR or better, 3/20 with CR, 6/20 with VGPR. Median time to response was 4.3 weeks (range: 2.1-11.3). Overall response rate (ORR) was 75% (15/20) combining all patients in part 1 and 2 and 92.3% (12/13) for part 1 patients, who had at least 2 months of follow-up or discontinued earlier. Conclusions: DARA+LEN+DEX has favorable safety profile with manageable toxicities in relapsed and RR MM. Encouraging early activity is seen with marked reduction in M-protein and majority of the patients (~75%) achieved PR or better. Results of approximately 30 patients from part 2 with at least 2 months of treatment exposure and 10 patients (out of 30 patients) with shortened duration of infusion will be presented. References Lokhorst et. al., EHA 2013 abstract #8512 van der Veer et. al., Haematologica 2011;96(2):284-90 Plesner et. al. J Clin Oncol 32:5s, 2014 (suppl; abstr 8533). Disclosures Plesner: Genmab: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Celegene: Membership on an entity's Board of Directors or advisory committees. Lokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Minnema:Janssen: Consultancy, Honoraria. Laubach:Onyx: Research Funding; Novartis: Research Funding; Millenium: Research Funding; Celgene: Research Funding. Ahmadi:Janssen: Employment. Yeh:Janssen: Employment. Guckert:Janssen: Employment. Feng:Janssen: Employment. Brun:Genmab: Employment. Lisby:Genmab: Employment. Basse:Genmab: Employment. Palumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding.

Introduction : Daratumumab (DARA), a human CD38 IgG1κ monoclonal antibody, has demonstrated single agent efficacy while being highly tolerable as a monotherapy, and significant efficacy in combination with standard-of-care regimens in pts with multiple myeloma (MM) who have received ≥1 prior lines of therapy (Lokhorst HM. NEJM 2015, 373(13):1207-19; Lonial S. Lancet 2016, 387:1551-60;Palumbo A. NEJM 2016, in press; Dimopoulos MA. NEJM 2016, in press). DARA is currently administered as an intravenous (IV) infusion. Subcutaneous (SC) delivery of DARA is being tested in combination with the recombinant human hyaluronidase enzyme (rHuPH20) to facilitate systemic absorption of DARA after SC infusion into the abdominal wall. PAVO is the first study to assess the safety, pharmacokinetics, and efficacy of SC administration of DARA plus rHuPH20 (DARA-PH20) in pts with relapsed or refractory MM (RRMM). Methods : Pts had RRMM with ≥2 prior lines of therapy including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD). Part 1 of the 2 part study enrolled sequential cohorts at 1200 mg and 1800 mg DARA dose levels to determine the recommended SC dose for Part 2. DARA-PH20 was administered in 4-week treatment cycles: QW for 8 weeks, Q2W for 16 weeks, and Q4W thereafter. DARA-PH20 was infused in 1200 mg doses in 60 mL over 20 min or 1800 mg in 90 mL over 30 min, via a syringe pump at rotating sites on the abdomen. Pre- and/or post-infusion medications included paracetamol, diphenhydramine, montelukast, and methylprednisolone. In part 2, pts will be randomized 1:1 to receive the recommended phase 2 dose (RP2D) of SC DARA-PH20 or IV DARA (16 mg/kg). The RP2D of DARA-PH20 will be selected based on a cumulative review of the pharmacokinetic and safety data obtained from part 1 and should achieve a maximum serum Ctrough during weekly dosing that is similar to or higher than that observed for the approved 16 mg/kg IV dose. Primary endpoints were Ctrough of DARA up to Cycle 3 Day 1 and safety. Secondary endpoints included overall response rate (ORR). Results : To date, 41 pts were treated in part 1 with SC DARA-PH20 at the 1200 mg (n=8) and 1800 mg (n=33) dose levels. Infusion related reactions (IRRs) were reported in 9/41 pts (22%) and were mostly grade 1/2 in severity including chills, fever, rigors, vomiting, itching, edema of the tongue, non-cardiac chest pain and wheezing. One pt developed grade 3 dyspnea and 1 pt required hospitalization due to fever and chills (both grade 2) after the first infusion. All IRRs developed during or within 6 hours of the first SC infusion and were controlled with antihistamine, corticosteroid, antiemetic, or bronchodilator treatment. No IRRs were reported with subsequent infusions. Overall, the adverse event profile of DARA-PH20 was consistent with that of IV DARA. Grade 3 or higher drug-related adverse events were reported in 5/41 (12%) pts including fatigue (2 pts), influenza, hypertension, dyspnea, and tumor lysis syndrome. SC administration of DARA-PH20 was well tolerated at the abdominal wall injection site with 3/41 (7%) pts reporting grade 1 erythema, induration, or burning sensation. Analysis showed a higher max Ctrough in the 1800 mg cohort in comparison to the max Ctrough achieved following IV DARA (16 mg/kg). In the 1200 mg cohort of 8 pts (median of 5 lines of prior therapy [range 2-10]; prior ASCT, 63%; PI refractory only, 0%; IMiD refractory only, 13%; double refractory to PI and IMiD, 63%) a 25% ORR was observed including 2 partial responses (PR). Median time to response was 14 (range 