non-Hodgkin Lymphoma Cell Lines Research Articles

A Novel Antibody-Cell Conjugation Method to Enhance Cellular Therapies for the Treatment of Hematological Malignancies

IntroductionCellular therapies including γδ T cells, NK-92 cells, and cytokine-induced killer (CIK) cells have shown modest anti-tumor activity against multiple hematological and solid malignancies in preclinical and phase I/II clinical trials. We investigate whether the antitumor activity of these cellular therapies can be augmented by using a novel method that rapidly affixes tumor-targeting antibodies directly to cell surface proteins.MethodsMonoclonal antibodies including rituximab and daratumumab were conjugated to the cell surface proteins using a three step approach. First, we conjugate single-stranded DNAs (ssDNAs) to the therapeutic monoclonal antibodies, as previously described (ref below). Next, we conjugate complementary ssDNAs to surface proteins of cells being used for cellular therapy. Finally, the modified antibodies are attached to the modified cells via hybridization of the complementary DNA strands.ResultsWe demonstrate that multiple anti-neoplastic monoclonal antibodies including rituximab and daratumumab can be conjugated to the surface proteins on a variety of cell including γδ T cells, NK-92 cells, CIK cells, and primary human T cells. Then we evaluated the killing activity of these conjugated cells in vitro, in vivo, and ex vivo . In vitro, Rituximab-armed γδ T cells and NK-92 cells demonstrated increased cytokine production (IFN-γ and granzyme B) and enhanced cytotoxic killing against of multiple human B-cell non-Hodgkin lymphoma (NHL) and multiple myeloma cell lines. Similar enhanced anti-tumor activity was seen with rituximab-armed or daratumumab-armed CIK cells, as well as increased CD107a expression (a marker of cytotoxicity activity) by the antibody-armed CIK cells. Rituximab-armed primary T cells also show enhanced cytotoxic killing against Raji cells, although not as pronounced as autologous rituximab-armed CIK cells. We next evaluated the therapeutic potential of antibody-armed cell therapies in vivo in a disseminated xenograft mouse model using SCID-beige mice. Rituximab-armed γδ T cells exhibited potent anti-lymphoma activity against Raji cells, which lead to improved overall survival; whereas unarmed γδ T cells alone or with rituximab co-infusion demonstrated limited anti-lymphoma activity. We also evaluated the therapeutic activity of antibody-armed cell therapies against a primary lymphoma target ex vivo. Rituximab-arm CIK cells demonstrated enhanced cytolytic activity against primary follicular lymphoma freshly collected from a patient via fine needle aspiration biopsy.DiscussionWe have demonstrated a novel approach to create tumor-specific cellular immunotherapies without the need for genetic engineering. The antibody to cell conjugation method is straight forward. The modified antibodies can be made and stored until needed. It takes approximately 1 hour to attaching these modified antibodies to the cell surface and is performed in standard aqueous buffers, such as phosphate-buffered saline (PBS). This method is also flexible being able to conjugate multiple different antibodies to the surface proteins of a variety of different cell types. We also see enhanced cell killing across multiple hematological targets using multiple cell populations including γδ T cells, NK-92 cells, CIK cells, and primary human T cells. Overall, our data demonstrate existing monoclonal antibodies can be used to “antibody program” cell therapies for the treatment of hematological malignancies.RefHsiao et al Langmuir 2009,25(12), 6985-6991 DisclosuresHsiao:Acepodia, Inc: Employment.

Charting Venetoclax Sensitivity and Synergism Ex Vivo in Multiple Myeloma

We have implemented a drug screening platform for evaluation of multiple myeloma (MM) relevant therapeutics alone or in combination, providing immediate actionable results. We assembled 80 assay-ready, MM relevant FDA approved drugs and clinical trial therapeutics. This panel was evaluated in 25 MM and 10 Non-Hodgkin lymphoma cell lines, and in 65 primary MM samples using a viability endpoint. The panel includes venetoclax, a selective BCL-2 inhibitor that is currently in clinical development for MM as a single agent or in combination with bortezomib and dexamethasone. Single agent clinical trial results have shown that venetoclax is active in 21% of all relapsed refractory MM patients, 40% of patients with a t(11;14) and only 9% of patients lacking this translocation. It is unclear why not all patients with this translocation respond and who the 9% of non-translocated responders are. We were interested to know if our assay exhibited the same targeted pattern of activity and in assembling a genomic database to interrogate these questions.The cellular efficacies of the MM drug panel, including venetoclax, were measured using the CellTiter Glo assay (Promega), in 25 MM cell lines using 7 drug doses covering a broad concentration range at both 24 and 72 hour time points. Concurrent with clinical observations, venetoclax presents a differential activity profile (Table 1). At 72h drug incubation, 75% of t(11;14) MM cell lines responded with an EC50<100nM but only 3 of 21 (14%) of translocation negative cell lines responded (overall response rate of 24%). Ex vivo sensitivity to venetoclax was also measured following 24h drug exposure in CD138+ selected tumor cells from 56 MM patients, 15 of which have a t(11;14). A majority of patient samples (85%) exhibit a dose response curve to venetoclax, but a broad EC50 range is observed (0.59 to 4900 nM). Twenty-seven patient samples (48% of all those tested) were inhibited by venetoclax at <100 nM concentrations, 10 of the 15 patients with t(11;14) responded at <100 nanomolar EC50, accounting for 67% of the samples with that translocation. Two patients with a t(6;14) also responded. For patients who lack either cyclin D1/D3 translocations (n=39), 15 responded with efficacies <100nM, or 38%. These trends are confirmed by median efficacies, with a median EC50 for t(11;14) samples of 29nM, much lower than the 118nM median observed for samples without this translocation. Nevertheless 17 of 41 patients lacking the t(11;14) are sensitive below 100nM EC50, a group which clearly deserves a deeper genomic analysis and clinical correlation.Using venetoclax as a base drug, we then studied all possible combinations with the 79 other agents of the MM drug panel in sensitive and resistant HMCLs. The cytotoxic response of the base and combined drugs was measured in 5-point 2-fold drug dilutions centered on their respective EC50s. Primary combination screen results were scored using consensus performance metrics from Chou-Talalay, Highest Single Agent (HSA), and Bliss independence models. Combinations predicted synergistic by at least 2 methods were confirmed in a matrix format. The screen combining venetoclax and the drug panel was designed to identify synergistic combinations that enhance venetoclax response in groups that lack t(11;14) and have either intermediate sensitivity (OPM2) or are resistant (AMO1). In OPM2, 25 drugs were found to synergize with venetoclax with a combination index CI < 0.2 (strong synergism), vs 1 non overlapping drug in the resistant HMCL, AMO1. The confirmation of these findings in a matrix format and in sensitive cell lines will be reported.In summary, our data corroborates the clinical observation of a strong pattern for venetoclax response and t(11;14) through drug screening in primary patient materials ex vivo with a 24 hour read out. Using this strategy we will begin to add drug combinations of high promise to our drug screening platform to develop the map of drug synergism in multiple myeloma.Acknowledgments: We thank the Multiple Myeloma Research Foundation for funding this project. [Display omitted] DisclosuresStewart:Bristol-Myers Squibb: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Roche: Consultancy.

