Increase of CIK cell efficacy by upregulating cell surface MICA and inhibition of NKG2D ligand shedding in multiple myeloma

2016 ◽  
Vol 35 (4) ◽  
pp. 719-725 ◽  
Author(s):  
Chidimma A. Nwangwu ◽  
Hans Weiher ◽  
Ingo G. H. Schmidt-Wolf
Keyword(s):  
Multiple Myeloma ◽  
Cell Surface ◽  
Nkg2d Ligand ◽  
Cik Cell ◽  
Ligand Shedding

137 Genomics of multiple myeloma influences the expression of CAR T-cell targets

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A150-A150
Author(s):  
Christina Yu ◽  
Brian Walker ◽  
G David Roodman ◽  
Kun Huang ◽  
Michel Sadelain ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
High Risk ◽  
Cell Surface ◽  
T Cell Therapy ◽  
Genomic Features ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T ◽  
Risk Patients

BackgroundMultiple Myeloma (MM) is an incurable disease, with a particularly poor prognosis for patients with refractory/relapsed MM or high-risk cytogenetics. Chimeric Antigen Receptor (CAR) T-cell therapy targeting BCMA can induce deep responses in highly pretreated RRMM; however, remissions are not sustained, and the majority of patients eventually relapse. We hypothesized that genomic determinants of MM play a role in dictating the expression of surface targets that can be of use for immune targeting.MethodsWe analyzed the gene expression of 24 immunotherapeutic targets in a combined dataset of 1900 MM patients from three independent expression datasets obtained from the Multiple Myeloma Research Foundation CoMMpass study and Gene Expression Omnibus. Given that CAR T-cell therapy may be especially important for patients with high-risk myeloma, we defined the expression of each target in high-risk MM patients by stratifying patients based on several genomic features impacting prognosis. Additionally, we conducted a gene co-expression network analysis and identified 30 gene modules highly correlated with 16 cell surface targets from our panel, further suggesting that genetic determinants of MM may shape a targetable cell surfaceome. In order to determine whether targeting any of these candidate antigens might cause major toxicity to normal cells, we utilized several repositories providing protein data1 to annotate their expression in several normal cell types.ResultsWe determined that a number of genomic factors could stratify the 24 targets into three general groups: 1) targets that show consistent overexpression in high-risk patients: IGF1R, ITGB7, GPRC5D and CD70, and are thus suitable for most high-risk patients; 2) targets that are down-regulated in patients with high-risk genomic features: CD200, CD19, CD40, CD1D and IGKC, perhaps playing a role in cancer immune escape; and 3) targets associated with one specific genetic abnormality, i.e. t(4;14): FUT3, SLAMF7, CD56, CD138 and BCMA, thus of use for precision CAR therapy in this high-risk patient subset.ConclusionsOur work provides a means of target selection for precision CAR therapy, by considering both patient genomic backgrounds and cancer cell surface profiles. Furthermore, our results provide a roadmap for immunotherapy of MM by unbiasedly comparing the expression of top MM cell surface targets in patient data and normal cells and suggest that the genetic landscape of MM may predict the expression of specific targets for precision immunotherapy. The quest for novel MM targets for immunotherapies remains open, and CAR target discovery driven by specific genetic events remains an active area of investigation.ReferencePerna F, Berman SH, Soni RK, et al. Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer Cell 2017;32(4):506–19.

scholarly journals Myeloma Cell Associated Therapeutic Protein Discovery Using Single Cell RNA-Seq Data

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Lijun Yao ◽  
Reyka G Jayasinghe ◽  
Tianjiao Wang ◽  
Julie O'Neal ◽  
Ruiyang Liu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Plasma Cell ◽  
Tissue Specificity ◽  
Plasma Cells ◽  
Expression Data ◽  
Intracellular Proteins

