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.

Triggering of Toll-Like Receptor-4 In Human Multiple Myeloma Cells Promotes Proliferation and Alters Cell Responses to Immune and Chemotherapy Drug Attack

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1905-1905
Author(s):  
Zhen Cai ◽  
Hanying Bao ◽  
Peilin Lu ◽  
Lijuan Wang ◽  
Donghua He ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Response To Chemotherapy ◽  
Tlr4 Ligand ◽  
Induced Apoptosis

Abstract Abstract 1905 Multiple myeloma (MM) is a fatal plasma cell malignancy mainly localized in the bone marrow. The clonal expansion of tumor cells is associated with the disappearance of normal plasma cells and with a marked depression in the production of normal immunoglobulin (Ig). This makes MM patients highly vulnerable to bacterial, fungal and viral infections and recurrent infections remain to be a major cause of death in MM patients. It has been shown that most primary myeloma cells and cell lines express multiple Toll-like receptors (TLRs). Among them, TLR4 is most frequently expressed. To investigate TLR-initiated responses in MM cells including proliferation, anti-apoptosis and immune escape, we first screened four commonly used human myeloma cell line (HMCL) for the expression of major TLRs by RT-PCR. Surprisingly, all the HMCL expressed multiple TLRs. We also examined primary myeloma cells from 4 patients with MM and our results showed that TLR4 was expressed by all the tumor cells. We incubated myeloma cells with LPS, the natural ligand for TLR4, and found that cell proliferation increased significantly. Targeting TLRs on malignant B cells can induce resistance to chemotherapeutic agents but can also be exploited for combined therapeutic approaches. As mechanisms involved in the resistance to apoptosis play a major role in MM escape to therapies, we sought to determine the capacity of TLR4 ligand to promote the survival of HMCL cells. Myeloma cells were pretreated for four hours with LPS before being induced apoptosis by adriamycin. Results showed that LPS pretreatment partially protected the cells from adriamycin-induced apoptosis. The TLR signaling pathway activates several signaling elements, including NF-kB and ERK/JNK/p38 MAPKs, which regulate many immunologically relevant proteins. Time-dependent MAPK phosphorylation was measured to assess the activation of these kinases upon treatment with LPS in cell lines. ERK1/2, p38, and JNK phosphorylation and NF-kB were significantly up-regulated following LPS treatment. Moreover, our findings demonstrated that LPS-induced cell proliferation was dependent on JNK, ERK and p38 signaling. IL-18, a recently described member of the IL-1 cytokine superfamily, is now recognized as an important regulator of innate and acquired immune responses. In this study, we found that LPS induced IL-18 secretion and activated MAPK and NF-kB signaling simultaneously. Therefore, our results suggest that activation of the MAPK signaling and secretion of IL-18 are interconnected. Tumors evade immune surveillance by multiple mechanisms, including the production of factors such as TGF-β and VEGF, which inhibit and impair tumor-specific T cell immunity. Our study also showed that T cell proliferation induced by allostimulatory cells decreased when the HMCL were pre-treated with LPS. Moreover, immunoregulatory molecules on HMCL, such as B7-H1, B7-H2 and CD40, were upregulated after treatment with LPS, suggesting that TLR4 ligand LPS facilitates tumor cell evasion of the immune system. Our results show that TLRs are functional on myeloma tumor cells, and the ligands to these TLRs have a functional role in affecting myeloma cell proliferation, survival, and response to chemotherapy and immune attacks. Disclosures: No relevant conflicts of interest to declare.

