IntroductionImmature dendritic cells (DCs), specialized in the uptake of antigen, 1 reside as sentinels in almost every tissue. Under steadystate conditions, a small fraction of DCs acquire a semimature state and migrate to the draining secondary lymphoid organs. There, these semimature DCs exert tolerogenic functions by inducing apoptosis, anergy, or even a regulatory state in interacting autoreactive T cells that escaped elimination during negative thymic selection. In addition to natural regulatory T cells (Treg's), which originate in the thymus, induced Treg's, activated by tolerogenic DCs, are essential for maintainance of peripheral tolerance 2 and ensure tolerance to harmless environmental antigens. 3 In contrast, during infection, DCs engulf and process pathogenic material. Exposure to pathogen-derived molecules like lipopolysaccharide (LPS) or proinflammatory cytokines produced by cells in the microenvironment induces the full maturation of DCs, characterized by strong up-regulation of expression of costimulatory molecules and the production of proinflammatory cytokines. 4 Mature DCs constitute the most potent antigen-presenting cells, which are capable of stimulating naive antigen-specific T cells and thereby inducing a primary immune reaction.Several DC subsets with distinct phenotypes and functions have been identified. 5 In mice, CD11c has been acknowledged as a useful marker for DCs. DC subsets of myeloid origin (mDCs) mature in response to bacterial products and proinflammatory cytokines, and fully mature mDCs are inducers of strong immune responses. However, following treatment with anti-inflammatory cytokines (IL-10, transforming growth factor-␤ [TGF-␤]) or pharmacologic agents like dexamethasone (DEX) and vitamin D 3 , mDCs differentiate into a tolerogenic state. 6,7 Plasmacytoid DCs (pDCs) express markers like GR-1 and, in mice, B220, although a distinct lymphoid origin of pDCs is still a matter of debate. 8,9 pDCs have been ascribed a profound tolerogenic potential both under homeostatic conditions and even upon activation, mainly induced by DNA and RNA viruses. 10 DC functions are mainly studied using bone-marrow-derived DCs (BM-DCs) which are as potent as primary DCs isolated from spleen. 11 BM-DCs exhibit myeloid characteristics upon generation from progenitors in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas a major fraction displays a pDC-like phenotype when progenitors are cultivated with Flt3-L. 12 However, the generation of BM-DCs is time consuming, cultures may contain contaminating cell types, and BM-DCs display a heterogenous phenotype concerning the expression of major histocompatibility complex (MHC) II and costimulatory molecules, and therefore T-cell stimulatory capacity. 13 To circumvent these problems, several cell lines with DC-like characteristics have been derived either from long-term cultures of murine skin Langerhans cells 14,15 or spleen cell suspensions, 16 or by An Inside Blood analysis of this article appears at the front of this...

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M3258 is an orally bioavailable, potent, selective, reversible inhibitor of the large multifunctional peptidase 7 (LMP7, β5i, PSMB8) proteolytic subunit of the immunoproteasome; a crucial component of the cellular protein degradation machinery, which is highly expressed in malignant hematopoietic cells including multiple myeloma. M3258 was previously shown to deliver strong in vivo preclinical efficacy in multiple myeloma xenograft models, as well as a more benign non-clinical safety profile compared to approved pan-proteasome inhibitors, exemplified by a lack of effects on the central and peripheral nervous systems and cardiac and respiratory organs. Here we describe preclinical PK/PD and PK/efficacy modelling which led to a prediction of the PK profile, and the efficacious and safe dose ranges of M3258 in human which were used to guide the design of the phase I dose-escalation trial of M3258 in >3 line relapsed/refractory multiple myeloma (RRMM) patients. Mouse, rat, dog and monkey PK, plasma protein binding and intrinsic clearance data were used to estimate a half-life of approximately 6 hours for M3258 in human. The human total clearance and volume of distribution for M3258 were predicted to be 0.033 L/h/kg and 0.28 L/kg, respectively, whilst oral bioavailability was estimated to be above 80%. LMP7 proteolytic activity was assessed as a PD readout in human multiple myeloma tumor cells xenografted to mice as well as in dog peripheral blood mononuclear cells (PBMCs). A strong PK/PD relationship was observed for M3258 across both species. LMP7 inhibition by M3258 also correlated strongly with anti-tumor efficacy in multiple myeloma xenografts, with maximal efficacy observed at M3258 exposure delivering sustained inhibition of tumor LMP7 activity. Quantitative PK/PD/efficacy modeling predicted the biologically efficacious dose (BED) of M3258 upon oral application to be between 10 - 90 mg daily in human. By incorporating data from nonclinical safety studies, these data suggest an attractive human PK profile of M3258, enabling oral application, as well as an improved human therapeutic index compared to approved pan-proteasome inhibitors. M3258 is being investigated in a phase I, first-in-man, 2-part, open label clinical study designed to determine the safety, tolerability, PK, PD and early signs of efficacy of M3258 as a single agent (dose-escalation) and co-administered with dexamethasone (dose-expansion) in participants with RRMM whose disease has progressed following > 3 prior lines of therapy and for whom no effective standard therapy exists. Integration of these data will guide the selection of the BED for potential further clinical development of M3258. Disclosures Lignet: Merck Healthcare KGaA: Employment. Esdar:Merck Healthcare KGaA: Employment. Friese-Hamim:Merck Healthcare KGaA: Employment. Becker:Merck Healthcare KGaA: Employment, Other: Holding shares with a value below 1000-USD. Drouin:EMD Serono Research and Development Institute: Employment. El Bawab:Merck Healthcare KGaA: Employment. Goodstal:EMD Serono Research and Development Institute: Employment. Gimmi:Merck Healthcare KGaA: Employment. Haselmayer:Merck Healthcare KGaA: Employment. Jährling:Merck Healthcare KGaA: Employment. Sanderson:Merck Healthcare KGaA: Employment. Sloot:Merck Healthcare KGaA: Employment. Stinchi:Merck Healthcare KGaA: Employment. Victor:Merck Healthcare KGaA: Employment. Walter:Merck Healthcare KGaA: Employment. Rohdich:Merck Healthcare KGaA: Employment.