Multiple myeloma (MM) is a hematological cancer of the antibody-secreting plasma cells. Despite therapeutic advancements, MM remains incurable due to high incidence of drug-resistant relapse. In recent years, targeted immunotherapies, which take advantage of the immune system's cytotoxic defenses to specifically eliminate tumor cells expressing certain cell surface and intracellular proteins have shown promise in combating this and other B cell hematologic malignancies. A major limitation in the development of these therapies lies in the discovery of optimal candidate targets, which require both high expression in tumor cells as well as stringent tissue specificity. In an effort to identify potential myeloma-specific target antigens, we performed an unbiased search for genes with specific expression in plasma and/or B cells using single-cell RNA-sequencing (scRNAseq) of 53 bone marrow samples taken from 42 patients. By comparing >40K plasma cells to >97K immune cells across our cohort, we were able to identify a total of 181 plasma cell-associated genes, including 65 that encode cell-surface proteins and 116 encoding intracellular proteins. Of particular interest is that the plasma cells from each patient were shown to be transcriptionally distinct with unique sets of genes expressed defining each patient's malignant plasma cells. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to B-Cell receptor (BCR) signaling, protein transport, and endoplasmic reticulum (ER) stress, involving genes such as DERL3, HERPUD1, PDIA4, PDIA6, RRBP1, SSR3, SSR4, TXNDC5, and UBE2J1. To note, our strategy successfully captured several of the most promising MM therapeutic targets currently under pre-clinical and clinical trials, including TNFRSF17(BCMA), SLAMF7, and SDC1 (CD138). Among these, TNFRSF17 showed very high plasma cell expression, with concomitant sharp exclusion of other immune cell types. To ascertain tissue specificity of candidate genes outside of the bone marrow, we analyzed gene and protein expression data from the Genotype-Tissue Expression (GTEx) portal and Human Protein Atlas (HPA). We found further support for several candidates (incl. TNFRSF17,SLAMF7, TNFRSF13B (TACI), and TNFRSF13C) as being both exclusively and highly expressed in lymphoid tissues. While several surface candidates were not found to be lymphocyte-restricted at the protein level, they remain relevant considerations as secondary targets for bi-specific immunotherapy approaches currently under development. To further investigate potential combinatorial targeting, we examine sample-level patterns of candidate co-expression and mutually-exclusive expression using correlation analysis. As the majority of our detected plasma cell-specific genes encode intracellular proteins, we investigated the potential utility of these epitopes as therapeutic targets via MHC presentation. Highly expressed candidates include MZB1, SEC11C, HLA-DOB, POU2AF1, and EAF2. We analyzed protein sequences using NetMHC and NETMHCII to predict high-affinity peptides for common class-I and class-II HLA alleles. To correlate MHC allelic preference with candidate expression in our cohort, we performed HLA-typing for 29 samples using Optitype. To support our scRNAseq-driven findings, we cross-referenced gene expression data with 907 bulk RNA-sequencing samples, including 15 from internal studies and 892 from the Multiple Myeloma Research Foundation (MMRF), as well as bulk global proteomics data from 4 MM cell lines (TIB.U266, RPMI8226, OPM2, MM1ST) and 