Interleukin-16 Is An Important Growth-Promoting Factor and a Novel Diagnostic and Therapeutic Target for Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4056-4056
Author(s):  
Djordje Atanackovic ◽  
York Hildebrandt ◽  
Tim Luetkens ◽  
Axel R. Zander ◽  
Carsten Bokemeyer ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Antibody Array ◽  
Western Blots ◽  
Myeloma Cells ◽  
Growth Promoting

Abstract Abstract 4056 Background: Multiple myeloma (MM) is a malignancy characterized by the expansion of a plasma cell (PC) clone that localizes to the bone marrow (BM). Myeloma cells and BM stromal cells both produce soluble factors promoting the survival and progression of MM. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers. Methods: Using an antibody array we screened supernatants of myeloma cell lines for the presence of a variety of cytokines/chemokines. We confirmed IL-16 expression in myeloma cell lines as well as in malignant PC and BM plasma from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We applied inhibitory RNA to analyze IL-16 function and we used anti-IL-16 antibodies to evaluate possible therapeutic options for MM. Results: We found IL-16 to be strongly overexpressed in the BM of myeloma patients. Myeloma cell lines as well as primary tumor cells from MM patients constitutively expressed IL-16 RNA and protein and spontaneously secreted soluble IL-16. Functional analyses revealed that IL-16 supports the proliferation of myeloma cells. Accordingly, silencing of IL-16 expression had an anti-proliferative effect on the tumor cells. Most importantly, the application of a monoclonal antibody directed against IL-16had a strong growth-inhibiting influence on myeloma cells. Conclusions: These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent a novel diagnostic and therapeutic target for this incurable human malignancy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Receptor Tyrosine Kinase AXL: A Potential Strategy to Counter Immune Suppression and Dormancy in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4335-4335
Author(s):  
Kim De Veirman ◽  
Siyang Yan ◽  
Ken Maes ◽  
Nathan De Beule ◽  
Sylvia Faict ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Overall Survival ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Receptor Tyrosine Kinase ◽  
Single Agent ◽  
Myeloid Cells ◽  
Myeloma Cell ◽  
Myeloma Cells

Introduction The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies revealed a crucial role of AXL signaling in proliferation, survival, dormancy and therapy resistance in different cancers including lung cancer, hepatocellular cancer and AML. In this study, we aimed to investigate the role of AXL in Multiple Myeloma (MM), focusing on myeloma cell dormancy and AXL expression in different cellular components of the bone marrow microenvironment. Material & Methods To investigate dormancy, we used the syngeneic murine 5TGM1 MM model. 5TGM1-GFP+cells were DiD-labeled and injected intravenously in naïve C57BL/KaLwRij mice. At end-stage, GFP+DiD+('dormant', non-proliferating) and GFP+DiD-('proliferating') MM cells were analyzed by flow cytometry for AXL expression. In addition, AXL expression was also analyzed in CD11b+ myeloid cells and in in vitrogenerated macrophages from the 5TMM model. The effects of AXL inhibition by R428 (BGB324|Bemcentinib, Sigma-Aldrich), a highly potent and AXL-specific small molecular inhibitor, on viability and induced apoptosis of MM cells was determined by Cell Titer Glo and AnnexinV/7AAD staining respectively. AXL expression in human myeloma cell lines (HMCL) (JJN3, U266 and LP-1) and murine 5TGM1 cells was analyzed by qRT-PCR and cytospin stainings. Patient cohorts (TT2/TT3) were used to correlate AXL expression and overall survival. Plasma of healthy donors and MM patients was analyzed by ELISA (R&D). Results Using the in vivo5TGM1 dormancy model, we demonstrated an increased expression of AXL (4x higher) in dormant MM cells compared to proliferating MM cells (n=3, p<0,05). Myeloma cell lines (JJN3, U266, 5TGM1) had a very low AXL expression, however, treatment with melphalan induced a more than twofold increase in AXL expression (n=3, p<0.05). The combination of melphalan and R428 significantly increased apoptosis of JJN3 (>10%), U266 (>20%) and LP-1 (>10%) cells compared to single agent therapy (n=6) (p<0.01). Using patient cohorts, we observed that AXL expression correlated with a good overall survival (p=0.006). In addition, plasma samples of patients (n=31) showed a decreased expression of AXL compared to samples of healthy controls (n=9) (p<0.001). This confirms our hypothesis that AXL is associated with dormancy and therefore correlates with a better overall survival. In a second part, we investigated AXL expression in 5TMM-derived myeloid cells and macrophages (n=3). We observed a high expression of AXL in myeloid derived suppressor cells and tumor associated macrophages compared to myeloma cells. In addition, we observed that myeloid cells were much more sensitive to R428 compared to MM cells (n=5, p>0.01). Conclusion We observed that AXL is highly expressed in dormant MM cells and environmental myeloid cells. Despite its association with a good prognosis in MM, AXL serves as an interesting target to eradicate dormant myeloma cells as AXL inhibitors affect viability and induce apoptosis of myeloma cells, especially in combination with melphalan. Therefore, AXL can be considered as a new therapeutic strategy, to target both the immunosuppressive myeloid cells and the residual cancer cells in MM patients. Disclosures No relevant conflicts of interest to declare.

