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1.  Therapeutic monoclonal antibodies for multiple myeloma: an update and future perspectives 
Multiple myeloma (MM) still remains incurable in most of the patients. Despite of treatments with high-dose chemotherapy, stem cell transplantation and other novel therapies, most patients will become refractory to the therapies and relapse. Thus, it is urgent to develop new approaches for MM treatment. Currently, antibody-targeted therapy has been extensively utilized in hematological malignancies, including MM. Several novel monoclonal antibodies (mAbs) against MM have been generated and developed over the past several years. These mAbs aim to target not only tumor cells alone but also tumor microenvironment, including interaction of tumor-bone marrow stromal cells and the components of bone marrow milieu, such as cytokines or chemokines that support myeloma cell growth and survival. These include mAbs specific for CD38, CS1, CD40, CD74, CD70, HM1.24, interleukin-6 and β2-microglobulin (β2M). We have shown that anti-β2M mAbs may be a potential antitumor agent for MM therapy due to their remarkable efficacy to induce myeloma cell apoptosis in tumor cell lines and primary myeloma cells from patients in vitro and in established myeloma mouse models. In this article, we will review advances in the development and mechanisms of MM-targeted mAbs and especially, anti-β2M mAbs. We will also discuss the potential application of the mAbs as therapeutic agents to treat MM.
PMCID: PMC3207269  PMID: 22065141
Multiple myeloma; monoclonal antibodies; anti-β2M mAbs; therapy
2.  Combinatorial efficacy of anti-CS1 monoclonal antibody elotuzumab (HuLuc63) and bortezomib against multiple myeloma 
Molecular cancer therapeutics  2009;8(9):2616-2624.
Monoclonal antibody (mAb) therapy for multiple myeloma, a malignancy of plasma cells, has not been clinically efficacious in part due to a lack of appropriate targets. We recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319), was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, is currently in a Phase I clinical trial in relapsed/refractory myeloma. In this report we investigated whether the activity of elotuzumab could be enhanced by bortezomib, a reversible proteasome inhibitor with significant activity in myeloma. We first showed that elotuzumab could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model. We next showed that CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly enhanced elotuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), both for OPM2 myeloma cells using natural killer (NK) or peripheral blood mononuclear cells (PBMC) from healthy donors and for primary myeloma cells using autologous NK effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo anti-tumor activity. These findings provide the rationale for a clinical trial combining elotuzumab and bortezomib, which will test the hypothesis that combining both drugs would result in enhanced immune lysis of myeloma by elotuzumab and direct targeting of myeloma by bortezomib.
doi:10.1158/1535-7163.MCT-09-0483
PMCID: PMC2748787  PMID: 19723891
multiple myeloma; CS1; bortezomib; antibody therapy; natural killer cells
3.  Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue 
Diagnostic Pathology  2014;9:30.
Background
A T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 is a multifunctional molecule associated with various proteins such as adenosine deaminase, caveolin-1, CXCR4, collagen, and fibronectin, while playing an important role in the regulation of inflammatory responses and tumor biology. We have focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and have developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which is currently being evaluated in a phase I clinical trial for patients with CD26-expressing tumors, including malignant mesothelioma. Since detection of tumor CD26 expression is required for determining potential eligibility for YS110 therapy, the development of anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in the formalin-fixed paraffin-embedded tissues is critical.
Methods
To develop novel anti-CD26 mAbs capable of binding to the denatured CD26, we immunized mice with CD26 protein denatured in urea buffer. After the fusion of splenocytes and myeloma cells, the mAbs were screened for specific reactivity with human CD26 by flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemistry. The binding competitiveness of novel anti-CD26 mAbs with the humanized anti-CD26 mAb YS110 was also examined.
Results
We have succeeded in developing novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in formalin-fixed tissue sections with reliable clarity and intensity. Importantly, some of these mAbs exhibit no cross-reactivity with the humanized anti-CD26 mAb.
Conclusions
These novel mAbs are potentially useful as companion diagnostic agents to analyze CD26 expression in the clinical setting while advancing future CD26-related research.