4 patients. We see consistent trends across both cohorts, with high positive correlation (Pearson R ranging between 0.60 and 0.99) for a majority of genes when comparing scRNA and bulk RNA expression in the same samples. Our experimental design and analysis strategies enabled the efficient discovery of myeloma-associated therapeutic target candidates. In conclusion, this study identified a set of promising myeloma CAR-T targets, providing novel treatment options for myeloma patients. Disclosures Goldsmith: Wugen Inc.: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Inhibition of Multiple Myeloma Cell Adhesion to Fibronectin by Ephrin Ligation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2360-2360
Author(s):  
Stuart Ratner ◽  
Charles A. Schiffer ◽  
Jeffrey A. Zonder
Keyword(s):  
Multiple Myeloma ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Myeloma Cell ◽  
Separate Experiment ◽  
Partial Recovery ◽  
Multiple Myeloma Cell ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) cell adhesion to fibronectin (FN), mediated via VLA-4 and VLA-5, has been shown to induce resistance to several chemotherapeutic drugs. Disruption of MM cell adhesion to FN and other marrow microenvironment elements might therefore enhance the effects of therapy. We now present the first evidence that Eph-ephrin signaling may be exploited to inhibit MM cell binding to fibronectin. Ephs are transmembrane tyrosine kinases and ephrins are their cell-surface ligands. There are two classes of Ephs and ephrins, A and B. Both Ephs and ephrins can transduce repulsive signals that cause interacting cells to lose contact with each other and with extracellular matrix. We are not aware of any previous systematic study of Eph and ephrin expression or function in MM cells. We have found MM cell lines H929, U266, and RPMI 8226 express members of the A classes of both Ephs and ephrins, but not the B classes. First, we demonstrated ligation with commercially available anti-ephrin A3 antibody was followed by ephrin capping and shedding from the cell surface. We next explored whether ephrin ligation affects MM cell adhesiveness in culture. Whereas H929, U266, and RPMI 8226 cells adhered rapidly to fibronectin-coated plastic surfaces, all three cell lines failed completely to adhere to a mixed coating of FN and rabbit anti-ephrin A3 antibody for a period of 2 hrs. This effect was not seen with FN + normal rabbit Ig. This suggests binding of ephrin A3 (or another cross-reacting A-class ephrin) by solid-state antibody triggers intracellular signals that interfere with initial steps of integrin-mediated adhesion. After 2 hr, spontaneous partial recovery of adhesion occurred, reaching a plateau of approximately 30% of control values by 24 hr. We postulate this recovery occurs via clipping of the extracellular ephrin domain by transmembrane metalloproteases, since recovery of FN adhesion was partially prevented by the metalloprotease inhibitor GM6001 (25 uM). Also consistent with this theory, we found in a separate experiment that GM6001 reduced the shedding of cross-linked A-class ephrins from MM cell lines. In summary, we have demonstrated that manipulation of EPH-ephrin signaling can impair MM-cell adhesion to FN, and that this effect is enhanced by simultaneous inhibition of metalloprotease activity. We are currently studying the effect of A-class ephrin ligation on adhesion-mediated drug resistance in MM cell lines. We also intend to evaluate EPH-ephrin expression in marrow specimens from patients with MM.