Fully Human CD38 Antibodies Efficiently Trigger ADCC of Multiple Myeloma Cell Lines and Primary Tumor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3377-3377 ◽  
Author(s):  
Matthias Peipp ◽  
Michel de Weers ◽  
Thomas Beyer ◽  
Roland Repp ◽  
Paul Parren ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Treatment Modalities ◽  
Potential Candidate ◽  
Isolated Tumor Cells ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract Although new treatment modalities have recently been added to the standard regimens for multiple myeloma, the clinical outcome for patients with advanced disease is often limited. Monoclonal antibodies are increasingly used for tumor therapy, and may also represent interesting options for multiple myeloma patients. CD38 is one of the most promising target antigens on malignant plasma cells, which are evaluated in preclinical and early clinical studies as targets for antibody therapy. CD38 is a type II transmembrane protein with ectoenzymatic activity, which is involved in calcium mobilization. Human CD38 is predominantly expressed by bone marrow precursor cells and by terminally differentiated plasma cells. Multiple myeloma cells show moderate to high expression levels - making CD38 a potential candidate as target for immunotherapy. A panel of 42 fully human CD38 antibodies was generated by immunizing human Ig transgenic mice. Immunofluorescence studies with CD38 transfected cells demonstrated antigen-specific, high affinity binding, and cross-blocking experiments revealed four distinct epitope groups. Seven antibodies, representing each of the four groups, were selected for further analyses. ADCC and CDC activity against CD38-positive myeloma cell lines (AMO-1 and JK6), and against freshly-isolated primary multiple myeloma cells was investigated. Human whole blood served as effector source, which was then fractionated into plasma (containing human complement), mononuclear (MNC) or granulocytic (PMN) effector cells. All antibodies mediated concentration-dependent killing of both multiple myeloma cell lines - using human mononuclear cells as effector source. Also complement-dependent killing of freshly isolated myeloma cells was observed. However, none of the antibodies recruited PMN for tumor cell lysis. Importantly, CD38 antibodies also killed freshly isolated tumor cells from a rare patient with a CD38/138- positive plasma cell leukemia, which was chemotherapy- refractory at the time of analysis. Furthermore, CD38 antibodies effectively prevented outgrowth of CD38-positive tumor cells in SCID mouse xenograft models. Antibody 005 was significantly more effective in these assays compared to the remaining panel of CD38 antibodies. In conclusion, CD38 antibodies efficiently mediated killing of multiple myeloma cell lines as well as freshly isolated tumor cells and prevented tumor outgrowth in xenografted SCID mice. Antibody 005 was superior in mediating CDC and ADCC via MNC - particularly at low antibody concentrations.

Human Monoclonal Antibodies to TRAIL Receptors R1 and R2 Effectively Induce Apoptosis in Human Myeloma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3366-3366 ◽  
Author(s):  
Shuji Ozaki ◽  
Etsuko Sekimoto ◽  
Yoichi Tanaka ◽  
Takashi Ohshima ◽  
Hironobu Shibata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Apoptosis Induction ◽  
Human Monoclonal Antibodies ◽  
Myeloma Cells ◽  
Trail Receptors ◽  
Induced Apoptosis

