Effects of Integrin-Linked Kinase (ILK) Inhibitor QLT0267 on Multiple Myeloma Cells and Evaluation of ILK as a Therapeutic Target in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3529-3529
Author(s):  
Thorsten Stühmer ◽  
Torsten Steinbrunn ◽  
Evelyn Grella ◽  
Ralf C. Bargou
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Treatment Options ◽  
Therapeutic Window ◽  
Culture Dish ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells

Abstract Multiple myeloma (MM) is a fatal plasma cell tumor that accounts for about 1% of cancers. A hallmark of the disease is its location in the bone marrow where the tumor cells receive prosurvival support from the microenvironment and cause extensive osteolytic damage. Novel drugs are currently being developed into a range of new treatment options. However, because the problems of cancer relapse and eventual selection of therapy-resistant offspring remain, additional therapeutic targets should still be investigated. ILK is a multifunctional protein that, as an adaptor and/or as a kinase, may relay adhesion- and growth factor receptor-mediated signals to downstream signaling cascades that promote growth and survival. We have analysed the expression of ILK in MM cells and have tested the effects of a novel small molecule ILK-inhibitor (QLT0267; QLT Inc., Vancouver, Canada) in MM cell lines, primary MM tumor cells and healthy cells, respectively. ILK expression at either cDNA or protein level was detectable in virtually every MM sample tested. Treatment with QLT0267 for up to 3 days resulted in extensive apoptotic death in MM cell lines (EC50 values below 10 microM in 8/9 MM cell lines tested) and in a majority of primary (anti-CD138-purified) MM samples (EC50 values below 10 microM in 8/14 primary MM samples tested). Drug treatment led to rapid decreases in the levels of phospho-STAT3, phospho-GSK3beta and total Akt protein, whereas levels of ILK and of phospho-ERK were unaffected or, in the latter case, showed a slight increase. Similar to other current pharmacologic approaches, targeting ILK may have several detrimental impacts on the signaling network that sustains MM cells. Such pleiotropic effects could prove valuable for combination treatments. The survival of peripheral blood mononuclear cells and of bone marrow stromal cells (BMSCs) at 20 microM QLT0267 was just slightly affected, indicating that the scope for establishment of a therapeutic window in MM might exist. High (20 microM) concentrations of QLT0267 gradually (and reversibly) promoted detachment of BMSCs from the culture dish, indicating that the drug might be useful to temporarily impair their effectiveness to support myeloma cells. Taken together, these experiments provide a rationale to further explore the utility of ILK-inhibition for the treatment of MM.

Signaling Profile and Anti-Tumor Activity of the Novel HSP90 Inhibitor NVP-AUY922 in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4788-4788
Author(s):  
Thorsten Stühmer ◽  
Angela Zöllinger ◽  
Daniela Siegmund ◽  
Manik Chatterjee ◽  
Evelyn Grella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Primary Tumor ◽  
Hsp90 Inhibitor ◽  
Dose Effect ◽  
Therapeutic Window ◽  
Large Set ◽  
Molecular Effects

Abstract We have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumor cell survival. Pharmacologic blockade of Hsp90 has consistently been shown to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumor cells is lacking. Furthermore, these investigations have so far exclusively relied on geldanamycin derivatives. Here, we analyzed the anti-tumor effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) in a large set of primary MM samples and in MM cell lines. The majority of cell lines (n = 8), as well as most primary samples (n = 20), displayed profound apoptotic responses and steep dose-effect curves with EC50 values in the range of 5–15 nM and EC90 values between 8 and 25 nM. This effect was not attenuated in coculture with cells from the bone marrow microenvironment. Some cell lines and about a quarter of primary MM samples displayed greater resilience to drug treatment, with EC50 values but not EC90 values reached at concentrations up to 50 nM. Sensitivity of MM cells to the Hsp90 inhibitor was not correlated with TP53 mutation or Hsp70 induction levels. Western analyses of MM cell lines and flow cytometric analyses of antibody-stained Hsp90 client proteins in primary tumor cells showed that NVP-AUY922-treatment entailed molecular effects and pharmacodynamic properties consistent with abrogation of Hsp90 function. Consequently, downregulation of multiple signaling and survival pathways was detectable through, for example, decreases in the phosphorylated (activated) forms of extracellular signal-regulated kinase (ERK) 1 and 2, signal transducer and activator of transcription (STAT) 3 and glycogen synthase kinase-3beta. All samples treated displayed strong upregulation of Hsp70. Importantly, peripheral blood mononuclear cells as well as primary bone marrow stromal cells were much less affected by high (50–100 nM) concentrations of NVP-AUY922, showing that a therapeutic window might be established for the treatment of multiple myeloma. Taken together, NVP-AUY922 could be a promising new drug for the treatment of a majority of myeloma patients.