Potential Therapeutic Role of the Selective Adhesion Molecule (SAM) Inhibitor Natalizumab in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1850-1850 ◽  
Author(s):  
Klaus Podar ◽  
Alexander Zimmerhackl ◽  
Ursula Hainz ◽  
Mariateresa Fulciniti ◽  
Sonia Vallet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Research Funding ◽  
Therapeutic Role ◽  
Potential Therapeutic Role

Abstract Abstract 1850 Poster Board I-876 Multiple Myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow. Despite current therapeutic approach and prolongation of the median survival, new therapies are urgently needed. Integrins are cell surface receptors which mediate both cell-cell adhesion and cell-extracellular matrix (ECM) protein adhesion. beta1-integrins, including very-late antigen-4 (VLA-4;á4β1), are typically expressed on MM cells. In MM, VLA-4-mediated binding to ECMS and bone marrow stromal cells (BMSCs) confers protection against drug-induced apoptosis and triggers transcription and secretion of IL-6, the major MM growth and survival factor. In addition to up-regulation of cell surface-clustering, integrin activity can also be triggered by multiple agonists through ‘inside-out’ signaling, independent of changes in integrin expression levels. Importantly, VEGF-induced migration of MM cells on fibronectin is also associated with β1-integrin- and PI3-kinase- dependent PKC activation. Targeting VLA-4 is therefore of potential high therapeutic interest in MM. Indeed, an antibody against murine á4 induces inhibition of MM growth in a murine model. Natalizumab is a recombinant humanized IgG4 monoclonal antibody, which belongs to a new class of molecules known as selective adhesion molecule (SAM) inhibitors and binds to á4-integrin. Clinically, Natalizumab has demonstrated activity in patients with multiple sclerosis and Crohn's disease. Here we tested the potential therapeutic role of Natalizumab on MM cell survival, and migration in the BM microenvironment. VLA-4 is expressed by all MM cell lines investigated (NCIH929, RPMI8226, INA-6, MM.1S, and OPM2). Functionally, Natalizumab but not a control antibody, triggered dose-dependent inhibition of MM cell adhesion to fibronectin, BMSCs, and endothelial cells (ECs). Importantly, inhibition of adhesion to fibronectin, BMSCs, or ECs was observed in MM cells pretreated with Natalizumab. Moreover, inhibition of MM cell adhesion to fibronectin, BMSCs, or ECs was also observed when Natalizumab was added to already adherent MM cells. Taken together, Natalizumab decreases adhesion of non-adherent MM cells as well as binding of already adherent MM cells to non-cellular and cellular components of the microenvironment. Given the protective role of the microenvironment on MM cell survival, we next sought to evaluate the chemosensitizing activity of Natalizumab. Specifically, we investigated dose- and time- dependent effects of Natalizumab, alone and when combined with conventional and novel therapies, on MM cells. Our results show that Natalizumab alone did not inhibit growth or survival of MM cells when cultured without components of the microenvironment. However, Natalizumab enhanced sensitivity of tumor cells to both bortezomib and dexamethasone in MM-BMSC and, MM-EC co-cultures. These data indicate a potential role of Natalizumab in bortezomib- and dexamethasone-containing treatment regimens including MPV. Moreover, Natalizumab decreases IL-6 and VEGF secretion triggered in MM-BMSC co-cultures. Consequently, angiogenesis triggered by supernatants of Natalizumab- treated MM-BMSC co-cultures was inhibited. Moreover, Natalizumab blocked MM cell migration on fibronectin triggered by both VEGF and IGF-1. Finally, our previous results implicate an PKC signaling in MM cell migration on fibronectin, and our current results show that Natalizumab inhibits phosphorylation of á4 integrins and PKC induced by co-stimulation with VEGF/ fibronectin, IGF-1/ fibronectin, and patient serum. Taken together, our data indicate a potential therapeutic role of Natalizumab in MM. Ongoing studies evaluating the effect of Natalizumab in a SCID-hu murine model of MM will also be reported. Disclosures: Podar: Biogen Idec: Research Funding. Off Label Use: natalizumab, integrin inhibitor. Zimmerhackl:Biogen Idec: Research Funding. Olsen:Biogen Idec: Employment.

Induction Of Proliferation and Survival Of Multiple Myeloma Cells By CD137 Ligand Reverse Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2537-2537
Author(s):  
Chengcheng Fu ◽  
Hui Liu ◽  
Juan Wang ◽  
Ling Ma ◽  
Songguang Ju ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Counting ◽  
Myeloma Cells ◽  
Reverse Signaling

