New treatment of multiple mieloma and anaplastic T cell lymphoma using C-fixing anti-CD162 antibodies

Complement C1q and C8beta deficiency in an individual with recurrent bacterial meningitis and adult-onset systemic lupus erythematosus-like illness.

Co-existing complement C8 deficiency ameliorated the SLE associated with C1q deficiency.

C1q as a novel player in angiogenesis with therapeutic implication in wound healing

We have previously shown that C1q is expressed on endothelial cells (ECs) of newly formed decidual tissue. Here we demonstrate that C1q is deposited in wound-healing skin in the absence of C4 and C3 and that C1q mRNA is locally expressed as revealed by real-time PCR and in situ hybridization. C1q was found to induce permeability of the EC monolayer, to stimulate EC proliferation and migration, and to promote tube formation and sprouting of new vessels in a rat aortic ring assay. Using a murine model of wound healing we observed that vessel formation was defective in C1qa(-/-) mice and was restored to normal after local application of C1q. The mean vessel density of wound-healing tissue and …

Distinct Roles of Classical and Lectin Pathways of Complement in Preeclamptic Placentae

Pre-eclampsia is a pregnancy complication characterized by defective vascular remodeling in maternal decidua responsible for reduced blood flow leading to functional and structural alterations in the placenta. We have investigated the contribution of the complement system to decidual vascular changes and showed that trophoblasts surrounding unremodeled vessels prevalent in preeclamptic decidua fail to express C1q that are clearly detected in cells around remodeled vessels predominant in control placenta. The critical role of C1q is supported by the finding that decidual trophoblasts of femaleC1qa-/-pregnant mice mated toC1qa+/+male mice surrounding remodeled vessels express C1q of paternal …

C1q induces in vivo angiogenesis and promotes wound healing

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We ha…

C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation

Complement C1q is the activator of the classical pathway. However, it is now recognized that C1q can exert functions unrelated to complement activation. Here we show that C1q, but not C4, is expressed in the stroma and vascular endothelium of several human malignant tumours. Compared with wild-type (WT) or C3- or C5-deficient mice, C1q-deficient (C1qa−/−) mice bearing a syngeneic B16 melanoma exhibit a slower tumour growth and prolonged survival. This effect is not attributable to differences in the tumour-infiltrating immune cells. Tumours developing in WT mice display early deposition of C1q, higher vascular density and an increase in the number of lung metastases compared with C1qa−/− mi…

Preeclampsia is associated with defective production of C1q by invasive trophoblast

Dynamics of complement activation in aHUS and how to monitor eculizumab therapy.

Atypical hemolytic-uremic syndrome (aHUS) is associated with genetic complement abnormalities/anti–complement factor H antibodies, which paved the way to treatment with eculizumab. We studied 44 aHUS patients and their relatives to (1) test new assays of complement activation, (2) verify whether such abnormality occurs also in unaffected mutation carriers, and (3) search for a tool for eculizumab titration. An abnormal circulating complement profile (low C3, high C5a, or SC5b-9) was found in 47% to 64% of patients, irrespective of disease phase. Acute aHUS serum, but not serum from remission, caused wider C3 and C5b-9 deposits than control serum on unstimulated human microvascular endotheli…

An alternative role of C1q in cell migration and tissue remodeling: contribution to trophoblast invasion and placental development.

Abstract Fetal trophoblast cells invading the decidua in the early phase of pregnancy establish complex interaction with the maternal extracellular matrix. We discovered that C1q was widely distributed in human decidual stroma in the absence of C4 and C3 and was actively synthesized by migrating extravillous trophoblasts. The cells expressed the messages for the three chains of C1q and secreted this complement component that interacted with the proteins of the decidual extracellular matrix. Solid phase-bound C1q promoted trophoblast adhesion and migration, and cell binding to C1q resulted in activation of ERK1/2 MAPKs. Ab inhibition experiments showed that the receptors for the globular hea…