scholarly journals Characteristics of a Novel Target Antigen against Myeloma Cells for Immunotherapy

2020 ◽  
Author(s):  
Maiko Matsushita ◽  
Saku Saito ◽  
Shinya Yokoe ◽  
Daiju Ichikawa ◽  
Yutaka Hattori
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Demethylating Agents ◽  
Specific Ctls

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by RT-PCR using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2'-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

scholarly journals Specific killing of multiple myeloma cells by (-)-epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2804-2810 ◽  
Author(s):  
Masood A. Shammas ◽  
Paola Neri ◽  
Hemanta Koley ◽  
Ramesh B. Batchu ◽  
Robert C. Bertheau ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Green Tea ◽  
Fas Ligand ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Growth Arrest ◽  
Laminin Receptor ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells

AbstractEpigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM). EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines including IL-6-dependent cells and primary patient cells, without significant effect on the growth of peripheral blood mononuclear cells (PBMCs) and normal fibroblasts. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. EGCG interacts with the 67-kDa laminin receptor 1 (LR1), which is significantly elevated in myeloma cell lines and patient samples relative to normal PBMCs. RNAi-mediated inhibition of LR1 resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that LR1 plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-κB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expression of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.

scholarly journals Characteristics of a Novel Target Antigen against Myeloma Cells for Immunotherapy

Vaccines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 579
Author(s):  
Maiko Matsushita ◽  
Saku Saito ◽  
Shinya Yokoe ◽  
Daiju Ichikawa ◽  
Yutaka Hattori
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Target Antigen ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Myeloma Cells ◽  
Demethylating Agents ◽  
Specific Ctls

Despite the availability of therapeutic treatments, multiple myeloma is an incurable haematological disorder. In this study, we aimed to clarify the role of CXorf48 as a therapeutic target in multiple myeloma. Based on a previously identified HLA-A*24:02-restiricted epitope from this novel cancer/testis antigen, we characterized the activities of cytotoxic T lymphocytes (CTLs) specific to this antigen against myeloma cells and evaluated the effects of demethylating agents in increasing antigen expression and enhancing the cytotoxic activity of CTLs. CXorf48 expression was examined by reverse transcription polymerase chain reaction (RT-PCR) using nine myeloma cell lines. Cell lines with low CXorf48 expression were treated by demethylating agents (DMAs), 5-azacytidine (5-aza), and 5-aza-2’-deoxycytidine (DAC) to evaluate gene expression using quantitative RT-PCR. Furthermore, CXorf48-specific CTLs were induced from peripheral blood mononuclear cells of HLA-A*24:02-positive healthy donors to evaluate antigen recognition using ELISpot and 51Cr cytotoxicity assays. CXorf48 was widely expressed in myeloma cells, and gene expression was significantly increased by DMAs. Furthermore, CXorf48-specific CTLs recognized DMA-treated myeloma cells. These findings suggest that CXorf48 is a useful target for immunotherapy, such as vaccination, in combination with demethylating agents for the treatment of patients with myeloma.

Inhibition of Multiple Myeloma Cells Growth by AS602868, a Pharmacological Inhibitor of IKK2.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3472-3472 ◽  
Author(s):  
Michel Jourdan ◽  
Jerome Moreaux ◽  
Dirk Hose ◽  
Jean-Francois Rossi ◽  
Karène Mahtouk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Mononuclear Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Hematopoietic Stem ◽  
Myeloma Cells

Abstract The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p <.01). Thus, an autocrine production of APRIL confers a resistance to the AS602868 IKK2-I. This can be explained since APRIL has been shown to activate the alternative NF-κB pathway which implicates an IKK complex composed of IKK1 homodimers instead of the IKK1/IKK2/NEMO complex involved in the canonical NF-κB pathway. The results demonstrate the important role of NF-κB in maintaining survival of MM cells and suggest that a pharmacological inhibition of the NF-κB pathway by an IKK2-I, AS602868, can efficiently kill myeloma cell lines or primary myeloma cells and might represent an innovative approach for treating MM patients.

scholarly journals Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Youngil Koh ◽  
Woo-June Jung ◽  
Kwang-Sung Ahn ◽  
Sung-Soo Yoon
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Clonal Evolution ◽  
Myeloma Cell ◽  
Mouse Bone Marrow ◽  
Single Patient ◽  
U266 Cell