Abstract CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively, regulate cell activation and proliferation of immune system. CD137L, in addition to its ability to costimulate T cells by triggering CD137 receptor, also signals back into antigen presenting cells inducing proliferation, prolonging survival and enhancing secretion of proinflammatory cytokines. The expression of CD137L and its function on multiple myeloma cells is unknown. We identified the constitutive expression of CD137L by flow cytometry on U266, RPMI 8226, LP1, MY5 and KMS-11 of Multiple myeloma (MM) cell lines as high as 96%, 97.5%, 89%, 93% and 94%.But, CD137 expressed on the cell surface was low as 4%, 5%, 1%, 2%, 5% respectively. Now that, CD137L was expressed very strongly on MM cell lines, next, we investigated CD137L expression of MM cells from 85 BM samples of patients seen in the hematological Dept of the First Affiliated Hosp. of Soochow University between January 2012 and June 2013 and diagnosed of active multiple MM, including the patients of newly-diagnosed (n=35), relapsed (n=5) and after 2- 4 prior therapies (n=45). The BM samples were examined using antibodies against CD45RO PE-Cy7, CD138 APC-H7, CD38 FITC and CD137L PE, according to standard protocols for surface staining. Indeed, CD137L protein was expressed by a select group of CD45-CD38++CD138+cells as higher than 95%, the same, CD38 and CD138 are expressed more than 90% of the cells of CD45-CD137L+.There were 22 samples from 11 cases collected before and after treatment and this was further evidence that CD137L molecule was consistently expressed on the MM cell surface. However, CD137L expression was not or hardly detectable on normal plasma cells confirmed by CD45+CD38++CD138+ CD56- CD19+, indicating that CD137L was ectopically expressed by MM cells and probably a specific marker of MM cells. The ectopic CD137L expression was not a mere epiphenomenon but was selected for, what function of it? We hypothesized that it would also act as a growth stimulus for B cell cancers. Then we selected U266-a MM cell line to explore the biological effect of CD137L reverse signaling and its underlying mechanism. As a result, in vitro study, U266 cells(2X105/ml))were cultured plate pre-coated with mAb 1F1 or irrelevant mouse IgG at l ug/ml in PBS and at 400 ul per well of 24-well plate or 80 ul per well of 96-well plate and washed twice after overnight incubation at 4°C. The proliferation and survival of U266 was enhanced by stimulating- CD137L mAb (1F1) than those induced by control mouse IgG by cell counting (4.2 X105/ml VS 3.3 X105/ml), WST-8(1.15 VS 0.81) and CFSE assay (930 VS 991) at incubation for 48h. In addition, the cell cycle analysis showed that CD137L induces proliferation and increases the number of cells in the S phase from 36.1% to 42.5% after 72h incubation. The percentage of apoptosis cells (Annexin V+ and PI+) was 19.6% VS 21.2% with no statistical significance. In order to explore the mechanism of the function of CD137L on MM cells, we surveyed the cytokine profiles during the incubation of U266 cells cultured for 2 days with different stimuli with mAb 1F1 compared with the control group. Intracellular cytokine staining showed that treatment of cells with 1F1 increased the production of IL-6 from 3.8% to 63.9% by Flow cytometry. When neutralizing anti-IL-6 mAb (5 ug/ml) was added to the culture medium, the cells(2X105/ml))were cultured for 48 h in pure medium or plus 10 ng/ml Fc or CD137–Fc protein and the cell proliferation measured by WST-8 was 0.79 VS 0.80 VS 0.72.1F1-induced cell proliferation was effectively inhibited. IL-6 can promote cell proliferation and survival of MM. An increase of these cytokines might explain why CD137L expression could stimulate the proliferation of U266. Finally, the U266 cells were treated with bortezomib and the growth of cells was analyzed by WST-8 assay. It demonstrated that bortezomib could inhibit the function of 1F1 and the inhibition ratio of bortezomib was 22%, 51% and 58% at 24h, 48h and 72h. MM is a B-cell malignancy characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. In our study, CD137L is not only a novel ectopic constitutive marker of MM, but also a promoting proliferation factor. This suggests the possibility that its expression on MM cells may be directly target for immunomodulatory therapy for MM. Disclosures: No relevant conflicts of interest to declare.

T Cell Receptor Gene Therapy Targeting the Intracellular Transcription Factor Bob1 for the Treatment of Multiple Myeloma and Other B Cell Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3002-3002 ◽  
Author(s):  
Lorenz Jahn ◽  
Renate S. Hagedoorn ◽  
Pleun Hombrink ◽  
Michel G.D. Kester ◽  
Dirk M. van der Steen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Gene Transfer ◽  
Cell Surface ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Malignancies ◽  
T Cell Clone