Abstract Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of apoptotic pathway in a variety of tumor cells but not normal cells. Therefore, TRAIL and its receptors have been considered as possible therapeutic targets in cancer treatment. However, several myeloma cells were resistant to TRAIL-induced apoptosis depending on the expression patterns of TRAIL receptors including TRAIL-R1 and -R2 death receptors, and TRAIL-R3 and -R4 decoy receptors. To explore the contribution of each TRAIL receptor to apoptosis induction of myeloma cells, we generated fully human monoclonal antibodies (MoAbs) that bind specifically to TRAIL-R1 and TRAIL-R2 using KM mice that possess the human chromosome fragments containing human immunoglobulin heavy chain loci and YAC transgene containing human kappa light chain gene. Several myeloma cell lines as well as freshly isolated myeloma cells were cultured with TRAIL or these MoAbs in the presence of F(ab′)2 goat anti-human IgG. Soluble TRAIL (1000 ng/mL) showed more than 80% of cytotoxicity in RPMI 8226 and KMS12-BM cells within 24 hours. However, U266, HS-Sultan, and ARH-77 cells were relatively resistant to TRAIL with maximal cytotoxicity of only 3–31%. In contrast, anti-TRAIL-R1 MoAb (R1-B12) effectively induced apoptosis even in TRAIL-resistant myeloma cell lines in a time- and dose-dependent manner with maximal cytotoxicity of 65-99%. This apoptotic response of myeloma cells was confirmed by caspase activation and annexin V binding. On the other hand, anti-TRAIL-R2 MoAb (R2-E11) was less effective against these myeloma cell lines showing 1–33% of cytotoxicity. Among 10 primary myeloma cells, R1-B12 induced at least 10% of cytotoxicity in 7 samples and R2-E11 showed in 3 samples. Flow cytometric analysis demonstrated that TRAIL-R1 was expressed at a higher level than TRAIL-R2 on these myeloma cell lines and specific mean fluorescence intensity (MFI) was 3.8–7.9 and 1.6–4.2, respectively. TRAIL-R1 and -R2 were also detected on primary myeloma cells at low levels and specific MFI was 1.0–2.0 and 1.0–1.6, respectively. Thus, the sensitivity to R1-B12 and R2-E11 was correlated with the expression level of TRAIL-R1 and -R2 on cell surface. Treatment of proteasome inhibitor bortezomib and histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) did not increase the cell surface expression of TRAIL-R1 and -R2 in myeloma cells. However, bortezomib and SAHA induced reduction of cellular FLICE inhibitory protein (c-FLIP) and synergistically enhanced the effect of R1-B12 but not of R2-E11 on apoptosis induction of TRAIL-resistant U266 cells. These results suggest that TRAIL-R1 mainly contribute to TRAIL-induced apoptosis in myeloma cells and that R1-B12 may have the therapeutic potential in combination with bortezomib or HDAC inhibitors in patients with multiple myeloma.

DKK-1 Is a Widely Expressed, Potent Tumor-Associated Antigen in Multiple Myeloma Recognized by Cytotoxic T Lymphocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3467-3467
Author(s):  
Qing Yi ◽  
Jianfei Qian ◽  
Jing Yang ◽  
Siqing Wang ◽  
Fenghuang Zhan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Tumor Cells ◽  
Cell Lines ◽  
Wnt Signaling Pathway ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Tumor Associated Antigen ◽  
Potent Tumor

