Mice were divided in four groups of ten animals each and were treated with MEN1309/OBT076 (2.5 mg/kg IV, D1 and D12), or rituximab (3 mg/kg IV on D1; 5 mg/kg IV on D12), or MEN1309/OBT076 plus rituximab (same schedule as single agents), or with vehicle only (IV). Single agents and combinations were more active (both single agent arms and tumor eradication. drug conjugates (ADC) represent a class of anti-cancer agents that can have an important impact on the clinical outcome of cancer patients, as exemplified by brentuximab vedotin for Hodgkin lymphoma patients or ado-trastuzumabemtansine (T-DM1) for breast cancer patients.1 In lymphomas, different surface proteins (CD19, CD37, CD79B, CD25) have provided targets for active ADC.1 CD205, encoded by the LY75 gene (Lymphocyte antigen 75, DEC-205), is a surface multilectin receptor with a cytoplasmatic domain containing protein motifs crucial for endocytosis and internalization upon ligation. Although its Zosuquidar biologic role has not been fully defined, it is known to act as a surface receptor for apoptotic and necrotic cells,2,3 leading to antigen uptake and processing. CD205 is expressed in hematopoietic cells, mainly by antigen presenting cells (APC), but also in other tissues, including solid tumors.4-8 CD205 presents a rapid internalization rate and a favorable profile in terms of differential expression between neoplastic and healthy Zosuquidar tissues, and is a potential new target for ADC.7 MEN1309/OBT076 is a novel humanized IgG1 antibody directed against CD205, with Goat polyclonal to IgG (H+L)(FITC) no direct anti-tumor effect, conjugated through a cleavable N-succinimidyl-4-(2- pyridyldithio) butanoate linker to the potent maytansinoid microtubule disruptor DM4.7 The ADC has shown potent anti-tumor activity with durable responses and complete tumor regressions in many models derived from triple negative breast cancer, pancreatic and bladder cancer cell lines and primary cells.7 MEN1309/OBT076 is now under early clinical investigation for patients with solid tumors and lymphoma (CD205-Shuttle study; and status were defined as previously described.11 Compounds MEN1309/OBT076, MBH1309 and IgG-DM4 for proliferation assay and MEN1309-PE for FACS analysis were provided by Menarini. Idelalisib, bortezomib, lenalidomide, venetoclax were purchased from Selleckchem (Houston, TX, USA) and rituximab from Roche (Basel, Switzerland). Proliferation and apoptosis assays Anti-proliferative activity of MEN1309/OBT076 or IgG-DM4 was assessed as before12 and are described in the combinations were assessed as previously described.10,12 Based on the Chou-Talalay Combination Index,13 the effect of the combinations was defined as beneficial if synergistic ( 0.9) or additive (0-9-1.1). Western blotting analysis Protein extraction, separation and immunoblotting were performed as previously described.10 The following antibodies were used: anti–Tubulin (cst-2146, Cell Signaling Technology), anti- PARP1 (sc-8007, Santa Cruz Biotechnology), anti-BCL2 (sc-492), anti-MCL1 (cst-5453), anti-BCLXL (sc-8392), and anti-CD20 (ab9475, Abcam). Immunohistochemistry Immunohistochemical staining was performed as described in the anti-tumor activity in diffuse large B-cell lymphoma Based on the expression data, we exposed 42 B-cell lymphoma cell lines derived from DLBCL, MCL, MZL, and CLL, to the anti-CD205 ADC MEN1309/OBT076 (translocation or translocation or inactivation (106 of the OCI-LY-10) (gene, coding for CD205, as measured by gene expression profiling or real-time polymerase chain reaction (RT-PCR) (gene, coding for CD205, is known to give rise to intergenically spliced transcripts with its centromeric neighboring gene anti-tumor activity in DLBCL To confirm the observed MEN1309/OBT076 anti-lymphoma activity, we performed an xenograft experiment with the OCI-LY-10 model of DLBCL. Cohorts of mice were treated with vehicle control, IgGDM4 (5 mg/kg, once every 3 weeks), or MEN1309/OBT076 (1.25, 2.5 or 5 mg/kg once every 3 weeks) (Figure 2A). Low activity was observed with MEN1309/OBT076 1.25 mg/kg (day [D] 7, control (D21, control mice (D7, D21, D28; antitumor activity in the activated B-cell like diffuse large B-cell lymphoma OCILY10 xenograf t mo del. Trea tme Zosuquidar nt with MEN1309/OBT 076 a n d IgG-DM4 st arted when tumors became visible (100mm3). (A) Control (CTR) group, n=4; IgG-DM4 group, n=4; MEN1309/OBT076 1.25 mg/kg, n=8; MEN1309/O BT076 2 .5 mg/kg, n=7; MEN1309/OBT076 5 mg/kg, n=8. In each box-plot, the line in the middle of the box represents the median and the box extends from the 25th to the 75th percentile (IQ: interquartile range). Each dot represent a mouse. xaxis: days of treatment. y -axis: tumor volume expressed in mm3. Zosuquidar (B) Kaplan-Meier LSS (lymph oma specific sur vival ). MEN 1309/OBT076 (5 mg /k g) all other groups together. anti-tumor activity than the single agents The synergism with rituximab was validated using an ABC-DLBCL lymphoma model, OCI-LY-10 (Figure 4). Mice were divided in four groups of ten animals each and were treated with MEN1309/OBT076 (2.5 mg/kg IV, D1 and D12), or rituximab (3 mg/kg IV on D1; 5 mg/kg IV on D12), or MEN1309/OBT076 plus rituximab (same schedule as single agents), or with vehicle only (IV). Single agents and combinations were more active (both single agent arms and tumor eradication. At the used doses, MEN1309/OBT076 presented higher anti-lymphoma activity compared to rituximab, although this was not statistically significant (and anti-tumor activity in lymphomas. The novel ADC had an anti-tumor activity that was highly correlated with expression of its target CD205 and reached synergism.