Abstract Therapeutic reactivity of CD20-specific monoclonal antibodies (mAb) or CD19-specific chimeric antigen receptor (CAR)-transduced T cells is exerted by targeting extracellular antigens. In contrast to mAbs and CARs, T cell receptors (TCRs) recognize antigen-derived peptides that are bound to human leukocyte antigen (HLA) molecules on the cell surface. Since HLA molecules constantly sample the entire endogenous proteome of a cell, extracellular and intracellular antigens are presented and can thus be recognized by a TCR. Here, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for immunotherapy. Bob1 is highly expressed in CD19+ B cells, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM) and is absent in the non-B lineages including CD34+ hematopoietic progenitor cells (HPCs), T cells, fibroblasts, keratinocytes and gastrointestinal tract. Bob1 is localized intracellularly but HLA-presented Bob1-derived peptides are accessible on the cell surface to TCRs and can thus be recognized by T cells. From the HLA-presented ligandome (Mol Cell Proteomics, 2013;12:1829) we identified naturally processed Bob1-derived peptides displayed in HLA-A*0201 (HLA-A2) and in HLA-B*0702 (HLA-B7). Since auto-reactivity towards self-antigens such as Bob1 is prevented by depleting high-avidity T cells recognizing self-antigens in self-HLA, we exploited the immunogenicity of these peptides presented in allogeneic HLA. From a HLA-A2/B7-negative healthy individual we isolated T cell clone 4G11 demonstrating high sensitivity and specificity for Bob1-derived peptide Bob144 presented in HLA-B7. Bob1-dependent recognition was demonstrated by transduction of Bob1 into cell lines that otherwise lack Bob1 expression. No harmful toxicities of clone 4G11 were observed against a wide panel of Bob1-negative stimulator cells including HLA-B7-positive CD34+ HPCs, T cells, monocytes, immature and mature dendritic cells, and fibroblasts even under simulated inflamed conditions. Furthermore, stringent HLA-B7-restricted recognition was observed for clone 4G11 when tested against a stimulator panel expressing a wide range of common and rare HLA class I and II molecules. Clone 4G11 demonstrated clinical applicability by efficiently recognizing HLA-B7+ primary ALL, CLL and MCL. Furthermore, reproducible strong recognition of purified primary HLA-B7+ MM could be demonstrated. Therefore, the TCR of clone 4G11 may be used for immunotherapy by administering TCR-transduced T cells to patients suffering from B cell malignancies including multiple myeloma. Retroviral gene transfer of TCR 4G11 led to efficient cell surface expression demonstrated by binding of TCR-transduced CD8+ T cells to pMHC-tetramer composed of peptide Bob144 bound to HLA-B7. TCR-modified CD8+ T cells strongly recognized Bob1-expressing HLA-B7+ multiple myeloma cell lines U266 and UM9, and ALL cell lines. TCR-modified T cells efficiently lysed HLA-B7+ primary ALL, CLL and MCL at very low effector-to-target ratios. In addition, highly purified primary multiple myeloma samples were also readily lysed. Furthermore, TCR-transduced T cells strongly proliferated in an antigen-specific manner when stimulated with primary malignant cell samples including ALL, CLL, and MCL or MM cell lines. As expected, TCR-transduced T cells also lysed autologous primary and CD40L-stimulated B cells since these targets cells also express Bob1. In contrast, no lysis of Bob1-negative autologous primary and activated T cells, or monocytes was observed when co-cultured with TCR-transduced T cells. In summary, we identified the intracellular transcription factor Bob1 encoded by gene POU2AF1 as a suitable target for TCR-based immunotherapies of B cell malignancies. Bob1-specific T cell clone 4G11 efficiently recognized primary B cell leukemia and multiple myeloma. Gene transfer of TCR of clone 4G11 installed Bob1-reactivity and specificity onto recipient T cells shown here by cytolytic capacity and proliferation upon antigen encounter. TCR gene transfer approaches using this Bob1-specific TCR can bring novel treatment modalities and possibly curative therapy to patients with B cell malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Posttranslational regulation of the NKG2D ligand Mult1 in response to cell stress

2009 ◽  
Vol 206 (2) ◽  
pp. 287-298 ◽  
Author(s):  
Timothy J. Nice ◽  
Laurent Coscoy ◽  
David H. Raulet
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Posttranslational Regulation ◽  
Nkg2d Ligand ◽  
Normal Tissues ◽  
Distinct Disease ◽  
Ligand Expression ◽  
Infected Cells ◽  
Cellular Control

NKG2D is a major stimulatory receptor expressed by natural killer (NK) cells and some T cells. The receptor recognizes major histocompatability complex class I–like cell surface ligands that are poorly expressed by normal tissues but are often induced in transformed and infected cells. The existence of several NKG2D ligands in each individual, some with strikingly divergent protein sequences, raises the possibility that different ligands are regulated by distinct disease-associated stresses. The transcripts for some ligands, including murine UL16-binding proteinlike transcript 1 (Mult1), are abundant in certain normal tissues where cell surface expression is absent, suggesting the existence of translational or posttranslational regulation. We report here that under normal conditions, Mult1 protein undergoes ubiquitination dependent on lysines in its cytoplasmic tail and lysosomal degradation. Mult1 degradation and ubiquitination is reduced in response to stress imparted by heat shock or ultraviolet irradiation, but not by other forms of genotoxicity, providing a novel mechanism for stress-mediated cellular control of NKG2D ligand expression.