8-20) weeks. Among 17 response evaluable pts in the 1800 mg cohort with cycle 3 day 1 assessments (median of 4 prior lines of therapy [range 2-7]; prior ASCT, 76%; PI refractory only, 6%; IMiD refractory only, 12%; double refractory to PI and IMiD, 65%) a 41% ORR was observed consisting of 3 very good partial responses and 4 PRs. Median time to response was 4 (range 4-8) weeks. Conclusions : SC DARA-PH20 was well tolerated and achieved serum trough concentrations similar to or greater than IV DARA with a lower rate of IRRs compared to IV DARA over a significantly shorter infusion time. Preliminary data suggest that in this pt population SC DARA-PH20 may enable similar response rates to IV DARA monotherapy. The 1800 mg dose level of DARA-PH20 was selected as the RP2D for part 2 of the study. These early data support further study of SC DARA in clinical trials. Additional data on pts from part 1 will be presented at the meeting. Disclosures Usmani: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Research Funding; Britsol-Myers Squibb: Consultancy, Research Funding; Skyline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Array: Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau. Mateos:Janssen, Celgene, Amgen, Takeda, BMS: Honoraria. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Chari:Pharmacyclics: Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding. Kaufman:Celgene: Consultancy, Research Funding; Pharmacyclics: Consultancy; Novartis: Consultancy, Research Funding; Incyte: Consultancy. Moreau:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau. Oriol:Janssen: Honoraria, Other: Expert board committee; Amgen: Honoraria, Other: Expert board committee. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hellemans:Janssen: Employment, Equity Ownership. Masterson:Janssen: Employment. Clemens:Janssen: Employment. Ahmadi:Janssen: Employment. Liu:Merck: Equity Ownership; Janssen: Employment; J&J: Equity Ownership. San-Miguel:Amgen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Introduction: Daratumumab (DARA) is a human CD38-targeting monoclonal antibody that induces deep clinical responses in MM pts through multifaceted mechanisms of action (MOA) including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and induction of apoptosis. Flow cytometry analysis revealed a previously unknown immunomodulatory role of DARA, via T-cell induction expansion, T-cell activity enhancement, and reduction of immune suppressive cell populations including CD38+ myeloid-derived suppressor cells, CD38+ regulatory T cells (TRegs), and CD38+ regulatory B cells (BRegs). Next-generation mass cytometry (CyTOF), which allows high parameter evaluation of the immune system, was used to assess the effects of DARA alone or in combination on a more comprehensive profile of immune cell subpopulations. Methods: Relapsed/refractory MM pt samples from a subset of single agent studies; SIRIUS (32 pts; whole blood [WB] only; Lonial S et al. The Lancet, 2016) and GEN501 (5 pts; WB and bone marrow [BM], Lokhorst HM et al. NEJM, 2015) along with GEN503, a study of DARA plus lenalidomide and dexamethasone (9 pts; WB and BM; Plesner T et al. ASH 2015) were analyzed. Fluorochrome or metal-conjugated antibody panel stained samples were evaluated by flow cytometry or cytometry by time-of-flight (CyTOF®) platforms, respectively. FACS analyses were performed and analyzed by FACS Canto II flow cytometers and FACSDiva software. For CyTOF analysis, events were clustered by phenotype by a spanning tree progression of density normalized events (SPADE) algorithm, and each cluster was associated with an immune population via Cytobank® software. Differential analysis of population fractions and marker intensity, over time and between response groups, derived raw P values from t-tests and single cell level bootstrap adjusted P values corrected for multiple dependent hypothesis testing. Results were visualized using SPADE trees (Figure) and Radviz projections, a new method that allows for the comparison of populations and conditions while preserving the relation to original dimensions. Results: Flow cytometry and high-dimensional CyTOF analyses confirmed previous findings including higher CD38 expression on plasma cells compared with other immune populations of natural killer (NK), monocytes, B and T cells, and depletion of both plasma cells and NK cells upon DARA treatment. Interestingly, while NK cells were significantly reduced with DARA treatment, remaining active NK cells (CD16+CD56dim) demonstrated increased expression of activation markers CD69, CD25 and CD137 while also decreasing granzyme B and increasing naive marker CD27. Though functionality tests weren't performed, the ability to evaluate several markers simultaneously suggests these cells possess limited cytotoxicity. Additionally, these studies indicated depletion of CD38 positive immune suppressive subsets of Tregs and Bregs. CD38+ basophil reductions occurred independent of response and may provide insight to short-lived infusion related reactions. Several observations