Virtual slides
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5987140221097729
doi:10.1186/1746-1596-9-30
PMCID: PMC3944398  PMID: 24502396
CD26/dipeptidyl peptidase 4; Immunohistochemical staining; Companion diagnostic drug; Malignant mesothelioma; T cell costimulation
4.  Antibody-Based Therapies in Multiple Myeloma 
Bone Marrow Research  2011;2011:924058.
The unmet need for improved multiple myeloma (MM) therapy has stimulated clinical development of monoclonal antibodies (mAbs) targeting either MM cells or cells of the bone marrow (BM) microenvironment. In contrast to small-molecule inhibitors, therapeutic mAbs present the potential to specifically target tumor cells and directly induce an immune response to lyse tumor cells. Unique immune-effector mechanisms are only triggered by therapeutic mAbs but not by small molecule targeting agents. Although therapeutic murine mAbs or chimeric mAbs can cause immunogenicity, the advancement of genetic recombination for humanizing rodent mAbs has allowed large-scale production and designation of mAbs with better affinities, efficient selection, decreasing immunogenicity, and improved effector functions. These advancements of antibody engineering technologies have largely overcome the critical obstacle of antibody immunogenicity and enabled the development and subsequent Food and Drug Administration (FDA) approval of therapeutic Abs for cancer and other diseases.
doi:10.1155/2011/924058
PMCID: PMC3200112  PMID: 22046572
5.  Targeting the Biophysical Properties of the Myeloma Initiating Cell Niches: A Pharmaceutical Synergism Analysis Using Multi-Scale Agent-Based Modeling 
PLoS ONE  2014;9(1):e85059.
Multiple myeloma, the second most common hematological cancer, is currently incurable due to refractory disease relapse and development of multiple drug resistance. We and others recently established the biophysical model that myeloma initiating (stem) cells (MICs) trigger the stiffening of their niches via SDF-1/CXCR4 paracrine; The stiffened niches then promote the colonogenesis of MICs and protect them from drug treatment. In this work we examined in silico the pharmaceutical potential of targeting MIC niche stiffness to facilitate cytotoxic chemotherapies. We first established a multi-scale agent-based model using the Markov Chain Monte Carlo approach to recapitulate the niche stiffness centric, pro-oncogenetic positive feedback loop between MICs and myeloma-associated bone marrow stromal cells (MBMSCs), and investigated the effects of such intercellular chemo-physical communications on myeloma development. Then we used AMD3100 (to interrupt the interactions between MICs and their stroma) and Bortezomib (a recently developed novel therapeutic agent) as representative drugs to examine if the biophysical properties of myeloma niches are drugable. Results showed that our model recaptured the key experimental observation that the MBMSCs were more sensitive to SDF-1 secreted by MICs, and provided stiffer niches for these initiating cells and promoted their proliferation and drug resistance. Drug synergism analysis suggested that AMD3100 treatment undermined the capability of MICs to modulate the bone marrow microenvironment, and thus re-sensitized myeloma to Bortezomib treatments. This work is also the first attempt to virtually visualize in 3D the dynamics of the bone marrow stiffness during myeloma development. In summary, we established a multi-scale model to facilitate the translation of the niche-stiffness centric myeloma model as well as experimental observations to possible clinical applications. We concluded that targeting the biophysical properties of stem cell niches is of high clinical potential since it may re-sensitize tumor initiating cells to chemotherapies and reduce risks of cancer relapse.
doi:10.1371/journal.pone.0085059
PMCID: PMC3903473  PMID: 24475036
6.  The Therapeutic Value of Monoclonal Antibodies Directed Against Immunogenic Tumor Glycoproteins 
Journal of Cancer  2010;1:209-222.
Monoclonal antibodies developed against immunogenic proteins (Tumor Specific Antigens/TSA's) that are expressed in human cancers, display a unique behavioral pattern. They appear to serve in a dual role. This includes the early recognition of these immunogenic membrane proteins that can serve as diagnostic markers, and the targeting of such markers for the destruction of the tumor, primarily thru ADCC.