scholarly journals Blocking the Don't Eat Me Signal (CD47-SIRPα Axis) to Improve Antibody-Based Immunotherapy of Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2684-2684
Author(s):  
Katja Klausz ◽  
Carina Lynn Gehlert ◽  
Ammelie Svea Boje ◽  
Marta Lustig ◽  
Steffen Krohn ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloid Cells ◽  
Myeloma Cell ◽  
Cell Phenotype ◽  
Blocking Antibody ◽  
Myeloma Cells

Abstract The addition of monoclonal antibodies daratumumab, elotuzumab and isatuximab to the treatment of patients with multiple myeloma significantly improved the outcome and prolonged survival. Unfortunately, although many patients benefit, depth and duration of response are a problem. In order to improve efficacy of antibody-based immunotherapy, we aimed to combine CD38-directed antibodies daratumumab and isatuximab as well as SLAMF7-targeting elotuzumab with a CD47 blocking antibody to enhance phagocytosis of myeloma cells. Antibody-dependent cellular phagocytosis (ADCP) of malignant plasma cells is described to be one important mode of action of daratumumab, isatuximab and elotuzumab, respectively. Of note, CD47 is highly expressed on myeloma cells and allows evading immune recognition by myeloid cells, i.e. monocytes, macrophages and neutrophils. Binding of CD47 to SIRPα expressed on myeloid cells provides a strong 'don't eat me' signal and diminishes phagocytosis of tumor cells. Blocking the CD47-SIRPα axis, by a monoclonal antibody against CD47 or a SIRPα-Fc fusion protein can restore recognition of tumor cells by macrophages and enhance phagocytosis. In patients with Non-Hodgkin's lymphoma the combination of CD20 antibody rituximab with CD47 antibody magrolimab was clinically successful (Advani et al., NEJM 379:1711, 2018). To test the applicability of blocking the CD47-SIRPα axis and improve ADCP of myeloma cells by CD38-targeting or SLAMF7-directed myeloma antibodies, we generated a CD47 IgG2σ antibody carrying an engineered Fc domain not binding to Fcγ receptors (FcγR). This CD47 antibody was subsequently used in phagocytosis experiments in combination with antibodies daratumumab, isatuximab as well as elotuzumab and various myeloma cell lines. The cell lines AMO-1, JK-6L, L363, RPMI-8226, and U266 express different levels of CD47, CD38 and SLAMF7 as determined by quantitative flow cytometry. M0 macrophages expressing FcγRs were generated from healthy donor PBMC monocytes by cultivation with M-CSF for 10-14 days prior use in 6 hour real-time live cell imaging phagocytosis experiments with pHrodo-labeled myeloma cells - turning red only when engulfed by macrophages. Macrophages and myeloma cells were used at an effector-to-target cell ratio of 1:1. Importantly, ADCP of myeloma cells induced by all three monoclonal antibodies, daratumumab, isatuximab or elotuzumab, can be enhanced by the addition of the CD47 blocking antibody. However, improvement in phagocytosis strongly differs between myeloma cell lines although all have high CD47 level on their cell surface. In responsive myeloma cell lines, ADCP mediated by CD38 antibodies daratumumab or isatuximab was found more efficient than that by SLAMF7 antibody elotuzumab. This may be related to the significantly higher CD38 than SLAMF7 expression at the myeloma cell surface. Our findings demonstrate that ADCP of approved IgG antibodies targeting CD38 or SLAMF7 can be enhanced by blocking the CD47-SIRPα axis and this may depend on the particular malignant plasma cell phenotype. The inhibition of this myeloid 'don't eat me' signal with a CD47 blocking antibody may open a new avenue for powerful myeloma immunotherapy. Since combination treatments with proteasome inhibitors and IMiDs are commonly used, these interactions also require attention. Initial data indicate that pre-treatment of myeloma cells with proteasome inhibitor carfilzomib did not negatively impact improvement of ADCP by blocking the CD47-SIRPα axis in responsive cell lines. Taken together, particularly CD38-targeting antibodies may have a significant potential to further improve immunotherapy in multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document