within the T-cell compartment were indicative of a DARA-mediated adaptive response in both WB and BM samples. T cells displayed increases in total numbers and shifted towards higher CD8:CD4 and effector:naïve ratios after 2 months of DARA treatment. Responders had higher expression levels of several activation markers including CD69 and HLA-DR along with increased production of cytolytic enzyme granzyme B in CD8+ T cells following DARA treatment. Interestingly, in the GEN503 sample set, pts who achieved a complete response presented with a distinct BM CD4 T-cell phenotype of high granzyme B positivity versus those that achieved a partial response or very good partial response. This observation suggests pts with an active immune phenotype may achieve deeper responses to DARA in combination with standard of care agents lenalidomide and dexamethasone. Conclusion: CyTOF analysis of pt samples from both single agent and combination DARA studies agree with flow cytometry and support the pharmacodynamics and immune modulatory MOA of DARA while providing additional insight into changes in T-cell subtypes and activation status. Future CyTOF analyses of clinical samples from phase 3 combination studies aim to confirm these observations and expand the understanding of the MOA of DARA. Disclosures Adams: Janssen Research & Development, LLC: Employment. Stevenaert:Janssen: Employment. Van der Borght:Janssen: Employment. Casneuf:Janssen R&D, Beerse, Belgium: Employment; Johnson & Johnson: Equity Ownership. Smets:Janssen: Employment. Bald:Janssen: Employment. Abraham:Janssen: Employment. Ceulemans:Janssen: Employment. Vanhoof:Janssen: Employment; Johnson & Johnson: Equity Ownership. Ahmadi:Janssen: Employment. Usmani:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array: Research Funding; BioPharma: Research Funding; Pharmacyclics: Research Funding; Takeda: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees; Skyline: Membership on an entity's Board of Directors or advisory committees. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lonial:Janssen: Consultancy; BMS: Consultancy; Merck: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Onyx: Consultancy; Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mutis:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Celgene: Research Funding. van de Donk:Janssen: Research Funding; BMS: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Sasser:Janssen Pharmaceuticals R&D: Employment; Johnson & Johnson: Equity Ownership.

Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody (ab)-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells endowed with tumor-reactive CARs are highly promising tools for immunotherapy of cancer. There are however only a few truly tumor specific molecules that can be targeted by CARs, a drawback for the broad application of CAR T cell therapy. Indeed, when we recently aimed at targeting CD38high multiple myeloma (MM) with T cells transduced with high affinity CD38CARs, we observed that they not only lysed the CD38high MM cells but also CD38+ normal hematopoietic cells, pointing towards potential safety issues of such tumor-associated, but not entirely tumor-specific CARs. Therefore, using CD38 as a model for antigen we now tested whether it would be possible to reduce the on target, off-tumor effects of such CARs by optimizing their target cell affinity. To this end, we generated a new panel of CD38 abs through the "light chain exchange" method, in which heavy chains of two high affinity CD38 abs were combined with 176 different germ line light chains. This approach revealed around 100 new abs, which displayed 10- >1000 fold lower affinity to CD38 as compared to the parental abs. After categorizing them in three classes based on CD38 binding affinity, we used 8 abs from each class to generate 24 different CD38-CAR constructs. Testing the cytotoxic activity of T cells transduced with these CD38-CARs against CD38++ MM cell lines, primary MM cells and CD38+ normal hematopoietic cells in vitro and in vivo demonstrated that CD38-CAR T cells with ca. 1000 fold lower affinity to CD38 could still effectively lyse CD38++ MM cells while there was little or no cytotoxicity against CD38+ healthy hematopoietic cells. The results of this study reveal that it is possible to reduce the on-target off-tumor effects of CARs by optimizing their affinity. Thus, tailored affinity of the ab binding domain may open up new roads for CAR therapy. Disclosures van de Donk: Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Patients with recurrent or refractory low-grade non-Hodgkin's lymphoma (NHL) are increasingly treated with myeloablative therapy and autologous stem cell transplantation. However, allogeneic bone marrow transplantation (BMT) is only sporadically performed in such patients. Therefore, we wish to compare treatment results of patients with recurrent or refractory low-grade NHL who underwent allogeneic BMT with those who underwent autologous BMT in our center. Twenty-eight patients were studied. The patients had received 2 