Near-Infrared Fluorescence In-Vivo Imaging of Non-Hodgkin Lymphoma Using Cy7-Bevacizumab

Introduction: Tumor angiogenesis is necessary for tumor growth and metastasis. Of the many known inducers that promote tumor angiogenesis, vascular endothelial growth factor (VEGF) plays the most important role. Increased VEGF expression has been found in many tumor types, including non-Hodgkin lymphoma (NHL). Bevacizumab is a humanized monoclonal antibody that recognizes all known isoforms of VEGF. This study aims to design a diagnostic molecular probe, Cy7-Bevacizumab, in which Cy7 is a near-infrared fluorescent dye designed to in-vivo non-invasively identify VEGF expression in NHL tumors.Methodology: VEGF expression was confirmed using flow cytometry on NHL cell line Toledo. Cy7-Bevacizumab was synthesized through nucleophilic substitution reaction between monofunctional N-hydroxysuccinimide ester (Cy7-NHS) and Bevacizumab [1]. After purification, the conjugate was characterized by SDS-PAGE and spectrophotometry. For in-vivo imaging, Cy7-Bevacizumab (1 nmol) was injected intravenously in NHL tumor-bearing Balb/c nude mice (n=3) and was evaluated with near-infrared fluorescence (NIRF) at 2, 6, 24, 48, 72 and 96 h post-injection. After 96 h the lymph nodes accumulation was analyzed by ex-vivo NIRF images.Results: Flow cytometry showed that NHL Toledo cell line expresses high levels of VEGF. Bevacizumab was successfully labeled with Cy7, as shown by SDS-PAGE and spectrophotometry. Non-invasively NIRF in-vivo imaging of NHL tumor-bearing Balb/c nude mice allowed identification of tumors up to 96 h post-injection. Ex-vivo NIRF images confirmed tracer deposition at tumors.Conclusions: NHL Toledo cell line expresses VEGF. Cy7 labeled Bevacizumab has the potential to become an optical imaging agent for VEGF expressing tumors such as NHL, being possibly useful in guiding surgical excision of tumors and biopsies, which merits further evaluation.Acknowledgments: Agencia Nacional de Innovación e Investigación - Uruguay (ANII), Roche Laboratories, Pro.In.Bio (Uruguay), PEDECIBA Química (Uruguay)) and Comisión Sectorial de Investigación Científica-Universidad de la República-Uruguay (CSIC, UdelaR) I+D Grupos Oncología Nuclear.Disclosures: No relevant conflicts of interest to declare.

Deregulated miRNAs Contribute to Silencing of B-Cell Specific Transcription Factors and Activation of NF-κB in Classical Hodgkin Lymphoma.

Simple SummaryThe role of transcriptionally deregulated miRNAs (microRNAs) in classical Hodgkin lymphoma (cHL) is still not fully understood. To address this issue, we have performed global miRNA expression profiling of commonly used cHL cell lines and we present a complete cHL miRNome (microRNome). Within this group, we identify miRNAs recurrently deregulated in cHL cell lines, and compare them to non-Hodgkin lymphoma cell lines and sorted normal CD77+ germinal centre B-cells. Moreover, we show that several of the recurrently overexpressed miRNAs in cHL cell lines, and also primary microdissected HRS (Hodgkin and Reed-Sternberg) cells, target known B-cell-related transcription factors and NF-κB inhibitors. These findings provide evidence that deregulated miRNAs contribute to the loss of B-cell phenotype and NF-κB activation observed in this lymphoma.A hallmark of classical Hodgkin lymphoma (cHL) is the attenuation of B-cell transcription factors leading to global transcriptional reprogramming. The role of miRNAs (microRNAs) involved in this process is poorly studied. Therefore, we performed global miRNA expression profiling using RNA-seq on commonly used cHL cell lines, non-Hodgkin lymphoma cell lines and sorted normal CD77+ germinal centre B-cells as controls and characterized the cHL miRNome (microRNome). Among the 298 miRNAs expressed in cHL, 56 were significantly overexpressed and 23 downregulated (p < 0.05) compared to the controls. Moreover, we identified five miRNAs (hsa-miR-9-5p, hsa-miR-24-3p, hsa-miR-196a-5p, hsa-miR-21-5p, hsa-miR-155-5p) as especially important in the pathogenesis of this lymphoma. Target genes of the overexpressed miRNAs in cHL were significantly enriched (p < 0.05) in gene ontologies related to transcription factor activity. Therefore, we further focused on selected interactions with the SPI1 and ELF1 transcription factors attenuated in cHL and the NF-ĸB inhibitor TNFAIP3. We confirmed the interactions between hsa-miR-27a-5p:SPI1, hsa-miR-330-3p:ELF-1, hsa-miR-450b-5p:ELF-1 and hsa-miR-23a-3p:TNFAIP3, which suggest that overexpression of these miRNAs contributes to silencing of the respective genes. Moreover, by analyzing microdissected HRS cells, we demonstrated that these miRNAs are also overexpressed in primary tumor cells. Therefore, these miRNAs play a role in silencing the B-cell phenotype in cHL.