The monoclonals (mAbs) that we have developed against specific immunogenic tumor membrane proteins have been studied in detail. These tumor proteins, when first defined, were referred to as tumor associated antigens. With the ability of the mAbs to demonstrate therapeutic antitumor activity in those patients with relatively advanced malignancies, the term tumor specific was introduced. Monoclonals that we were able to develop from tumor specific proteins derived from colon and pancreas cancer were found capable of targeting those tumors to induce apoptosis. We were also able to define immunogenic membrane proteins from lung (squamous and adenoCa) as well as prostate neoplasms. Monoclonals developed from these tumor antigens are in the initial phases of investigation with regard to their specificity and antitumor activity.
Mabs capable of targeting the malignancies noted above were produced following immunization of BALBc mice with the Tumor Specific Antigens. The hybridomas that were screened and found to express the antibodies of interest appeared for the most part as IgG2a's. It became apparent after a short period of time that stability of the Fab CDR loops as well as the therapeutic efficacy of the hybridoma mAbs could be lost. Stability was achieved by chimerization and or humanization. The resulting mAbs were found to switch their isotypes to an IgG1 subsequent to chimerization and or humanization, when expressed in CHO cells. The monoclonals, so produced, were not only more efficient in controlling tumor growth but minimized the development of a HAMA response.
Because of 1) the specificity of this group of monoclonal antibodies in targeting well defined immunogenic proteins that were expressed on the tumor cell membrane,2) their lack of cross reactivity to normal tissue, 3) relatively low toxicity when delivered intravenously, 4) rapid targeting of tumor cell populations (4-6 hrs in vitro) and their 5) ability to destroy xenograft transplants (in vivo) within days of delivery, these mAbs were felt to be ideal for possible use in the treatment of patients with recurrent and or metastatic tumors.
Initial clinical studies have been planned for following the filing of an IND. It is required by FDA that the potential effects of tumor control and toxicity be defined using the naked antibodies produced under GMP conditions, In those situations where patients with recurrent malignancies are to be studied we have come to realize that a number of factors can influence the response to monoclonal therapy. This includes the amount of shed antigen in the serum at the time of treatment that could initiate immune complex formation as well as the shedding of inhibitory material into the serum possibly effecting an immune response. As such we plan to eventually employ the therapeutic mAbs in combination with chemotherapy as a means of enhancing the immunogenicity of the tumor system being treated and to possibly weaken the malignant growth for easier destruction by the mAb. We will also look at the combination of mAbs with immunostimulants such as GMCSF and IL-2 (fusion proteins) and eventual conjugation of the mAbs with alpha and possibly B-emitters to help in targeting bystander cells. The present paper reviews the potential therapeutic value of such mAbs in the treatment of recurrent malignancies, especially those having failed chemotherapy in established clinical trials.
PMCID: PMC2974238  PMID: 21060731
Monoclonal antibodies; hybridomas; chimeric antibodies; ADCC; apoptosis; tumor specific antigens (TSA).
7.  Human-like mouse models for testing the efficacy and safety of anti-β2-microglobulin monoclonal antibodies to treat myeloma 
Purpose
We recently demonstrated that anti-β2-microglobulin (β2M) mAbs have remarkably strong apoptotic effects on myeloma cells in vitro and in SCID(-hu) mice. However, whether the mAbs will be therapeutic and safe in the treatment of myeloma patients, in whom every tissues express low densities of MHC class I molecules and elevated levels of soluble β2M are present, remains to be determined.
Experimental Design
In this study, human-like myeloma mouse models (HLA-A2-transgenic NOD/SCID mice) were developed, which express mature and functional human MHC class I (HLA-A2 and human β2M) on murine organs and present high levels of circulating human β2M derived from human myeloma cells. Myeloma-bearing mice were treated intraperitoneally with anti-β2M mAbs, and the distribution and effects of the mAbs on normal organs and established tumors were examined.
Results
Our results show that anti-β2M mAbs were effective in suppressing myeloma growth in treated mice. The therapeutic efficacy of the mAbs in these mice are comparable to those observed in myeloma-bearing nontransgenic NOD/SCID mice in which no human MHC class I is expressed on murine organs. Furthermore, although the mAbs can be detected on different organs, no tissue damage or cell apoptosis was observed in the mice.
