Multiple Myeloma Tumor Cell Lines Research Articles

Bivalent BCMA Binding and Low Affinity CD3 T-Cell Engagement By Abbv-383 Drives Sustained Activation with Reduced T-Cell Exhaustion in Preclinical Models of Multiple Myeloma

Introduction: ABBV-383 is an IgG4-based CD3-bispecific T-cell engager (TCE) that has shown promising efficacy and a favorable safety profile in heavily pre-treated patients with relapsed/refractory multiple myeloma (MM). ABBV-383 possesses a unique combination of a low-affinity CD3δε antibody coupled to a bivalent, high-affinity BCMA engager arm in a 2+1 format. Prior pre-clinical studies demonstrated that ABBV-383 induces potent redirected T-cell killing of MM tumors with reduced cytokine release. Here, we provide additional evidence that bivalent BCMA binding by ABBV-383 reduces the negative impact of soluble BCMA and low affinity CD3 T-cell engagement drives sustained T-cell activation with reduced immune exhaustion. Methods: Structural variants of ABBV-383 were created, including 1) the bivalent BCMA targeting antibody of ABBV-383 replaced by proximal and distal monovalent VH targeting molecules, and 2) BCMA antibody structures coupled to high-and low affinity CD3 variants ( Figure). For functional testing, ABBV-383 and the structural variants were added to PBMCs or T-cells from healthy donors to serve as effector cells in co-culture with BCMA-positive MM tumor cell lines. Tumor cell killing and T-cell activation were determined by flow cytometry, and cytokine production was assessed using Luminex instrumentation. Recombinant soluble BCMA (sBCMA) was utilized in the experimental models to evaluate the extent of BCMA TCE suppression. To define the relationship between CD3-affinity and T-cell exhaustion, multi-dimensional flow cytometry was performed in long-term, 28-day co-cultures with irradiated tumor cells replated every 7 days. Results: T-cell binding was shifted by >100x with substitution of the high affinity CD3 antibody compared to ABBV-383. While all antibody variants tested induced T-cell specific MM cell killing in a BCMA-dependent manner, the negative impact of soluble BCMA on tumor killing was reduced with ABBV-383 due to its bivalent interaction with tumor cells compared to the monovalent TCE formats. In addition, the low CD3 binding affinity of ABBV-383 lowered T-cell activation antigen expression and reduced inflammatory cytokine production but resulted in similar maximal cytotoxicity of tumor cells in co-cultures of healthy and MM patient samples compared to high-affinity CD3 targeting. While maximal tumor cytotoxicity was similar, ABBV-383 was found to significantly delay the induction of a critical exhaustion-inducing transcription factor, TOX, in long-term assays. T-cell population changes were observed as early as 3 days with decreased effector memory cells (CD45RA-CD62L-) and increased naïve T-cell populations (CD62L+CD45RA+) compared to high-affinity CD3 targeting. On day 21, the high-affinity CD3 x bivalent BCMA molecule resulted in 85% of the CD8+ T-cell population expressing the transcription factor TOX compared to only 15% for ABBV-383 co-cultures. TOX induces several gene families with chromatin-modulating capacity that drive the distinct transcriptional trajectory leading to T-cell dysfunction during chronic stimulation. Simultaneous expression of multiple checkpoint inhibitory proteins is indicative of T-cell exhaustion. Triple positive CD8+ T-cells expressing LAG3, PD-1 and CTLA4 were significantly reduced by ABBV-383 versus the high-affinity CD3 variant ( Figure). Conclusions: High avidity BCMA binding coupled to low affinity T-cell engagement distinguishes the resulting mechanism of action for ABBV-383 in MM. Here, we show that the presence of bivalent-BCMA reduced the negative impact of soluble BCMA, reduced inflammatory cytokine production, and dampened the emergence of TOX+ CD8+ T-cells indicating that ABBV-383 structure maximizes its clinical potential as a safe and effective MM therapy.