A phase 3 study to evaluate the efficacy and safety of tafasitamab plus lenalidomide and rituximab versus placebo plus lenalidomide and rituximab in patients with relapsed/refractory (R/R) follicular lymphoma (FL) or marginal zone lymphoma (MZL).

TPS7568 Background: Most patients with the indolent non-Hodgkin lymphoma (NHL) subtypes FL or MZL respond to first-line treatment but relapse is common, and there is no single standard treatment for patients with R/R FL or MZL. Tafasitamab is an Fc-engineered humanized monoclonal antibody (mAb) against CD19 which is broadly expressed in FL and MZL, and regulates B-cell proliferation via B-cell receptor signaling. In preclinical studies, tafasitamab has shown activity against NHL cell lines in combination with rituximab (anti-CD20 mAb) and lenalidomide (LEN). Tafasitamab monotherapy has shown promising clinical activity in a phase 2a study in patients with R/R NHL (NCT01685008), with an ORR of 29% (n/N = 10/34) in patients with FL and 33% (n/N = 3/9) in patients with MZL. In an ongoing phase 2, single-arm study (L-MIND, NCT02399085), tafasitamab plus LEN followed by tafasitamab alone demonstrated an ORR of 57.5% (n/N = 46/80) in patients with R/R diffuse large B-cell lymphoma (FDA approved indication). These preclinical and clinical observations from phase 2 trials suggest a potential clinical benefit of tafasitamab plus LEN and rituximab for patients with R/R FL or MZL. Methods: This phase 3 double-blind, placebo-controlled, randomized study is designed to investigate whether tafasitamab plus LEN and rituximab provides improved clinical benefit compared with LEN and rituximab in patients with R/R FL or R/R MZL. Patients will be randomized 1:1 to receive tafasitamab (12 mg/kg IV on days 1, 8, 15, and 22 of a 28-day cycle [cycles 1–3], then days 1 and 15 [cycles 4–12]) plus LEN (20 mg PO QD, days 1–21/ cycle for 12 cycles) and rituximab (375 mg/m2 IV on days 1, 8, 15, and 22 of cycle 1, then day 1 of cycles 2–5), or placebo (0.9% saline solution IV) plus LEN and rituximab. The primary study endpoint is PFS (investigator assessed [INV] by Lugano 2014 criteria) for patients with FL. Key secondary endpoints are PFS (INV) in overall population (FL and MZL), PET-CR rate (INV) at end of treatment (4–8 weeks after last treatment) and OS in patients with FL. Inclusion criteria include age ≥18 y, histologically confirmed FL (grade 1, 2, or 3a) or MZL (nodal, splenic, or extranodal), documented R/R disease, ≥1 prior systemic anti-CD20 therapy (including anti-CD20 refractory disease), ECOG PS ≤2, adequate systemic organ function, and high tumor burden (per GELF criteria). Exclusion criteria include prior rituximab plus LEN treatment, history of radiotherapy for other diseases (≥25% of bone marrow), nonhematologic malignancy, congestive heart failure (LVEF &lt; 50%), active systemic infection, known CNS lymphoma, or severe immunocompromised state. inMIND (NCT04680052, EudraCT2020-004407-13) is currently enrolling patients; planned enrollment is 528 patients with R/R FL and 60–90 patients with R/R MZL. Clinical trial information: NCT04680052.

Detection of novel and recurrent conjoined genes in non-Hodgkin B-cell lymphoma.

For this study, we investigated comprehensive expression of conjoined genes (CGs) in non-Hodgkin B-cell lymphoma (B-NHL) cell line KPUM-UH1 by using paired-end RNA sequencing. Furthermore, we analyzed the expression of these transcripts in an additional 21 cell lines, 37 primary samples of various malignancies and peripheral blood mononuclear cells of four normal individuals. Seventeen CGs were detected in KPUM-UH1: CTBS-GNG5, SRP9-EPHX1, RMND5A-ANAPC, OTX1-EHBP1, ATF2-CHN1, PRKAA1-TTC33, LARP1-MRPL22, LOC105379697-BAK1, TIAM2-SCAF8, SPAG1-VPS13B, WBP1L-CNNM2, NARS2-GAB2, CTSC-RAB38, VAMP1-CD27-AS1, LRRC37A2-NSF, UBA2-WTIP and ZNF600-ZNF611. To our knowledge, 10 of these genes have not been previously reported. The various characteristics of the CGs included in- and out-of-frame fusions, chimeras involving non-coding RNA and transcript variants. A finding of note was that LARP1-MRPL2 was characterized as in-frame fusion and was recurrently expressed in B-NHL samples. In this study, variety of CGs was expressed both in malignant and normal cells, some of which might be specific to lymphoma.

Polatuzumab Vedotin Plus Venetoclax with Rituximab in Relapsed/Refractory Diffuse Large B-Cell Lymphoma: Primary Efficacy Analysis of a Phase Ib/II Study

Polatuzumab Vedotin Plus Venetoclax with Rituximab in Relapsed/Refractory Diffuse Large B-Cell Lymphoma: Primary Efficacy Analysis of a Phase Ib/II Study

A Phase 1, Open-Label, Dose-Escalation Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of Multiple Intravenous Doses of IGN002 Administered Weekly to Subjects with Refractory Non-Hodgkin Lymphoma