Conclusion
Based on the antimyeloma efficacy and low toxicity in the mice, our study suggests that anti-β2M mAbs may be safe and the tissue-expressing and soluble β2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for MM.
doi:10.1158/1078-0432.CCR-08-1823
PMCID: PMC2659684  PMID: 19188166
Multiple myeloma; anti-β2 mAbs; mouse models; HLA-A2-transgenic
8.  Potent systemic therapy of Multiple Myeloma utilizing Oncolytic Vesicular stomatitis virus coding for Interferon-beta 
Cancer Gene Therapy  2012;19(7):443-450.
Multiple myeloma (MM) is an incurable malignancy of plasma secreting B-cells disseminated in the bone marrow. Successful utilization of oncolytic virotherapy for myeloma treatment requires a systemically administered virus that selectively destroys disseminated myeloma cells in an immune-competent host. Vesicular stomatitis virus (VSV) expressing Interferon-β (IFNβ) is a promising new oncolytic agent that exploits tumor-associated defects in innate immune signaling pathways to specifically destroy cancer cells. We demonstrate here that a single, intravenous dose of VSV-IFNβ specifically destroys subcutaneous and disseminated 5TGM1 myeloma in an immune competent myeloma model. VSV-IFN treatment significantly prolonged survival in mice bearing orthotopic myeloma. Viral murine IFNβ expression further delayed myeloma progression and significantly enhanced survival compared to VSV expressing human IFNβ. Evaluation of VSV-IFNβ oncolytic activity in human myeloma cell lines and primary patient samples confirmed myeloma specific oncolytic activity but revealed variable susceptibility to VSV-IFNβ oncolysis. The results indicate that VSV-IFNβ is a potent, safe oncolytic agent that can be systemically administered to effectively target and destroy disseminated myeloma in immune competent mice. IFNβ expression improves cancer specificity and enhances VSV therapeutic efficacy against disseminated myeloma. These data show VSV-IFNβ to be a promising vector for further development as a potential therapy for treatment of Multiple myeloma.
doi:10.1038/cgt.2012.14
PMCID: PMC3380174  PMID: 22522623
Oncolytic; virotherapy; myeloma; Vesicular stomatitis virus; systemic
9.  Combination Immune Therapies to Enhance Anti-Tumor Responses by NK Cells 
Natural killer (NK) cells are critical innate immune lymphocytes capable of destroying virally infected or cancerous cells through targeted cytotoxicity and further assisting in the immune response by releasing inflammatory cytokines. NK cells are thought to contribute to the process of tumor killing by certain therapeutic monoclonal antibodies (mAb) by directing antibody-dependent cellular cytotoxicity (ADCC) through FcγRIIIA (CD16). Numerous therapeutic mAb have been developed that target distinct cancer-specific cell markers and may direct NK cell-mediated ADCC. Recent therapeutic approaches have combined some of these cancer-specific mAb with additional strategies to optimize NK cell cytotoxicity. These include agonistic mAb targeting NK cell activating receptors and mAbs blocking NK cell inhibitory receptors to enhance NK cell functions. Furthermore, several drugs that can potentiate NK cell cytotoxicity through other mechanisms are being used in combination with therapeutic mAb. In this review, we examine the mechanisms employed by several promising agents used in combination therapies that enhance natural or Ab-dependent cytotoxicity of cancer cells by NK cells, with a focus on treatments for leukemia and multiple myeloma.
doi:10.3389/fimmu.2013.00481
PMCID: PMC3870292  PMID: 24391651
NK cells; immunotherapy of cancer; antibodies; monoclonal; ADCC; multiple myeloma
10.  New Strategies in the Treatment of Multiple Myeloma 
Multiple myeloma (MM) is the second most common hematologic malignancy affecting terminally differentiated plasma cells. Although high-dose chemotherapy and autologous stem cell transplantation improved survival in younger patients, the natural history of MM has been changed with the availability of five new agents approved in last 10 years (thalidomide, bortezomib, lenalidomide, liposomal doxorubicin and carfilzomib). Despite this significant improvement in overall outcome, MM remains incurable in majority of patients prompting continued search for additional therapeutic options. Extensive molecular and genomic characterization of MM cells in its bone marrow milieu, which affects myeloma cell growth and survival, has provided number of novel drugable targets and pathways. Perturbation of protein catabolism at multiple levels has become an important target in MM. Similarly with improvements in monoclonal antibody generation and vaccine development along with identification of number of cell surface and cellular targets have led to development of various strategies including antibodies and antibody-drug conjugates which are under investigation both preclinically as well as in early clinical studies. We propose that eventually, molecularly-informed multi-agent combination therapies will be required to eliminate the MM cell clone for a long-term disease control.