Abstract Idiotype proteins have been used for immunotherapy in multiple myeloma (MM); and the results thus far have been disappointing. Therefore, the identification and use of novel and more potent tumor-associated antigens are urgently needed to improve the efficacy of the treatment. Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most myeloma patients and may be responsible for suppressed osteoblast formation. Based on these data and the fact that DKK1 is not expressed in normal tissues except placenta, prostate, and mesenchymal stem cells, we hypothesized that DKK1 is a novel tumor-associated antigen in MM. In this study we examined the capacity of DKK1 to trigger the induction of antimyeloma cytotoxic T lymphocyte (CTL) responses. Using DKK-1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK-1 peptide (DKK120-29 and DKK166-74V)-specific CTL lines and clones from HLA-A2+ MM patients and healthy individuals. In our experiments, mature DCs obtained from cultures of blood monocytes were pulsed with HLA-A2-restricted DKK1 peptides. Autologous T cells were then stimulated weekly with these DCs, and cytotoxicity was examined against DKK1 peptide-pulsed T2 cells, myeloma cell lines, and primary myeloma cells isolated from patients. After 4 cycles of in vitro stimulation and subsequent T cell cloning and expansion, specific CD8+ CTL lines and clones were obtained. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed HLA-A2+ MM cell line U266 and primary tumor cells in vitro. No killing was observed against HLA-A2+ normal lymphocytes including B cells or HLA-A2− myeloma cell lines or primary myeloma cells from patients. The CTL response could be inhibited by anti-HLA-A2 antibody indicating that the response was indeed restricted by HLA-A2 molecules. IFN-γ, but not IL-4, was secreted by the T cells indicating that the effector cells were type-1 CD8+ T cells. Further functional studies are underway. Thus, these results demonstrate that DKK1-specific CTLs are able to lyse myeloma tumor cells including primary myeloma cells from patients, and identify DKK1 as a potentially important antigenic target for antimyeloma immunotherapeutic strategies

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 610-618 ◽  
Author(s):  
Inge Tinhofer ◽  
Ingrid Marschitz ◽  
Traudl Henn ◽  
Alexander Egle ◽  
Richard Greil
Keyword(s):  
Multiple Myeloma ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Constitutive Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cytotoxic Treatment ◽  
Interleukin 15 ◽  
Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

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.

Induction of Multiple Myeloma-Specific Cytotoxic T Lymphocytes Using HLA-A2.1-Specific CD19 and CD20 Peptides.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2477-2477
Author(s):  
Jooeun Bae ◽  
Jeff A. Martinson ◽  
Hans G. Klingemann ◽  
Steven Treon ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Lymphocytes ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cytotoxic T Lymphocytes ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Specific Ctls ◽  
Ifn Γ ◽  
Hla A2.1

Abstract We have identified novel CD19 and CD20 antigen-derived HLA-A2.1-specific immunogenic peptides, CD19150–158 (KLMSPKLYV) and CD20188–196 (SLFLGILSV), for generating cytotoxic T lymphocytes (CTLs) against malignant B-cell diseases. Initial testing showed that the CTLs displayed antigen-specific and HLA-A2.1-restriced cytotoxic activity against both Burkitt’s lymphoma and chronic lymphoid leukemia cell lines. The observed cytotoxic activity of the CTLs was shown to be specific to the CD19150–158 or the CD20188–196 peptides. Additionally, the CTLs displayed a distinct phenotype (majority CD69+/CD45RO+) along with a significant (p&lt;0.05) increase in cell proliferation and IFN-γ release following re-stimulation with HLA-A2.1+/CD19+/CD20+ tumor cell lines. Based on emerging information that clonogenic myeloma cells express CD19 and/or CD20, we evaluated the activity of the CD19 and CD20 peptide specific-CTLs against several multiple myeloma cell lines. Five of 10 myeloma cell lines evaluated were HLA-A2.1-positive and expressed both CD19 and CD20 antigens. CD19 peptide specific-CTLs generated from normal donors were able to specifically lyse CD19+/HLA-A2.1+ MM cell lines (30% lysis; 10:1 E:T ratio) but did not lyse CD19−/HLA-A2.1+ or CD19+/HLA-A2.1− cell lines. Similarly, the CD20-specific CTLs generated from normal donors lysed CD20+/HLA-A2.1+ MM cell lines (25% lysis; 10:1 E:T ratio), in a manner restricted to HLA-A2.1 and specific to antigens. We next showed IFN-γ production by the CTLs after exposure to CD19+/HLA-A2.1+ or CD20+/HLA-A2.1+ MM cells. Moreover, we have demonstrated the ability to expand CD20-CTLs under serum-free culture conditions while maintaining their cytotoxic activity (28–49%). In ongoing studies, we are evaluating the ability of CD19- and CD20-specific CTLs to eliminate clonogenic myeloma cells in vitro and in vivo in a SCID mouse model of myeloma. These preclinical studies strongly suggest that immunogenic CD19 and CD20 peptide-based vaccines represent a promising immunotherapeutic approach in myeloma.

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