Background:Non-Hodgkin lymphoma (NHL) is the most common, adult hematologic malignancy. Despite available treatment, relapse rates are high and novel therapies are warranted. IGN002 consists of an anti-CD20 monoclonal antibody (mAb) fused to interferon-alpha (IFNα) via a short peptide linker. The mAb portion of IGN002 is a chimeric IgG1 antibody with the same amino acid sequence as rituximab. The interferon amino acid sequence is human IFNα2b (hIFNα2b). In nonclinical pharmacology studies using CD20-positive human NHL cells, IGN002 demonstrates compelling anti-tumor effects. When compared to rituximab, IGN002 demonstrated enhanced complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) effector functions against CD20-positive NHL cells. Moreover, compared to rituximab against normal human primary B-cells, IGN002 displayed higher maximal and more potent ADCC activity. Finally, in xenograft studies with immunodeficient mice using 3 different human NHL cell lines comparing IGN002 to rituximab, IGN002 demonstrated superior median and overall survival in all 3 NHL xenograft tumor lines. Study Design and Methods :This open-label, non-randomized, first-in-human, dose finding Phase 1 study comprises a Dose-Escalation Stage and an Expansion Stage (NCT02519270). In the Dose-Escalation Stage, ascending dose cohorts will be treated in 2 periods until the maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D) is identified. In Period 1, subjects will receive 2 doses of IGN002 administered weekly. Subjects with no anti-drug antibodies to IGN002 at Week 6 in Period 1 will proceed to Period 2 and receive up to 24 additional doses of IGN002 administered weekly in three 8-week cycles, with no treatment-free interval. In the Expansion Stage, subjects will receive up to 24 doses of IGN002 at the MTD or RP2D administered weekly in three 8-week cycles, with no treatment-free interval. Cohorts 1-5 at dose level 0.1, 0.3, 1, 3, and 10 ug/kg, respectively, have been completed without any dose limiting toxicity (DLT) and enrollment for Cohorts 6-8 is impending. Clinical Endpoints:The primary outcome measures are the evaluation of safety and tolerability of multiple doses of IGN002 administered weekly as an IV infusion to subjects with refractory NHL and the determination of MTD or RP2D of IGN002. The secondary outcome measures include characterizing the pharmacokinetic profile of ascending doses of IGN002, the incidence of anti-IGN002 antibody formation, and assessment of anti-tumor activity (e.g., response rate, duration) of IGN002 using the Lugano Classification criteria for NHL. Eligibility Criteria:This study is enrolling patients &amp;gt; 18 years with an ECOG &amp;lt;2 who have a documented history of immunohistochemistry-confirmed CD20-positive B-cell NHL, including diffuse large B-cell, mantle cell, marginal zone, lymphoplasmacytic, follicular, transformed follicular, or primary mediastinal B-cell lymphoma that is refractory to at least 1 documented regimen containing rituximab or a similar anti-CD20 therapy. Patients must also have measurable disease and adequate organ function. The exclusionary criteria encompass treatment with an approved or investigational chemotherapy or anti-CD20 drug within 28 days of Day 1, treatment with an approved or investigational biologic drug that does not target CD20 within 90 days of Day 1, or radiation therapy within 4 weeks of Day 1. Statistical Methods:The sample size was not predicated on formal statistical considerations. Descriptive statistics will be used to summarize continuous variables. Dichotomous endpoints will be summarized using frequencies, proportions, and exact binomial 95% CI. For subjects treated at the MTD or RP2D in Period 2 of the Dose-Escalation Stage and the Expansion Stage, time-to-event variables will be summarized using the Kaplan-Meier method. Target Accrual:In the Dose-Escalation Stage and Expansion Stage, approximately 9-18 (~3-6 subjects per cohort) and 14 subjects, respectively, will be accrued. Enrollment for Cohort 6 is expected to commence in September 2020. Disclosures Mataveli: Spectrum Pharmaceuticals:Current Employment, Current equity holder in publicly-traded company.

Identification of a potent and selective phosphatidylinositol 3-kinase δ inhibitor for the treatment of non-Hodgkin's lymphoma

PI3Kδ has proved to be an effective target for anti-lymphoma drugs. However, the application of current approved PI3Kδ inhibitors has been greatly limited due to their specific immune-mediated toxicity and increased risk of infection, it is necessary to develop more PI3Kδ inhibitors with new scaffold. In this study, SAR study with respect to piperazinone-containing purine derivatives led to the discovery of a potent and selective PI3Kδ inhibitor, 4-(cyclobutanecarbonyl)-1-((2-(2-ethyl-1H-benzo[d]imidazol-1-yl)-9-methyl-6-morpholino-9H-purin-8-yl)methyl)piperazin-2-one (WNY1613). WNY1613 exhibits good antiproliferative activity against a panel of non-Hodgkin's lymphoma (NHL) cell lines by inducing cancer cell apoptosis and inhibiting the phosphorylation of PI3K and MAPK downstream components. In addition, it can also prevent the tumor growth in both SU-DHL-6 and JEKO-1 xenograft models without observable toxicity. WNY1613 thus could be developed as a promising candidate for the treatment of NHL after subsequent extensive pharmacodynamics and pharmacokinetics investigation.

Napabucasin, a novel inhibitor of STAT3, inhibits growth and synergises with doxorubicin in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL), the most common type of aggressive non-Hodgkin lymphoma (NHL), has highly heterogeneous molecular characteristics. Although some patients initially respond to standard R-CHOP therapy, 30-40% develop refractory disease or suffer relapse. Signal transducer and activator of transcription 3 (STAT3), which regulates multiple oncogenic processes, has been found to be constitutively activated in various cancers, including DLBCL, suggesting its potential as a therapeutic target. In this study, we determined that 34% (23/69) of DLBCL patients expressed pSTAT3 (Y705) in tumour tissues. Napabucasin, a novel STAT3 inhibitor, exhibited potent cytotoxicity against NHL cell lines in a dose-dependent manner. Mechanistic studies demonstrated that napabucasin induced intrinsic and extrinsic cell apoptosis, downregulated the expression of STAT3 target genes, including the antiapoptotic protein Mcl-1, and regulated the ROS-mediated mitogen-activated protein kinase (MAPK) pathway. Most importantly, in vivo studies revealed the suppressive efficacy of napabucasin as a monotherapy without obvious toxicity. Furthermore, preliminary combination studies of napabucasin with doxorubicin showed significant synergism both in vitro and in vivo. Thus, our studies provide evidence that napabucasin alone or in combination is a promising therapeutic candidate for DLBCL patients.