doi:10.1158/1078-0432.CCR-12-1881
PMCID: PMC4112820  PMID: 23515406
11.  Novel agents in Waldenström macroglobulinemia 
Clinical investigation  2011;1(6):815-824.
Waldenström macroglobulinemia (WM) is a B-cell disorder characterized by the infiltration of the bone marrow with lymphoplasmacytic cells and the detection of an IgM monoclonal gammopathy in the serum. WM is considered an incurable disease, with a median overall survival of 87 months. The success of targeted therapy in multiple myeloma has led to the development and investigation of more than 30 new compounds in this disease and in other plasma cell dyscrasias, including WM, both in the preclinical settings and as part of clinical trials. Among therapeutic options, first-line therapies have been based on single-agent or combination regimens with alkylator agents, nucleoside analogues and the monoclonal antibody anti-CD20. Based on the understanding of the complex interaction between WM tumor cells and the bone marrow microenvironment, and the signaling pathways that are deregulated in WM pathogenesis, a number of novel therapeutic agents are now available and have demonstrated significant efficacy in WM. The range of the overall response rate for these novel agents is between 25 and 96%. Ongoing and planned future clinical trials include those using protein kinase C inhibitors such as enzastaurin, new proteasome inhibitors such as carfilzomib, histone deacetylase inhibitors such as LBH589, humanized CD20 antibodies such as ofatumumab and additional alkylating agents such as bendamustine. These agents, when compared with traditional chemotherapeutic agents, may lead in the future to higher responses, longer remissions and better quality of life for patients with WM. This article will mainly focus on those novel agents that have entered clinical trials for the treatment of WM.
doi:10.4155/CLI.11.60
PMCID: PMC3199976  PMID: 22034589
novel agents; targeted therapies; Waldenström macroglobulinemia
12.  CSPG4 Protein as a New Target for the Antibody-Based Immunotherapy of Triple-Negative Breast Cancer 
Background
The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)–based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb.
Methods
CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided.
Results
CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell–derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm2; difference of mean = 121360.0 μm2, 95% confidence interval = 91010.7 to 151709.4 μm2; P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis.
Conclusions
This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.
doi:10.1093/jnci/djq343
PMCID: PMC2950168  PMID: 20852124
13.  Molecularly Targeted Therapies in Multiple Myeloma 
Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. Novel agents are currently in development for the treatment of relapsed/refractory MM, including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, cell signaling targeted therapies, and strategies targeting the tumor microenvironment. We have previously reviewed in detail the contemporary immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies therapies for MM. Therefore, in this review, we focused on the role of molecular targeted therapies in the treatment of relapsed/refractory multiple myeloma, including cell signaling targeted therapies (HDAC, PI3K/AKT/mTOR, p38 MAPK, Hsp90, Wnt, Notch, Hedgehog, and cell cycle) and strategies targeting the tumor microenvironment (hypoxia, angiogenesis, integrins, CD44, CXCR4, and selectins). Although these novel agents have improved the therapeutic outcomes for MM patients, further development of new therapeutic agents is warranted.
doi:10.1155/2014/976567
PMCID: PMC4009206  PMID: 24829804
14.  Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCS™ Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy 
PLoS ONE  2013;8(8):e71613.
Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients'sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo.
Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.
doi:10.1371/journal.pone.0071613
PMCID: PMC3753321  PMID: 23990965
15.  Targeting Proliferating Cell Nuclear Antigen and Its Protein Interactions Induces Apoptosis in Multiple Myeloma Cells 
PLoS ONE  2013;8(7):e70430.
Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA’s protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells’ sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.
doi:10.1371/journal.pone.0070430
PMCID: PMC3729839  PMID: 23936203
16.  Tumor-host cell interactions in the bone disease of myeloma 
Bone  2010;48(1):121-128.
Multiple myeloma is a hematological malignancy that is associated with the development of a destructive osteolytic bone disease, which is a major cause of morbidity for patients with myeloma. Interactions between myeloma cells and cells of the bone marrow microenvironment promote both tumor growth and survival and bone destruction, and the osteolytic bone disease is now recognized as a contributing component to tumor progression. Since myeloma bone disease is associated with both an increase in osteoclastic bone resorption and a suppression of osteoblastic bone formation, research to date has largely focused upon the role of the osteoclast and osteoblast. However, it is now clear that other cell types within the bone marrow, including cells of the immune system, mesenchymal stem cells and bone marrow stromal cells, can contribute to the development of myeloma bone disease. This review discusses the cellular mechanisms and potential therapeutic targets that have been implicated in myeloma bone disease.
doi:10.1016/j.bone.2010.06.029
PMCID: PMC3005983  PMID: 20615487
Multiple myeloma; osteolytic bone disease; osteoclast; osteoblast; bone marrow microenvironment
17.  Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia 
Molecular Cancer  2011;10:42.
Background
The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells.
Results
The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5.
Conclusions
Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.
doi:10.1186/1476-4598-10-42
PMCID: PMC3103468  PMID: 21504579
18.  Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo 
Neuro-Oncology  2010;12(7):687-700.
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas.
doi:10.1093/neuonc/nop069
PMCID: PMC2940669  PMID: 20511188
c-FLIPL; glioblastoma; monoclonal antibody; TRAIL; TRAIL-R2/DR5
19.  Engineering and Characterization of a Mouse/Human Chimeric Anti-Phencyclidine Monoclonal Antibody 
Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human κ and IgG2 constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5’s PCP-binding variable domains and human κ and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a KD of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a “more human” version of murine mAb6B5, retains mAb6B5’s unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse – introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs.
doi:10.1016/j.intimp.2007.09.020
PMCID: PMC2238695  PMID: 18068094
Phencyclidine; Arylcyclohexylamines; Substance abuse; Therapeutic antibody; Chimeric antibody
20.  Antibody-based targeting of FGFR3 in bladder carcinoma and t(4;14)-positive multiple myeloma in mice 
The Journal of Clinical Investigation  2009;119(5):1216-1229.
Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14)-positive multiple myeloma. While FGFR3 is frequently overexpressed and/or activated through mutations in bladder cancer, the functional importance of FGFR3 and its potential as a specific therapeutic target in this disease have not been elucidated in vivo. Here we report that inducible knockdown of FGFR3 in human bladder carcinoma cells arrested cell-cycle progression in culture and markedly attenuated tumor progression in xenografted mice. Further, we developed a unique antibody (R3Mab) that inhibited not only WT FGFR3, but also various mutants of the receptor, including disulfide-linked cysteine mutants. Biochemical analysis and 2.1-Å resolution crystallography revealed that R3Mab bound to a specific FGFR3 epitope that simultaneously blocked ligand binding, prevented receptor dimerization, and induced substantial conformational changes in the receptor. R3Mab exerted potent antitumor activity against bladder carcinoma and t(4;14)-positive multiple myeloma xenografts in mice by antagonizing FGFR3 signaling and eliciting antibody-dependent cell-mediated cytotoxicity (ADCC). These studies provide in vivo evidence demonstrating an oncogenic role of FGFR3 in bladder cancer and support antibody-based targeting of FGFR3 in hematologic and epithelial cancers driven by WT or mutant FGFR3.
doi:10.1172/JCI38017
PMCID: PMC2673861  PMID: 19381019
21.  MRl of Prostate Cancer Antigen Expression for Diagnosis and lmmunotherapy 
PLoS ONE  2012;7(6):e38350.