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

scholarly journals Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3017-3025 ◽  
Author(s):  
VS Goldmacher ◽  
LA Bourret ◽  
BA Levine ◽  
RA Rasmussen ◽  
M Pourshadi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Tumor Cell Line ◽  
Covalent Attachment ◽  
Myeloma Cells ◽  
Colony Forming Units

Abstract We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.

scholarly journals Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2269-2277 ◽  
Author(s):  
HM Lokhorst ◽  
T Lamme ◽  
M de Smet ◽  
S Klein ◽  
RA de Weger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Adherent Cells ◽  
Myeloma Cells ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Long-term bone marrow cultures (LTBMC) from patients with multiple myeloma (MM) and normal donors were analyzed for immunophenotype and cytokine production. Both LTBMC adherent cells from myeloma and normal donor origin expressed CD10, CD13, the adhesion molecules CD44, CD54, vascular cell adhesion molecule 1, very late antigen 2 (VLA-2), and VLA- 5, and were positive for extracellular matrix components fibronectin, laminin, and collagen types 3 and 4. LTBMC from myeloma patients and normal donors spontaneously secreted interleukin-6 (IL-6). However, levels of IL-6 correlated with the stage of disease; highest levels of IL-6 were found in LTBMC from patients with active myeloma. To identify the origin of IL-6 production, LTBMC from MM patients and normal donors were cocultured with BM-derived myeloma cells and cells from myeloma cell lines. IL-6 was induced by plasma cell lines that adhered to LTBMC such as ARH-77 and RPMI-8226, but not by nonadhering cell lines U266 and FRAVEL. Myeloma cells strongly stimulated IL-6 secretion in cocultures with LTBMC adherent cells from normal donors and myeloma patients. When direct cellular contact between LTBMC and plasma cells was prevented by tissue-culture inserts, no IL-6 production was induced. This implies that intimate cell-cell contact is a prerequisite for IL-6 induction. Binding of purified myeloma cells to LTBMC adherent cells was partly inhibited by monoclonal antibodies against adhesion molecules VLA-4, CD44, and lymphocyte function-associated antigen 1 (LFA-1) present on the plasma cell. Antibodies against VLA-4, CD29, and LFA-1 also inhibited the induced IL-6 secretion in plasma cell-LTBMC cocultures. In situ hybridization studies performed before and after coculture with plasma cells indicated that LTBMC adherent cells produce the IL-6. These results suggest that the high levels of IL-6 found in LTBMC of MM patients with active disease are a reflection of their previous contact with tumor cells in vivo. These results provide a new perspective on tumor growth in MM and emphasize the importance of plasma cell-LTBMC interaction in the pathophysiology of MM.

A Fully Human Antagonist Anti-CD40 Antibody Triggers Significant Antitumor Activity Against Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2414-2414
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Xia Tong2 ◽  
Laurence Catley ◽  
Daniel Santos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Specific Lysis ◽  
Growth And Survival ◽  
Human Multiple Myeloma ◽  
Dose Dependent

Abstract We previously demonstrated that CHIR-12.12, a fully human anti-CD40 mAb (IgG1) generated in XenoMouseÒ mice (Abgenix, Inc), blocks CD40/CD40 ligand (CD40L) interactions and has more potent anti-lymphoma activity than Rituximab both in vivo and in vitro (abstract #2386, ASH, San Diego, Dec. 2003). In this study, we assess the efficacy of CHIR-12.12 against human multiple myeloma (MM) using CD40-expressing MM cell lines and purified CD138+ patient cells. CHIR-12.12 binds to purified CD138+ MM cells in &gt;80% (10/12) of patient samples, as measured by flow cytometry: the mean fluorescence intensity (MFI) range was 1 to 20 for CHIR-12.12 vs 0.2–0.9 for control human IgG1. We next examined the antagonist activity of CHIR-12.12 in MM cells. CHIR-12.12 blocked CD40L-mediated proliferation of CD40-expressing MM lines and purified CD138+ patient cells from 2 MM patients in a dose-response manner. In contrast, CHIR-12.12 alone did not alter constitutive MM cell proliferation. Immunoblotting analysis demonstrated that PI3-K/AKT, NF-kB, and ERK activation induced by hCD40L in the 12BM MM cell line was significantly inhibited by CHIR-12.12 (5 μg/ml). Adhesion of MM cells to bone marrow stromal cells (BMSCs) confers growth and survival benefit for tumor cells. Since CD40 activation, either by stimulatory mouse anti-CD40 mAb G28.5 or formaldehyde-fixed CHO cells expressing hCD40L, induces MM cell adhesion to fibronectin (FN) or BMSCs, we next asked whether antagonist CHI12.12 abrogates this process. CHIR-12.12 inhibited CD40L-induced adhesion of MM cell lines to FN in a dose dependent manner (0.001-10 μg/ml), whereas control human IgG did not. Moreover, CHIR-12.12 (1 μg/ml) blocked hCD40L-induced adhesion of freshly isolated patient MM cells to BMSCs. Adhesion of MM cells to BMSCs induces IL-6 secretion, an important growth and survival cytokine for MM cells, and treatment of MM cells with hCD40L further augmented adhesion-induced IL-6 secretion. Conversely, pretreatment of CD40-expressing MM cell lines with CHIR-12.12 significantly decreased IL-6 secretion triggered by coculture of MM cells with BMSCs. We next examined whether CHIR-12.12 stimulates antibody-dependent cellular cytotoxicity (ADCC) against CD40-expressing MM cells. Human peripheral blood mononuclear cells and purified NK cells (CD56+CD3−) were used as effector cells. CHIR-12.12 triggered MM cell lysis in a dose dependent manner, as measured in CD40-expressing MM cell lines. The maximum specific lysis of 20–70 % was achieved at 10 μg/ml concentration of CHIR-12.12. CHIR-12.12 mediated lysis was specific to CD40-expressing MM cells, as CHIR-12.12 did not induce ADCC against CD40-negative MM cells. Importantly, CHIR-12.12 induced ADCC against CD138+ cells isolated from 2 MM patients. These results provide preclinical rationale for clinical evaluation of CHIR-12.12 with the goal of improving patient outcome in MM.