Abstract CT133: Targeting BCL-2 and MCL-1 overcomes treatment resistance in relapsed and refractory non-Hodgkin lymphoma: Pre-clinical rationale and results from an open-label phase 1b study

Abstract Purpose: Treatment of patients with relapsed or refractory lymphoid neoplasms represents a significant clinical challenge, largely due to the genetic heterogeneity of these malignancies. Our study goal was to design a rational treatment strategy that simultaneously targets multiple disease drivers to provide safe, efficacious, and durable responses in patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Experimental procedures and data summary: Venetoclax is a selective BCL-2 inhibitor that induces apoptosis and is efficacious in chronic lymphocytic leukemia; however, single-agent efficacy in other lymphoid neoplasms is limited. Using pharmacologic and genetic studies, we identified the pro-survival BCL-2 protein family member MCL-1 as a venetoclax resistance factor in NHL cell lines. We found that the monomethyl auristatin E (MMAE) payload of the antibody-drug conjugate polatuzumab vedotin promotes MCL-1 degradation via the ubiquitin/proteasome system, thus providing a strong rationale for combination with venetoclax. Mechanistically, polatuzumab vedotin combined with venetoclax promoted apoptotic NHL cell death. In NHL animal models, venetoclax, polatuzumab vedotin, and the anti-CD20 antibody obinutuzumab, that activates targeted antibody- and complement-dependent cell cytotoxicity, produced durable tumor regressions. In a phase 1b clinical trial, 33 patients with relapsed or refractory follicular lymphoma were evaluable for response over 6 dose levels with 19 complete responses and 6 partial responses (overall response rate 76%). All patients (8 of 8) treated with the recommended phase 2 regimen (venetoclax 800mg + polatuzumab vedotin 1.8mg/kg + obinutuzumab 1000mg) achieved complete responses at end of induction. The median duration of follow-up for all dose levels was 17.7 months. This triplet combination was well-tolerated by most patients and had an expected and acceptable safety profile. Conclusions: Our studies implicate MCL-1 as a key venetoclax resistance factor in NHL that can be overcome by polatuzumab vedotin, which promotes MCL-1 degradation. Pre-clinical NHL animal models and early clinical data confirmed that the combination of venetoclax, polatuzumab vedotin, and obinutuzumab is safe and promotes durable responses. Because MMAE and other anti-tubulin agents are likely not the only therapeutics that antagonize MCL-1, our study provides scientific rationale to pursue the broader strategy of identifying other therapeutics that similarly neutralize MCL-1 function. Such agents could be used in combination with venetoclax in other malignancies where MCL-1 is a venetoclax resistance factor. Our mechanism-based treatment regimen that directly targets oncogenic drivers in NHL patients may serve as a framework for treating other malignancies with complex etiologies. Citation Format: Dhara N. Amin, Rajat Bannerji, Raghuveer Singh Mali, Jason Oeh, Eva Lin, Anuradha Zindal, MaryAnn Go, Shang-Fan Yu, Maxwell Krem, Chris Arthur, Uwe Hahn, Anna M. Johnston, Vinit G. Karur, Nadia Khan, Paula Marlton, Tycel Phillips, Giuseppe Gritti, John F. Seymour, Monica Tani, Sam Yuen Yuen, Scott Martin, Matthew T. Chang, Christopher M. Rose, Victoria C. Pham, Elizabeth A. Lasater, Andrew G. Polson, YiMeng Chang, Jamie Hirata, Lisa Musick, Deepak Sampath, Christopher R. Flowers, Ingrid E. Wertz. Targeting BCL-2 and MCL-1 overcomes treatment resistance in relapsed and refractory non-Hodgkin lymphoma: Pre-clinical rationale and results from an open-label phase 1b study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT133.

Design, synthesis and structure-activity relationship study of piperazinone-containing thieno[3,2-d]pyrimidine derivatives as new PI3Kδ inhibitors

Two classes of piperazinone-containing thieno[3,2-d]pyrimidines were designed and synthesized as new PI3Kδ inhibitors in this study. Detailed SAR study with respect to the piperazinone substituents at the 6-position of thieno[3,2-d]pyrimidine core demonstrated that piperazinone-containing thieno[3,2-d]pyrimidines would be more potent and selective for PI3Kδ than their piperazine counterparts, which led to the discovery of several potent PI3Kδ inhibitors with comparable or better antiproliferative activity against a panel of non-Hodgkin lymphoma (NHL) cell lines as compared with idelalisib. Our study will promote the development of new PI3Kδ inhibitors based on piperazinone-containing thieno[3,2-d]pyrimidine scaffold.

B7-H6 Promotes Cell Proliferation, Migration and Invasion of Non-Hodgkin Lymphoma via Ras/MEK/ERK Pathway Based on Quantitative Phosphoproteomics Data.

PurposeB7 homologue 6 (B7-H6) has been found at an up-regulated level in multiple cancer cells and identified to be positively correlated with inferior clinical features. In non-Hodgkin lymphoma (NHL), however, the roles of B7-H6 and the underlying mechanism of action remain unclear. Through in vivo and in vitro experiments, the aim of this study was to explore the regulatory mechanism of B7-H6 in NHL in order to provide new therapeutic strategies that can potentially be applied in clinical practice.MethodsThe expression of B7-H6 in T-lymphoblastic lymphoma (TLBL), diffuse large B cell lymphoma (DLBCL) and lymph node reactive hyperplasia (LRH) tissues were compared by immunohistochemistry. A total of 10 NHL cell lines were screened by Western blot to evaluate the expression of B7-H6. The effects of B7-H6 knockdown on cell proliferation, migration and invasion of NHL cells were studied in vivo using a transplanted tumor mice model, and in vitro by Cell Counting Kit-8 (CCK-8) and Transwell assays. Quantitative phosphoproteomics was performed to identify the changes of protein phosphorylation and related pathways affected by B7-H6. The effects of B7-H6 on NHL were validated via B7-H6 overexpression and pathway inhibitor assays.ResultsThe expression levels of B7-H6 in NHL cell lines, and TLBL and DLBCL tissues were significantly increased compared with those in the control groups. Inhibition of cell proliferation, migration and invasion was observed in Jurkat and Raji cells with B7-H6 knockdown. The ability of B7-H6 in promoting tumorigenesis was further validated by in vivo experiments. In addition, Ras and HIF-1 signaling pathways were shown to be significantly affected by B7-H6 through quantitative phosphorylation proteomics analysis. Ras/MEK/ERK pathway was verified to be significantly inhibited after B7-H6 knockdown by Western blot analysis. Strikingly, MEK inhibitor AZD8330 was found to have the ability to sufficiently inhibit Ras/MEK/ERK pathway, partially reverse cell proliferation and completely reverse cell migration and invasion induced by B7-H6.ConclusionB7-H6 promotes cell proliferation, migration and invasion in NHL via Ras/MEK/ERK pathway. Hence, B7-H6 or Ras/MEK/ERK pathway targeting may be used as potential therapeutics for treating NHL.

CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T-cells.

The prognosis of many patients with chemotherapy-refractory or multiply relapsed CD30+ non-Hodgkin lymphoma (NHL) or Hodgkin lymphoma (HL) still remains poor, and novel therapeutic approaches are warranted to address this unmet clinical need. In light of this consideration, we designed and pre-clinically validated a chimeric antigen receptor (CAR) construct characterized by a novel anti-CD30 single- chain variable-fragment cassette, linked to CD3ζ by the signaling domains of two co-stimulatory molecules, namely CD28.4-1BB or CD28.OX40. We found that CAR.CD30 T cells exhibit remarkable cytolytic activity in vitro against both HL and NHL cell lines, with sustained proliferation and pro-inflammatory cytokine production, even after multiple and sequential lymphoma-cell challenges. CAR.CD30 T cells also demonstrated anti-lymphoma activity in two in vivo xenograft immune-deficient mouse models of metastatic HL and NHL. We observed that administration of CAR.CD30 T cells, incorporating the CD28.OX40 co-stimulatory domains and manufactured in the presence of interleukin 7 and interleukin 15, were associated with the best overall survival in the treated mice, along with establishment of a long-term immunological memory able to protect mice from further tumor re-challenge. Our data indicate that, in the context of in vivo systemic metastatic xenograft mouse models, the co-stimulatory machinery of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T cells upon tumor encounter. The CD28.OX40 co-stimulatory combination is ultimately responsible for the anti-tumor efficacy of the approach, paving the way to translate this therapeutic strategy into clinical use for patients with CD30+ HL and NHL.

177Lu-Lilotomab Satetraxetan Has the Potential to Counteract Resistance to Rituximab in Non-Hodgkin Lymphoma.

Patients with non-Hodgkin lymphoma (NHL) who are treated with rituximab may develop resistant disease, often associated with changes in expression of CD20. The next-generation β-particle–emitting radioimmunoconjugate 177Lu-lilotomab-satetraxetan (Betalutin) was shown to up-regulate CD20 expression in different rituximab-sensitive NHL cell lines and to act synergistically with rituximab in a rituximab-sensitive NHL animal model. We hypothesized that 177Lu-lilotomab-satetraxetan may be used to reverse rituximab resistance in NHL. Methods: The rituximab-resistant Raji2R and the parental Raji cell lines were used. CD20 expression was measured by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was measured by a bioluminescence reporter assay. The efficacies of combined treatments with 177Lu-lilotomab-satetraxetan (150 or 350 MBq/kg) and rituximab (4 × 10 mg/kg) were compared with those of single agents or phosphate-buffered saline in a Raji2R-xenograft model. Cox regression and the Bliss independence model were used to assess synergism. Results: Rituximab binding in Raji2R cells was 36% ± 5% of that in the rituximab-sensitive Raji cells. 177Lu-lilotomab-satetraxetan treatment of Raji2R cells increased the binding to 53% ± 3% of the parental cell line. Rituximab ADCC induction in Raji2R cells was 20% ± 2% of that induced in Raji cells, whereas treatment with 177Lu-lilotomab-satetraxetan increased the ADCC induction to 30% ± 3% of that in Raji cells, representing a 50% increase (P < 0.05). The combination of rituximab with 350 MBq/kg 177Lu-lilotomab-satetraxetan synergistically suppressed Raji2R tumor growth in athymic Foxn1nu mice. Conclusion: 177Lu-lilotomab-satetraxetan has the potential to reverse rituximab resistance; it can increase rituximab binding and ADCC activity in vitro and can synergistically improve antitumor efficacy in vivo.

Abstract B67: Development of MyD88 L265P mutation-specific TCR gene therapy for treatment of B-cell lymphoma and leukemia

Abstract Lymphoproliferative malignancies form a major group of cancers and are responsible for nearly 10% of all cancer deaths in developed countries. Adoptive transfer of engineered T cells has been shown to be a promising treatment approach; hence, screening and evaluation of cellular antigens for T-cell therapy with higher specificity and efficacy is a rapidly progressing area of research. Cancer-specific antigens derived by somatic mutations acquired during tumor development, so called “neoantigens,” represent a very advantageous target repertoire. Here we aimed to develop T-cell receptors (TCRs) targeting recurrent neoantigens in lymphoma and leukemia, for a truly cancer-specific immunotherapy. With this purpose, we have generated T-cell lines from multiple healthy donors targeting one of the most common driver mutations found in B-cell lymphomas: a missense mutation on adaptor protein MyD88 changing leucine at position 265 to proline (L265P). T cell lines generated by autologous in vitro priming were reactive selectively against the predicted mutant epitope restricted to HLA-B7, but not against the corresponding wild-type peptide. TCR alpha and beta nucleotide sequences from these T-cell lines were successfully cloned into a retroviral vector and expressed on T cells of healthy donors for further characterization. All cloned TCRs have shown mutation-specific and HLA-restricted reactivity with varying functional avidity. TCR-transduced T cells were able to selectively recognize and kill engineered target cells expressing MYD88 mutation, proving that the mutant epitope can be naturally processed by human proteasome and presented on HLA-B7. In order to further evaluate the therapeutic potency, we tested TCR-transduced T cells with non-Hodgkin lymphoma cell lines naturally harboring MyD88 L265P, and again observed HLA-restricted and mutation-specific reactivity and cytotoxicity, accompanied by antigen-induced proliferation. Taken together, our data suggest that mutation-specific TCRs can be used to target cancer cells with MyD88 L265P, and hold promise for immunotherapy of B-cell malignancies. Therefore, an application to the European Patent Office for variable region sequences of the TCRs has been made (EP19152801.7) in collaboration with Charité Technology Transfer Office. Preclinical safety screening of the TCRs and in vivo efficacy assessment in mice are in progress. Citation Format: Özcan Çinar, Antonia Busse, Antonio Pezzutto. Development of MyD88 L265P mutation-specific TCR gene therapy for treatment of B-cell lymphoma and leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B67.