Background
Tumor antigen (TA)–targeted monoclonal antibody (mAb) immunotherapy can be effective for the treatment of a broad range of cancer etiologies; however, these approaches have demonstrated variable clinical efficacy for the treatment of patients with prostate cancer (PCa). An obstacle currently impeding translational progress has been the inability to quantify the mAb dose that reaches the tumor site and binds to the targeted TAs. The coupling of mAb to nanoparticle-based magnetic resonance imaging (MRI) probes should permit in vivo measurement of patient-specific biodistributions; these measurements could facilitate future development of novel dosimetry paradigms wherein mAb dose is titrated to optimize outcomes for individual patients.
Methods
The prostate stem cell antigen (PSCA) is broadly expressed on the surface of prostate cancer (PCa) cells. Anti-human PSCA monoclonal antibodies (mAb 7F5) were bound to Au/Fe3O4 (GoldMag) nanoparticles (mAb 7F5@GoldMag) to serve as PSCA-specific theragnostic MRI probe permitting visualization of mAb biodistribution in vivo. First, the antibody immobilization efficiency of the GoldMag particles and the efficacy for PSCA-specific binding was assessed. Next, PC-3 (prostate cancer with PSCA over-expression) and SMMC-7721 (hepatoma cells without PSCA expression) tumor-bearing mice were injected with mAb 7F5@GoldMag for MRI. MRI probe biodistributions were assessed at increasing time intervals post-infusion; therapy response was evaluated with serial tumor volume measurements.
Results
Targeted binding of the mAb 7F5@GoldMag probes to PC-3 cells was verified using optical images and MRI; selective binding was not observed for SMMC-7721 tumors. The immunotherapeutic efficacy of the mAb 7F5@GoldMag in PC-3 tumor-bearing mice was verified with significant inhibition of tumor growth compared to untreated control animals.
Conclusion
Our promising results suggest the feasibility of using mAb 7F5@GoldMag probes as a novel paradigm for the detection and immunotherapeutic treatment of PCa. We optimistically anticipate that the approaches have the potential to be translated into the clinical settings.
doi:10.1371/journal.pone.0038350
PMCID: PMC3384648  PMID: 22761679
22.  PD-1/PD-L1 Blockade after Transient Lymphodepletion to Treat Myeloma 
Early phase clinical trials targeting the programmed death receptor-1/ligand-1 (PD-1/PD-L1) pathway to overcome tumor-mediated immunosuppression have reported promising results for a variety of cancers. This pathway appears to play an important role in the failure of immune reactivity to malignant plasma cells in multiple myeloma patients, as the tumor cells express relatively high levels of PD-L1 and T cells show increased PD-1 expression. In the current study, we demonstrate that PD-1/PD-L1 blockade with a PD-L1-specific antibody elicits rejection of a murine myeloma when combined with lymphodepleting irradiation. This particular combined approach by itself has not previously been shown to be efficacious in other tumor models. The anti-tumor effect of lymphodepletion/anti-PD-L1 therapy was most robust when tumor antigen-experienced T cells were present either through cell transfer or survival after non-myeloablative irradiation. In vivo depletion of CD4 or CD8 T cells completely eliminated anti-tumor efficacy of the lymphodepletion/anti-PD-L1 therapy, indicating that both T cell subsets are necessary for tumor rejection. Elimination of myeloma by T cells occurs relatively quickly as tumor cells in the bone marrow were nearly non-detectable by five days after the first anti-PD-L1 treatment, suggesting that anti-myeloma reactivity is primarily mediated by pre-activated T cells, rather than newly generated myeloma-reactive T cells. Anti-PD-L1 plus lymphodepletion failed to improve survival in two solid tumor models, but demonstrated significant efficacy in two hematologic malignancy models. In summary, our results support the clinical testing of lymphodepletion and PD-1/PD-L1 blockade as a novel approach for improving the survival of patients with multiple myeloma.
doi:10.4049/jimmunol.1202005
PMCID: PMC3891840  PMID: 23616570
23.  Antibody-maytansinoid conjugates for the treatment of myeloma 
mAbs  2009;1(6):548-551.