Abstract 4556: Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma

Abstract RO7297089 is a bispecific antibody (IgG-scFv) targeting Bcell maturation antigen (BCMA) and CD16a (FcγRIIIA) that is being developed for the treatment of multiple myeloma (MM). BCMA is exclusively expressed on plasmablasts and differentiated plasma cells (PCs), and is overexpressed on malignant PCs in MM patients. CD16a is expressed on natural killer (NK) cells, monocytes, mast cells, and macrophages. Herein, we characterized the mode of action and safety profile of RO7297089 in vitro and in vivo. RO7297089 showed potent cell killing when using BCMA+ MM tumor cell lines as target cells and human peripheral blood mononuclear cells, NK cells or macrophages as effector cells. Minimal increases in TNFα (2x) and IFNγ (4x), but not other cytokines/chemokines, were observed compared to the vehicle control treatment only in the presence of the BCMA+ MM tumor cell line up to the concentration tested. This suggests that, unlike T-cell engagers, the risk of cytokine release syndrome in patients receiving RO7297089 is low. Cynomolgus monkey is the only relevant nonclinical species for RO7297089 as it showed binding to both recombinant CD16 and BCMA with comparable affinity to human antigens. Following five weekly intravenous administrations to monkeys at 0, 15, and 50 mg/kg, RO7297089 was well tolerated. In line with the mechanism of action, there were no test article-related cytokine increases or adverse findings observed in both dose levels. Systemic exposure of RO7297089 was approximately dose proportional from 15 to 50 mg/kg. Anti-drug antibodies (ADA) were observed in some animals at both dose levels, and ADA-related decreases in concentrations were observed at only 15 mg/kg. To evaluate in vivo activity, RO7297089-related effects on total plasma sBCMA and PCs were assessed. Elevations of sBCMA levels (100x) were observed post dose at both dose levels, and these effects returned to predose levels in animals that did not maintain concentrations at 15 mg/kg, suggesting that RO7297089 bound to and stabilized circulating cynomolgus sBCMA. Time- and dose-dependent reductions in serum IgM levels were observed at both dose levels. Changes in PC numbers were not detected by immunophenotyping; however, gene expression analysis of PC markers was included and demonstrated clear reductions in mRNA expression levels of PC markers including BCMA and J-chain in blood at both dose levels, suggesting reductions in BCMA+ cells. Collectively, these studies suggest that RO7297089 selectively kills BCMA+ cells by engaging CD16a-positive immune cells and has a favorable safety profile. Citation Format: Satoko Kakiuchi-Kiyota, Melissa M. Schutten, Adeyemi O. Adedeji, Hao Cai, Robert Hendricks, Luna Liu, Sivan Cohen, Aaron M. Fullerton, Nicholas Corr, Lanlan Yu, Denise de Almeida Nagata, Shelly Zhong, Michael Dillon, Christoph Spiess, Steve R. Leong, Bing Zheng, Susanne Wingert, Uwe Reusch, Stefan Knackmuss, Thorsten Ross, Andrew Polson, Ayse M. Ovacik. Preclinical pharmacology and safety of RO7297089, a novel anti-BCMA/CD16a bispecific antibody for the treatment of multiple myeloma [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 4556.

Comprehensive characterization of the mutational landscape in multiple myeloma cell lines reveals potential drivers and pathways associated with tumor progression and drug resistance.

Human multiple myeloma tumor cell lines (HMCLs) have been a cornerstone of research in multiple myeloma (MM) and have helped to shape our understanding of molecular processes that drive tumor progression. A comprehensive characterization of genomic mutations in HMCLs will provide a basis for choosing relevant cell line models to study a particular aspect of myeloma biology, or to screen for an antagonist of certain cancer pathways.Methods: We performed whole exome sequencing on a large cohort of 30 HMCLs, representative of a large molecular heterogeneity of MM, and 8 control samples (epstein-barr virus (EBV)-immortalized B-cells obtained from 8 different patients). We evaluated the sensitivity of HMCLs to ten drugs.Results: We identified a high confidence list of 236 protein-coding genes with mutations affecting the structure of the encoded protein. Among the most frequently mutated genes, there were known MM drivers, such as TP53, KRAS, NRAS, ATM and FAM46C, as well as novel mutated genes, including CNOT3, KMT2D, MSH3 and PMS1. We next generated a comprehensive map of altered key pathways in HMCLs. These include cell growth pathways (MAPK, JAK-STAT, PI(3)K-AKT and TP53 / cell cycle pathway), DNA repair pathway and chromatin modifiers. Importantly, our analysis highlighted a significant association between the mutation of several genes and the response to conventional drugs used in MM as well as targeted inhibitors.Conclusion: Taken together, this first comprehensive exome-wide analysis of the mutational landscape in HMCLs provides unique resources for further studies and identifies novel genes potentially associated with MM pathophysiology, some of which may be targets for future therapeutic intervention.