Live Attenuated Salmonella enterica Expressing and Releasing Cell-Permeable Bax BH3 Peptide Through the MisL Autotransporter System Elicits Antitumor Activity in a Murine Xenograft Model of Human B Non-hodgkin's Lymphoma.

The survival of patients with non-Hodgkin's lymphoma (NHL) has substantially improved with current treatments. Nevertheless, the appearance of drug-resistant cancer cells leads to patient relapse. It is therefore necessary to find new antitumor therapies that can completely eradicate transformed cells. Chemotherapy-resistant cancer cells are characterized by the overexpression of members of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family, such as Bcl-XL, Bcl-2, and Mcl-1. We have recently shown that peptides derived from the BH3 domain of the pro-apoptotic Bax protein may antagonize the anti-apoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. In this study, we investigated the feasibility of releasing this peptide into the tumor microenvironment using live attenuated Salmonella enterica, which has proven to be an ally in cancer therapy due to its high affinity for tumor tissue, its ability to activate the innate and adaptive antitumor immune responses, and its potential use as a delivery system of heterologous molecules. Thus, we expressed and released the cell-permeable Bax BH3 peptide from the surface of Salmonella enterica serovar Typhimurium SL3261 through the MisL autotransporter system. We demonstrated that this recombinant bacterium significantly decreased the viability and increased the apoptosis of Ramos cells, a human B NHL cell line. Indeed, the intravenous administration of this recombinant Salmonella enterica elicited antitumor activity and extended survival in a xenograft NHL murine model. This antitumor activity was mediated by apoptosis and an inflammatory response. Our approach may represent an eventual alternative to treat relapsing or refractory NHL.

A New Promising Third Generation CAR-CD30 T-Cell Therapy for CD30+ Lymphoma

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Identification of FASN-Dependent Onco-Metabolic Regulation of the Pentose Phosphate Pathway (PPP) and Nucleotide Metabolism in Non-Hodgkin Lymphoma (NHL)

Introduction: FASN catalyzes de novo fatty acid (FA) biosynthesis, which is an oncogenic function observed in many cancers, including NHL. HIF1a inducible FASN activity is responsible for overcoming negative hypoxic influence and upregulation of glucose metabolism as observed during premalignant transformation. FASN catalytic activity is also dependent on precursors derived from glucose metabolism. However, implications of FASN targeted therapies on these interdependent metabolic interactions and the overall impact on cancer cell proliferation remains unknown. Methods: FASN small molecule inhibitors cerulenin, orlistat, TVB3657 and TVB3166 were evaluated using a diverse panel of B cell NHL lines and primary NHL cells for the impact on FASN inhibition signaling &amp; induction of cell death (MTT, caspases &amp; AnnexinV/PI). Global transcriptomics were done with Affymetrix Human 2.0 ST Genechip with Gene Set Enrichment and Ingenuity Pathway Analysis (GSEA &amp; IPA). Metabolomic profiling was performed using mass spectrometry. TXNRD1, GSR, NQO1 expression and activity were evaluated by western blot and using enzyme assay kits. Global DNA &amp; RNA synthesis were evaluated using ClickIt Edu and EU assay kits. RNAseq data available from 775 NHL patients were utilized for metabolic transcriptome mapping. Results: Treatment with all FASN inhibitors resulted in dose- and time-dependent reduction (&amp;gt;90%) in cell viability and cell death in all NHL cell lines. GSEA and IPA identified cell cycle &amp; RNA metabolism as downregulated biological processes with carbohydrate and oxidative stress as upregulated biological processes observed. "Key gene" analysis predicted TNF signaling as a prominent response to FASN inhibition, validated as increased TNF secretion by ELISA with cell fractionation and western blot analysis revealing activation of TNF-PI3K signaling. Activation of PI3K with FASN inhibition also corresponded with increased expression of PI3K-dependent metabolic genes associated with glucose and lipid metabolism. Furthermore, metabolomic profiling in SUDHL10 cells revealed accumulation of the FASN precursor acetyl-CoA with FASN inhibition that was accompanied by increased ketogenic glycolytic and citric acid cycle activity. In addition, NADPH accumulation (FASN substrate) occurred with FASN inhibition, which was accompanied with reduction in ribose-phosphate and nucleotide pools as these processes are relevant to NADPH-generating PPP function. G6PD, TXNRD1, GSR &amp; NQO1 were identified as "key genes" responsive to FASN inhibition. Interestingly, this cluster of "key genes" represented the entire enzymatic activity related to the first rate-limiting step in PPP. Subsequently, we determined that increased NADP(H) pools were responsible for impaired NADPH regenerating functions of TXNRD1 and GSR antioxidant enzymes, with PI3K-dependent activation of NQO1 and uptake of glucose facilitating de novo glutathione synthesis, which substituted for the loss of antioxidant functions in SUDHL10, SUDHL4 and Raji cells. All responses were sensitive to inhibition by PI3K inhibition (e.g., BKM120) with co-targeting via FASN &amp; PI3K inhibition resulting in markedly increased oxidative stress, loss of mitochondrial membrane potential &amp; synergistic cell death in NHL cell lines and primary NHL cells. Finally, FASN inhibition was associated with reduction in ribo/deoxy-ribonucleotide pools that decreased global transcriptional activity (de novo RNA synthesis, by EU labeling) and replication (by EdU incorporation in DNA) (Fig 1). Analysis of RNAseq data and mapping of metabolic transcriptome from 772 NHL patients showed consistently elevated expression of genes related to glycolysis, citric acid cycle, fatty acid and nucleotide metabolism in patients with mutations in p53, MYC, BCL2, mTOR, MYD88, PIM2 &amp; CREBP genes, suggesting that these onco-metabolic interactions may be important for lymphomagenesis. Conclusions: Taken together, FASN oncogenic activity appears to extend beyond de novo fatty acid biosynthesis, serving as a central onco-metabolic regulator of malignant cell proliferation vis-à-vis integrating glucose, nucleotides, and antioxidant metabolic functions in NHL. In addition, co-targeting FASN and PI3K induced synergistic cell death. Altogether, blocking FASN and the dependent onco-metabolic functions represent highly novel targets for therapeutic strategies in NHL. Disclosures Chen: Oncomics, LLC: Consultancy, Patents &amp; Royalties. Dave:Data Driven Bioscience: Equity Ownership. Evens:Seattle Genetics: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Tesaro: Research Funding; Verastem: Consultancy, Honoraria.