Despite recent advances in the treatment of multiple myeloma, new agents are still needed to improve the outcome for patients. The established success of monoclonal antibodies in the treatment of some cancers has promoted interest in developing antibody-based therapies for multiple myeloma. Efforts have included the development of antibodies conjugated to potent cytotoxic moieties that combine the specificity of anti-myeloma-targeting antibodies with highly active anti-tumor compounds. Two such immunoconjugates currently in clinical development are composed of antibodies that target cell surface proteins found on multiple myeloma cells, and are coupled to cytotoxic maytansinoids. IMGN901 targets the neural cell adhesion molecule, CD56, which is expressed on the majority of myeloma cells, as well as on other cancers, while BT062 targets CD138, a primary diagnostic marker for multiple myeloma. In this review, we discuss the preclinical and early clinical data for these two promising new antibody-based anti-myeloma agents.
PMCID: PMC2791311  PMID: 20068397
cancer; myeloma; antibody; immunoconjugate; CD56; CD138; maytansinoid; IMGN901; BT062
24.  Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells 
Many monoclonal antibodies (mAbs) have been extensively used in the clinic, such as rituximab to treat lymphoma. However, resistance and non-responsiveness to mAb treatment have been challenging for this line of therapy. Complement is one of the main mediators of antibody-based cancer therapy via the complement-dependent cytolysis (CDC) effect. CD59 plays a critical role in resistance to mAbs through the CDC effect. In this paper, we attempted to investigate whether the novel CD59 inhibitor, recombinant ILYd4, was effective in enhancing the rituximab-mediated CDC effect on rituximab-sensitive RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells. Meanwhile, the CDC effects, which were mediated by rituximab and anti-CD24 mAb, on the refractory multiple myeloma (MM) cell line ARH-77 and the solid tumor osteosarcoma cell line Saos-2, were respectively investigated. We found that rILYd4 rendered the refractory cells sensitive to the mAb-mediated CDC effect and that rILYd4 exhibited a synergistic effect with the mAb that resulted in tumor cells lysis. This effect on tumor cell lysis was apparent on both hematological tumors and solid tumors. Therefore, rILYd4 may serve as an adjuvant for mAb mediated-tumor immunotherapy.
doi:10.1038/cmi.2010.35
PMCID: PMC4003131  PMID: 21258360
CD59; complement; lymphoma; multiple myeloma; rituximab
25.  SST0001, a chemically modified heparin, inhibits myeloma growth and angiogenesis via disruption of the heparanase/syndecan-1 axis 
Purpose
Heparanase promotes myeloma growth, dissemination and angiogenesis through modulation of the tumor microenvironment, thus highlighting the potential of therapeutically targeting this enzyme. SST0001, a non-anticoagulant heparin with anti-heparanase activity was examined for its inhibition of myeloma tumor growth in vivo and for its mechanism of action.
Experimental Design
The ability of SST0001 to inhibit growth of myeloma tumors was assessed using multiple animal models and a diverse panel of human and murine myeloma cell lines. To investigate the mechanism of action of SST0001, pharmacodynamic markers of angiogenesis, heparanase activity, and pathways downstream of heparanase were monitored. The potential use of SST0001 as part of a combination therapy was also evaluated in vivo.
Results
SST0001 effectively inhibited myeloma growth in vivo, even when confronted with an aggressively growing tumor within human bone. In addition, SST0001 treatment causes changes within tumors consistent with the compound’s ability to inhibit heparanase; including down regulation of HGF, VEGF and MMP-9 expression and suppressed angiogenesis. SST0001 also diminishes heparanase-induced shedding of syndecan-1, a heparan sulfate proteoglycan known to be a potent promoter of myeloma growth. SST0001 inhibited the heparanase-mediated degradation of syndecan-1 heparan sulfate chains thus confirming the anti-heparanase activity of this compound. In combination with dexamethasone, SST0001 blocked tumor growth in vivo presumably through dual targeting of the tumor and its microenvironment.
Conclusions
These results provide mechanistic insight into the anti-tumor action of SST0001 and validate its use as a novel therapeutic tool for treating multiple myeloma.
doi:10.1158/1078-0432.CCR-10-2476
PMCID: PMC3060291  PMID: 21257720
Heparanase; myeloma; experimental therapeutics; tumor microenvironment; angiogenesis

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