to 5 lines of conventional chemotherapy before the BMT procedure. Eighteen patients, all with chemotherapy-sensitive disease at the time of transplantation, underwent autologous BMT and 10 patients, of whom 7 with chemotherapy-resistant disease at the time of transplantation, underwent allogeneic BMT. Furthermore, all allogeneic BMT patients had overt lymphoma infiltration of the BM at the time of transplantation. The conditioning regimen consisted of cyclophosphamide plus total body irradiation in all 28 patients. All allogeneic BMT patients achieved complete remission, 3 patients had a treatment-related death, and 7 patients are alive and disease-free with a median follow-up of 41 months. In contrast, none of the autologous BMT patients died of transplant-related complications. However, despite the fact that all autologous BMT patients had chemotherapy-sensitive disease and partial remission was converted to complete remission by the BMT procedure in 67% of them, only 3 of 18 patients are alive and disease-free. The probability of relapse or disease-progression among allogeneic BMT patients was 0% compared with 83% for autologous BMT patients (P = .002). Progression-free survival rates 2 years after BMT were 68% for allogeneic BMT patients and 22% for autologous BMT patients (P = .049). Although the numbers of patients are small, this study suggests that allogeneic BMT offers a better chance for cure than autologous BMT for patients with poor-prognosis low-grade lymphoma, and the difference in relapse or disease progression is strongly suggestive for the existence of a graft–versus–low-grade lymphoma effect.

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4+ or CD8+ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Results: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell–cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR. Clin Cancer Res; 19(20); 5591–601. ©2013 AACR.

Purpose: Regulatory T cells (Tregs) are potent tools to prevent graft-versus-host disease (GVHD) induced after allogeneic stem cell transplantation or donor lymphocyte infusions. Toward clinical application of Tregs for GVHD treatment, we investigated the impact of Tregs on the therapeutic graft-versus-tumor (GVT) effect against human multiple myeloma tumors with various immunogenicities, progression rates, and localizations in a humanized murine model. Experimental Design: Immunodeficient Rag2−/−γc−/− mice, bearing various human multiple myeloma tumors, were treated with human peripheral blood mononuclear cell (PBMC) alone or together with autologous ex vivo cultured Tregs. Mice were analyzed for the in vivo engraftment, homing of T-cell subsets, development of GVHD and GVT. In additional in vitro assays, Tregs that were cultured together with bone marrow stromal cells were analyzed for phenotype and functions. Results: Treatment with PBMC alone induced variable degrees of antitumor response, depending on the immunogenicity and the growth rate of the tumor. Coinfusion of Tregs did not impair the antitumor response against tumors residing within the bone marrow, irrespective of their immunogenicity or growth rates. In contrast, Tregs readily inhibited the antitumor effect against tumors growing outside the bone marrow. Exploring this remarkable phenomenon, we discovered that bone marrow stroma neutralizes the suppressive activity of Tregs in part via production of interleukin (IL)-1β/IL-6. We furthermore found in vitro and in vivo evidence of conversion of Tregs into IL-17–producing T cells in the bone marrow environment. Conclusions: These results provide new insights into the Treg immunobiology and indicate the conditional benefits of future Treg-based therapies. Clin Cancer Res; 19(6); 1467–75. ©2012 AACR.

Recently, cereblon (CRBN) expression was found to be essential for the activity of thalidomide and lenalidomide. In the present study, we investigated whether the clinical efficacy of thalidomide in multiple myeloma is associated with CRBN expression in myeloma cells. Patients with newly diagnosed multiple myeloma were included in the HOVON-65/GMMG-HD4 trial, in which postintensification treatment in 1 arm consisted of daily thalidomide (50 mg) for 2 years. Gene-expression profiling, determined at the start of the trial, was available for 96 patients who started thalidomide maintenance. In this patient set, increase of CRBN gene expression was significantly associated with longerprogression-free survival (P = .005). In contrast, no association between CRBN expression and survival was observed in the arm with bortezomib maintenance. We conclude that CRBN expression may be associated with the clinical efficacy of thalidomide. This trial has been registered at the Nederlands Trial Register (www.trialregister.nl) as NTR213; at the European Union Drug Regulating Authorities Clinical Trials (EudraCT) as 2004-000944-26; and at the International Standard Randomized Controlled Trial Number (ISRCTN) as 64455289.

Adoptive 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. © 2016 Ferrata Storti Foundation.