The Therapeutic CD38 Monoclonal Antibody Daratumumab Induces Programmed Cell Death via Fcγ Receptor-Mediated Cross-Linking.

Emerging evidence suggests that FcγR-mediated cross-linking of tumor-bound mAbs may induce signaling in tumor cells that contributes to their therapeutic activity. In this study, we show that daratumumab (DARA), a therapeutic human CD38 mAb with a broad-spectrum killing activity, is able to induce programmed cell death (PCD) of CD38(+) multiple myeloma tumor cell lines when cross-linked in vitro by secondary Abs or via an FcγR. By comparing DARA efficacy in a syngeneic in vivo tumor model using FcRγ-chain knockout or NOTAM mice carrying a signaling-inactive FcRγ-chain, we found that the inhibitory FcγRIIb as well as activating FcγRs induce DARA cross-linking-mediated PCD. In conclusion, our in vitro and in vivo data show that FcγR-mediated cross-linking of DARA induces PCD of CD38-expressing multiple myeloma tumor cells, which potentially contributes to the depth of response observed in DARA-treated patients and the drug's multifaceted mechanisms of action.

MAGE-A3 Specific T-Cell Receptor Adoptive Cell Transfer of Multiple Myeloma

Abstract 1843 Background:Multiple myeloma (MM) is a cancer of plasma cells and the second most common blood cancer. Current treatment strategies such as high dose chemotherapy, autologous stem cell rescue, and allogeneic transplantation have improved response rates and increased survival. However, these treatments often include high procedure-related morbidity and mortality and can only be applied to a small minority of myeloma patients. Therefore, safe broadly applicable immunologic strategies for myeloma, such as Adoptive Cell Therapy (ACT) are urgently needed. Methods:In this study we focused on aHLA-A*0201-restricted cancer testis antigen MAGE-A3:112–120, which is widely expressed in many forms of cancers such as metastatic melanoma, non-small cell lung cancer and MM, but not expressed in most normal tissues. To develop a system of effective strategies for T-cell therapy of multiple myeloma, we employed T-cell engineering technology using a MAGE-A3specific T-cell receptor (TCR)obtained from Dr. Steven Rosenberg at the National Cancer Institute. MAGE-A3 specific TCR was sub-cloned into a lentiviral vector and tranduced into purified CD8+ T-cells from human peripheral blood mononucleocytes (hPBMCs). To test the effector functionality of the MAGE-A3 specific TCR, the MAGE-A3 TCR-transduced CD8+ T-cells were subjected to cytokine release and chromium release assays after being co-cultured with MAGE-A3 peptide-loaded T2 cells, and U266 (MAGE-A3+/HLA-A*0201+), MM1.r (MAGE-A3+/HLA-A*0201-), KAS6 (MAGE-A3-/HLA-A*0201+), and KMS11(MAGE-A3-/HLA-A*0201-) MM tumor cell lines. Results:We observedcytokine production of INF-g and IL-2 in the MAGE-A3 TCR-transduced CD8+ T-cells generally in a dose-dependent manner to the MAGE-A3 peptide-loaded T2 cells. For example, the difference of INF-g secretion bythe MAGE-A3 TCR-transduced CD8+ T-cells wasa 10-fold increase from 0.001 uM to 0.02 uM of the loaded MAGE-A3 peptide. IL-2 secretion was also increasedby 7-fold from 0.001 uM to 0.1 uM of the MAGE-A3 peptide concentration. At 10uM of the peptide concentration, there was a 29-fold increase of the IL-2 production as compared to the 0.001 uM peptide concentration. Between 10uM and 100 uMof the peptide concentration, there was a decrease in IL-2 secretion by 2-fold, which is commonly observed at high peptide concentrations presumably due to cytotoxicity.Specific lysis of tumor cells by the MAGE-A3 TCR-transduced CD8+ T-cellswas observed in all four MM tumor cell lines, and we detected higher percentage of cell lysisin U266 (38%) and MM1.r (51%) cell lines as compared to the KAS6 (11%) and KMS11(21%) cell lines. Conclusions:Our findings suggest that the MAGE-A3 TCR-engineered CD 8+ T-cells are able to specifically recognize MAGE-A3 antigen, produce IL-2 and IFN-g, and destroy MM tumor cells loaded with the MAGE-A3 antigen. This potentially could further translate into effective MAGE-A3 specific targeted tumor rejection in vivo. We also plan to transduce the MAGE-A3 TCR into hematopoietic stem cells to and test the effector function of those cells against MM tumor cells and eventually against MM patient samples. Disclosures:No relevant conflicts of interest to declare.