CS1: A Potential New Therapeutic Target for the Treatment of Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3457-3457 ◽  
Author(s):  
Eric D. Hsi ◽  
Roxanne Steinle ◽  
Balaji Balasa ◽  
Aparna Draksharapu ◽  
Benny Shum ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cell ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Target Cells ◽  
Dependent Manner ◽  
Myeloma Cells ◽  
Effector Cells

Abstract Background: To identify genes upregulated in human memory B and plasma cells, naïve B cell cDNA was subtracted from plasma cell and memory B cell cDNA. One gene that was highly expressed in plasma cells encodes CS1 (CD2 subset 1, CRACC, SLAMF7), a cell surface glycoprotein of the CD2 family. CS1 was originally identified as a natural killer (NK) cell marker. Monoclonal antibodies (mAbs) specific for CS1 were used to validate CS1 as a potential target for the treatment of multiple myeloma (MM). Methods: Anti-CS1 mAbs were generated by immunizing mice with a protein comprising of the extracellular domain of CS1. Two clones, MuLuc63 and MuLuc90, were selected to characterize CS1 protein expression in normal and diseased tissues and blood. Fresh frozen tissue analysis was performed by immunohistochemistry (IHC). Blood and bone marrow analysis was performed using flow cytometry with directly conjugated antibodies. HuLuc63, a novel humanized anti-CS1 mAb (derived from MuLuc63) was used for functional characterization in non-isotopic LDH-based antibody-dependent cellular cytotoxicity (ADCC) assays. Results: IHC analysis showed that anti-CS1 staining occurred only on mononuclear cells within tissues. The majority of the mononuclear cells were identified as tissue plasma cells by co-staining with anti-CD138 antibodies. No anti-CS1 staining was detected on the epithelia, smooth muscle cells or vessels of any normal tissues tested. Strong anti-CS1 staining was also observed on myeloma cells in 9 of 9 plasmacytomas tested. Flow cytometry analysis of whole blood from both normal healthy donors and MM patients showed specific anti-CS1 staining in a subset of leukocytes, consisting primarily of CD3−CD(16+56)+ NK cells, CD3+CD(16+56)+ NKT cells, and CD3+CD8+ T cells. Flow cytometry of MM bone marrow showed a similar leukocyte subset staining pattern, except that strong staining was also observed on the majority of CD138+CD45−/dim to + myeloma cells. No anti-CS1 binding was detected to hematopoietic CD34+CD45+ stem cells. To test if antibodies towards CS1 may have anti-tumor cell activity in vitro, ADCC studies using effector cells (peripheral blood mononuclear cells) from 23 MM patients and L363 MM target cells were performed. The results showed that HuLuc63, a humanized form of MuLuc63, induced significant ADCC in a dose dependent manner. Conclusions: Our study identifies CS1 as an antigen that is uniformly expressed on normal and neoplastic plasma cells at high levels. The novel humanized anti-CS1 mAb, HuLuc63, exhibits significant ADCC using MM patient effector cells. These results demonstrate that HuLuc63 could be a potential new treatment for multiple myeloma. HuLuc63 will be entering a phase I clinical study for multiple myeloma.

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