Next, we compared the activity of loncastuximab tesirine against all cell lines with the activity of R-CHOP, which is used in the first-line treatment of DLBCL. to other agents, especially BCL2 and PI3K inhibitors. Our data support the further development of loncastuximab tesirine for use as a single agent and in combination for patients affected by mature B-cell neoplasms. The results also highlight the importance of CD19 expression and the existence of lymphoma populations characterized by resistance to multiple therapies. == Introduction == Despite recent improvements, current therapies are not yet curative for too many patients affected by lymphoid neoplasms,1-3and novel therapeutic strategies are still needed. Antibody-drug conjugates (ADC) represent one MI-503 of the most successful therapeutic approaches introduced into clinical practice in the last 25 years.4,5ADC are complex compounds that contain three components: an antibody, a warhead (i.e., a cytotoxic agent), and a linker that joins the two together. ADC enable targeted delivery of potent warheads into tumor cells using antibodies against tumor antigens. Due to its pattern of expression and its biological role in lymphocytes, the B-cell marker CD19 has been heavily exploited for antibody-based therapies, including ADC, and, more recently, for cellular therapies.4,6-10Loncastuximab tesirine (ADCT-402) is a CD19-targeting ADC, in which the CD19-specific antibody is stochastically conjugated through a protease cleavable dipeptide linker to a pyrrolobenzodiazepine (PBD) dimer warhead (SG3199).11Following binding to CD19-positive cells, loncastuximab tesirine is rapidly internalized and transported to lysosomes, where the linker is cleaved to release the PBD dimer SG3199.11In contrast to the microtubule-disrupting monomethyl auristatin E (MMAE) used in the CD30-targeting brentuximab vedotin and the CD79B-targeting polatuzumab vedotin ADC,12,13SG3199 belongs to a new generation of DNA cross-linking agents. It binds to guanine residues in the DNA minor groove, forming covalent cross-links of the two DNA strands.14,15Loncastuximab tesirine has been studied in various clinical trials16-18and, based on the results of a phase II study,16,19it was recently approved in the USA and Europe for the treatment of adult patients with relapsed/ refractory (R/R) large B-cell lymphoma after at least two prior lines of systemic therapy.20 Here, we assessed the anti-tumor activity of loncastuximab tesirine in a large panel of lymphoma cell lines, with a focus on the expression of its target and the identification of active combination partners. == Methods == Details on the cell lines and compounds used in this study, together with information on the Ly4.0 cancer personalized profiling (CAPP)-sequencing genomic DNA assay and variant calling are provided in theOnline Supplementary Materials and Methods. The full methods for the immunoblotting, and cell cycle analysis as well as the data mining are also described in theOnline Supplementary Materials and Methods. == In vitrocytotoxic activity == The cytotoxic activity of loncastuximab tesirine was assessedin vitro, as previously described.21Briefly, cells were exposed to each compound for 96 hours and assayed by MTT (3-[4,5-dimethylthiazolyl-2]-2, 5-diphenyltetrazoliumbromide). For R-CHOP treatment, cells were exposed for 72 hours to 1 1 g/mL CHOP + 100 g/mL rituximab at five different concentrations in 1:10 serial dilutions. Rituximab was diluted to clinically recommended serum levels22and CHOP represented a mix reflecting the clinical ratios of the drugs23,24(85%, 4-hydroperoxy-cyclophosphamide; 5.5%, doxorubicin; 0.16%, vincristine; 11.1%, prednisolone). Cells were also exposed in parallel to the PBD dimer SG3199 and the isotype-control ADC B12-SG3249.25 Synergism was assessed by exposing cells for 96 hours to increasing doses of loncastuximab tesirine and each of the other agents, either alone or in combination, followed by an MTT assay. The Chou-Talalay combination index (CTI) was determined as previously described.26Combinations were defined as synergistic (median CTI <0.9), additive (median CTI, 0.9-1.1), or of no benefit/antagonist (median CTI >1.1). == CD19 expression == Absolute cell surface CD19 expression was determined via quantification of the antigen on the surface of lymphoma cell lines using Quantum Simply Cellular MI-503 anti-human IgG beads (Bangs Laboratories) to create a MI-503 calibration curve. Antibody-binding capacity values were then normalized to those of the control isotype antibody B12. CD19 CNA1 RNA expression values were extracted from the datasetsGSE94669, previously obtained using a targeted RNA-sequencing approach (HTG EdgeSeq Oncology Biomarker panel) and microarray-based technology (Illumina HT-12 arrays),26andGSE221770, previously produced via total RNA.