The Multiple Myeloma Research Consortium Genomics Initiative.

The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative is a three-year program to analyze tumor tissue from hundreds of multiple myeloma (MM) patients via gene expression profiling (GEP), comparative genomic hybridization (aCGH), and exon re-sequencing. In addition, RNAi knockdown of selected genes in MM tumor cell lines is being evaluated to identify potential new targets. All genomic data generated is scheduled for placement in an open-access Multiple Myeloma Genomics Portal pre-publication and in near real-time (www.broad.mit.edu/mmgp). Additionally, samples are also destined for drug validation and correlative science on clinical protocols as this study moves forward. This comprehensive project is spearheaded by the MMRC and conducted via collaboration with the Eli and Edythe L. Broad Institute of MIT and Harvard, the Translational Genomics Research Institute (TGen), Mayo Clinic Arizona, and The Dana-Farber Cancer Center. The study is supported by the collection from member institutions of the MMRC of bone marrow aspirates and matched peripheral blood samples from over 1000 patients. Specific genomic technologies that are currently being employed across this sample set include GEP using Affymetrix Human Genome U133A 2.0 Plus Arrays, and, in parallel, efforts to identify regions of genomic gain and loss are using Agilent Human Genome CGH arrays. In contrast to other large-scale genomic projects based on exon-sequencing of targeted gene sets, this project will be the first to perform genome-scale single molecule sequencing (SMS) of DNA from patient specimens. Results will be targeted against candidate classes of genes (e.g. kinases, phosphatases, known oncogenes and tumor suppressors), and genes from GEP or within candidate regions of copy gain or loss identified by the aCGH experiments. Mutations will be further validated in an independent set of patient specimens. Finally we will attempt to identify points of vulnerability of MM through systematic loss-of-function screens in myeloma cell lines using high-throughput RNA interference (using both shRNA and siRNA platforms). Importantly, data generated from this genomics initiative will ultimately be made public pre-publication through the established MMRC Multiple Myeloma Genomics Portal. Data from all aspects of this project (sample collection and analyte isolation, GEP, aCGH, SMS, RNAi and bioinformatics) will be described in this presentation. The power of this study is the comprehensive collection of gene expression, CGH, and genome sequencing on a single reference set of clinically annotated samples. The addition of RNAi screens makes this a very important and unique data resource, which we hope will help expedite the discovery of novel targeted agents for MM scientific community.

The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell.

One of the distinguishing features of multiple myeloma (MM) is the proliferation of a clonal plasma cell population in the bone marrow (BM). It is of particular interest that the tumor plasma cells appear to be restricted to the microenvironment of the BM and are rarely detected in the peripheral system, yet the disease is found widely disseminated throughout the axial skeleton. Furthermore, isolation of MM tumor cell lines has proven to be quite problematic due to their slow growth rate. These observations have instigated the search for earlier cells in the B cell lineage that are clonally related to the plasma cell tumor and that may represent the growth fraction of the tumor. We used allele-specific oligonucleotides (ASO) derived from the third complementarity determining region of the rearranged tumor immunoglobulin heavy chain gene to detect isotypes clonally related to the plasma cell tumor. By reverse transcribing RNA from the BM with a panel of CH primers (mu, delta, alpha, and gamma), followed by ASO-polymerase chain reaction amplification, we demonstrate the existence of preswitch isotype species that are clonally related to the myeloma tumor. Furthermore, we show that separation of the BM cells into CD45+ and CD38+ cell populations results in a lineage-specific expression of the clonally related RNA molecules, with the C mu and C delta in the CD45+, and C gamma in the CD38+ population. Interestingly, clonally related C alpha transcripts are also derived from the CD45+ fraction. These results confirm the presence of B cell populations clonally related to the plasma cell tumor and are consistent with models that propose the existence of myeloma precursors.