We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3 T cell/myeloma cell crosslinking, followed by CD4/CD8 T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

Purpose: Knowledge about the mechanism of action (MoA) of monoclonal antibodies (mAb) is required to understand which patients with multiple myeloma (MM) benefit the most from a given mAb, alone or in combination therapy. Although there is considerable research about daratumumab, knowledge about other anti-CD38 mAbs remains scarce.Experimental Design: We performed a comprehensive analysis of the MoA of isatuximab.Results: Isatuximab induces internalization of CD38 but not its significant release from MM cell surface. In addition, we uncovered an association between levels of CD38 expression and different MoA: (i) Isatuximab was unable to induce direct apoptosis on MM cells with CD38 levels closer to those in patients with MM, (ii) isatuximab sensitized CD38 hi MM cells to bortezomib plus dexamethasone in the presence of stroma, (iii) antibody-dependent cellular cytotoxicity (ADCC) was triggered by CD38 lo and CD38 hi tumor plasma cells (PC), (iv) antibody-dependent cellular phagocytosis (ADCP) was triggered only by CD38 hi MM cells, whereas (v) complementdependent cytotoxicity could be triggered in less than half of the patient samples (those with elevated levels of CD38). Furthermore, we showed that isatuximab depletes CD38 hi B-lymphocyte precursors and natural killer (NK) lymphocytes ex vivo-the latter through activation followed by exhaustion and eventually phagocytosis.Conclusions: This study provides a framework to understand response determinants in patients treated with isatuximab based on the number of MoA triggered by CD38 levels of expression, and for the design of effective combinations aimed at capitalizing disrupted tumor-stroma cell protection, augmenting NK lymphocyte-mediated ADCC, or facilitating ADCP in CD38 lo MM patients.

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Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p <0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p <0.0001) and a shorter survival in MM patients with active disease requiring treatment (p ≤ 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM.

Abstract Granulocytic myeloid-derived suppressor cells (G-MDSCs) promote tumor growth and immunosuppression in multiple myeloma (MM). However, their phenotype is not well established for accurate monitoring or clinical translation. We aimed to provide the phenotypic profile of G-MDSCs based on their prognostic significance in MM, immunosuppressive potential, and molecular program. The preestablished phenotype of G-MDSCs was evaluated in bone marrow samples from controls and MM patients using multidimensional flow cytometry; surprisingly, we found that CD11b+CD14−CD15+CD33+HLADR− cells overlapped with common eosinophils and neutrophils, which were not expanded in MM patients. Therefore, we relied on automated clustering to unbiasedly identify all granulocytic subsets in the tumor microenvironment: basophils, eosinophils, and immature, intermediate, and mature neutrophils. In a series of 267 newly diagnosed MM patients (GEM2012MENOS65 trial), only the frequency of mature neutrophils at diagnosis was significantly associated with patient outcome, and a high mature neutrophil/T-cell ratio resulted in inferior progression-free survival (P &lt; .001). Upon fluorescence-activated cell sorting of each neutrophil subset, T-cell proliferation decreased in the presence of mature neutrophils (0.5-fold; P = .016), and the cytotoxic potential of T cells engaged by a BCMA×CD3-bispecific antibody increased notably with the depletion of mature neutrophils (fourfold; P = .0007). Most interestingly, RNA sequencing of the 3 subsets revealed that G-MDSC–related genes were specifically upregulated in mature neutrophils from MM patients vs controls because of differential chromatin accessibility. Taken together, our results establish a correlation between the clinical significance, immunosuppressive potential, and transcriptional network of well-defined neutrophil subsets, providing for the first time a set of optimal markers (CD11b/CD13/CD16) for accurate monitoring of G-MDSCs in MM.

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty-four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4 1 , CD8 1 , and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range 5 8.5-22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P 5 0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P 5 0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n 5 7 for an interval > 10 years and an SI < 20), 100% reached tolerance; in the second group (n 5 10 for an interval > 10 years and an SI > 20 or an interval < 10 years and an SI < 20), 60% reached tolerance; and in the third group (n 5 7 for an interval < 10 years and an SI > 20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables-the time from transplantation and the SI-may help to identify these patients. Liver Transpl 19:937-944, 2013. V C 2013 AASLD. Received February 25, 2013 accepted May 19, 2013. Although the survival of liver transplantation (LT) patients has improved since the early 1980s with refinements in immunosuppression therapy and surgical techniques, the morbidity and mortality rates of these patients are still greater than those of the general population. 1,2 The quality of life and survival are still diminished in comparison with those of ageand sex-matched controls. 3,4 As long-term survival after transplantation has improved, side effects See Editorial on Page 933

IntroductionThe vascular system is a bipolar complex network of arteries that transport oxygen-rich blood to all tissues and veins that bring oxygendeprived blood back to the heart. 1 Because of this bipolar set-up, arteries and veins feature anatomic and physiological differences. Unlike venous endothelium, arterial endothelium is surrounded by several layers of smooth muscle cells (SMCs), separated by elastic laminae, and embedded in a thick layer of fibrillar collagen. 2 Moreover, both vessel types have a differential susceptibility to atherosclerotic disease, possibly due to exposure to different levels of shear stress. Arterial and venous endothelial cells (ECs) also have a distinct molecular signature, and such molecular specification occurs before the onset of blood flow. 3 Indeed, arteriovenous (AV) specification of ECs is accomplished early in development and is associated with the expression of a specific complement of factors: venous endothelium is characterized by the expression of EphB4, 4 Lefty-1, 5 Lefty-2, 5 COUP-TFII, 6 and MYO1-␤, 5 and arterial ECs express high levels of Notch 1 and 4, 7 Dll-4, 8 EphrinB1 and EphrinB2, 4 Jagged-1 and -2, 7 connexin-40, and Hey-2 (gridlock zebrafish ortholog). 9,10 Studies in Xenopus, zebrafish, and mice have revealed that, besides blood flow, 11 vessel-intrinsic cues and-later in development-signals from outside the vasculature 12,13 are implicated in defining arterial or venous fate such as members of the TGF-␤ pathway, 14,15 VEGF isoforms,13,[16][17][18]17 angiopoietins, 19 the Notch pathway, 7,9,20-22 the patched pathway, 20 and COUP-TFII, a member of the orphan nuclear receptor superfamily. 6 Although it has been shown that some of these pathways are well conserved from zebrafish to mouse, less information is available on whether they have a similar role in humans. Because these molecular differences between arterial and venous ECs exist independently of blood flow and some of these factors work in an EC-intrinsic way, 2 it should be possible to manipulate some or all of these to endow ECs with an arterial or venous fate. Consistent with this notion, recent studies have suggested that arterial markers can be induced in primary mature ECs. 5,13,21,23,24 Many different stem cell populations, including bone marrow (BM) mononuclear cells, AC133 ϩ endothelial progenitor cells, and embryonic stem cells have the potential to differentiate in vitro and in vivo into mature and functional ECs. 4,[25][26][27][28] We have recently described another stem cell population, multipotent adult progenitor cells (MAPCs), that differentiates into most somatic cell types, including functional ECs, in vitro and in vivo. [29][30][31][32][33] The question of whether and how these stem cells can be coaxed into arterial or venous ECs has thus far not been addressed. In this study, we analyzed the in vitro and in vivo arterial and venous endothelial differentiation of human MAPCs (hMAPCs) and hAC133 ϩ cells. Materials and methodsAdditional and extended descriptions of methods are inc...

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