Targeting B-Cell Maturation Antigen (BCMA) with CC-93269, a 2+1 T Cell Engager, Elicits Significant Apoptosis in Diffuse Large B-Cell Lymphoma Preclinical Models

B-cell maturation antigen (BCMA), a member of the TNF receptor superfamily, serves as the cell surface receptor for B-cell activating factor (BAFF). Upon binding of BAFF to BCMA, an intracellular signaling cascade is initiated resulting in upregulation of JNK pathway signaling events and NFkB mediated transcription. The canonical expression pattern of BCMA begins on germinal center B-cells and becomes maximally expressed on mature cells such as plasma cells. Given the high degree of expression of BCMA on multiple myeloma (MM) cells, the malignant counterpart to plasma cells, it has become a target of interest for CAR T and antibody mediated modalities such as antibody drug conjugates and bispecific molecules. Recent clinical data from clinical trials employing BCMA targeted CAR T cells or T cell engager (TCE) antibodies have demonstrated significant responses in heavily pretreated myeloma patients with overall response rates ranging from 70% to 80%. As BCMA is known to be expressed in earlier B-cell lineages, we sought to explore the expression of BCMA in non-Hodgkin lymphoma (NHL) and its sensitivity to CC-93269, a 2+1 TCE currently being clinically investigated in MM. NHL is a heterogeneous collection of lymphomas that can be classified into two major subgroups; aggressive lymphomas of which diffuse large B-cell (DLBCL) is the most prevalent subtype and indolent lymphomas of which follicular lymphoma is the largest constituent. We first sought to directly quantitate cell surface expression of BCMA utilizing a flow cytometry system based on a logarithmic dilution of phycoerythrin beads of a known quantity. In a panel of 43 NHL cell lines, we determined that BCMA expression ranged from 43 to 17,048 molecules per cell (median, 420). An isogenic pair of K562 that is null for BCMA expression and K562 constitutively overexpressing BCMA (K562-BCMA) (15,866 molecules/cell) served as negative and positive controls, respectively. Additionally, the MM cell line H929 was profiled to serve as an additional control with a BCMA expression level of 7,065 molecules/cell. Next, utilizing quantitative PCR we found that relative BCMA mRNA expression in the lymphoma cell lines ranged from 0.001 to 0.17-fold when normalized to the H929 MM cell line. Furthermore, we were able to determine that in the lymphoma cells there is a poor correlation between protein expression (mean fluorescent intensity) and mRNA expression (r2, 0.33). We next examined if there was any correlation between BCMA surface expression and T-cell mediated cytotoxicity after administration of CC-93269 in a co-culture assay. We selected 11 DLBCL cell lines with a surface expression ranging from 45 molecules to 17,000 molecules per cells and incubated them in a co-culture system with a defined 1:5 target:effector ratio with CC-93269 (0-200 ng/ml) for five days. Significant apoptosis as measured by annexin V and ToPro-3 staining of CFSE positive target cells was observed in 10 of the 11 cell lines profiled with an IC50 of 0.1 to 38 ng/ml for CC-93269. As controls, the K562 isogenic pair were also profiled with the K562-BCMA cell line exhibiting an IC50 of 0.5 ng/ml and no activity observed against the parental K562 cell line. Additionally, a bispecific antibody where the two binding domains for BCMA was altered to target HEL (hen egg lysozyme) demonstrated no activity against any of the cell lines profiled at a defined dose of 200 ng/ml. No association between CC-93269 activity and BCMA expression or cell of origin was found. To determine the expression of BCMA in primary DLBCL biopsy samples, we developed a novel monoclonal BCMA immunohistochemistry antibody (clone: G12). The antibody and IHC staining protocol were validated to have good on-target specificity in both cell lines and tissues, including MM and DLBCL biopsies, with a range of stain intensity (1-3+) observed in both the golgi and on the plasma membrane. A proof of concept study on a cohort of 110 commercial DLBCL samples is currently underway. Cumulatively, our data demonstrate that BCMA is expressed on the cell surface of a broad panel of NHL cell lines and in primary DLBCL lymph node biopsies. Additionally, the expression levels of BCMA in these preclinical cell line models was sufficient to elicit significant CC-93269 mediated cytotoxicity. These data highlight the potential for the treatment of DLBCL patients with a 2+1 T-cell engager targeting BCMA. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership, Patents &amp; Royalties. Waldman:Celgene: Employment, Equity Ownership, Patents &amp; Royalties. Gray:Celgene: Employment, Equity Ownership. Yura:Celgene: Employment, Equity Ownership. Hersey:Celgene: Employment, Equity Ownership. Chan:Celgene: Employment, Equity Ownership. Zhang:Celgene: Employment, Equity Ownership. Boss:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents &amp; Royalties.