Selection and characterization of a novel agonistic human recombinant anti-Trail-R2 minibody with anti-leukemic activity

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising natural anticancer therapeutic agent because through its “death receptors”, TRAIL-R1 and TRAIL-R2, it induces apoptosis in many transformed tumor cells, but not in the majority of normal cells. Hence, agonistic compounds directed against TRAIL death receptors have the potential of being excellent cancer therapeutic agents, with minimal cytotoxicity in normal tissues. Here, we report the selection and characterization of a new single-chain fragment variable (scFv) to TRAIL-R2 receptor isolated from a human phage-display library, produced as minibody (MB), and characterized for the in vitro anti-leukemic tumoricid…

In vivo targeting of human neutralizing antibodies against CD55 and CD59 to lymphoma cells increases the antitumor activity of rituximab.

AbstractAn in vivo model of human CD20+ B-lymphoma was established in severe combined immunodeficiency mice to test the ability of human neutralizing miniantibodies to CD55 and CD59 (MB55 and MB59) to enhance the therapeutic effect of rituximab. The miniantibodies contained single-chain fragment variables and the hinge-CH2-CH3 domains of human IgG1. LCL2 cells were selected for the in vivo study among six B-lymphoma cell lines for their high susceptibility to rituximab-dependent complement-mediated killing enhanced by MB55 and MB59. The cells injected i.p. primarily colonized the liver and spleen, leading to the death of the animals within 30 to 40 days. Thirty percent of mice receiving bio…

Is local complement activation involved in renal damage in patients with atypical haemolytic uraemic syndrome?

ABSTRACTS: 9
Phenotype characterization of the anti-inflammatory behavior of decidual endothelial cells

Problem:  During pregnancy, an adequate inflammatory control is essential for normal intrauterine foetus development. Vascular endothelium is known to play a critical role in regulation of inflammatory processes. We hypothesize a different ability of decidual endothelial cells (DEC) compared to endothelium from other districts to respond to inflammatory stimuli. Material and Methods:  DEC were isolated and activated with inflammatory stimuli and their response in term of adhesion molecules expression, vascular leakage and cytokines production was evaluated. These data were compared to the data obtained with HUVEC and microvascular endothelial cells isolated from human skin (ADMEC) and from …

OP0023 Targeted Polymeric Nanoparticles as Diagnostic and Therapeutic Tool for Rheumatoid Arthritis

Background Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease affecting joints due to the persistent synovial tissue inflammation. RA treatment has dramatically evolved in the last 20 years due to the production of biological Disease Modifying Antirheumatic Drugs (bDMARDs). However, side effects and the high costs of biological drugs are holding back their widespread usage. Moreover, some patients fail to respond to bDMARDs, for all of these reasons, DMARDs remains the main desired strategy for the treatment of RA, and Methotrexate (MTX) is still the “anchor” drug to treat RA. A successful treatment depends also on the early diagnosis, treating patients as soon as possible …

Complement Protein C1q Binds to Hyaluronic Acid in the Malignant Pleural Mesothelioma Microenvironment and Promotes Tumor Growth

C1q is the first recognition subcomponent of the complement classical pathway, which acts towards the clearance of pathogens and apoptotic cells. C1q is also known to modulate a range of functions of immune and non-immune cells, including their involvement in placental development and sensorial synaptic pruning. We have recently shown that C1q can promote tumour by encouraging their adhesion, migration and proliferation in addition to angiogenesis and metastasis. In this study, we have examined the role of C1q in the microenvironment of malignant pleuric mesothelioma (MPM), a rare form of cancer commonly associated with exposure to asbestos. We found that C1q was highly expressed in all MPM…

Development of a human-SCID lymphoma as a model to evaluate the therapeutic effect of Rituximab

Pathogenic Role of Complement in Antiphospholipid Syndrome and Therapeutic Implications

Antiphospholipid syndrome (APS) is an acquired autoimmune disease characterized by thromboembolic events, pregnancy morbidity, and the presence of antiphospholipid (aPL) antibodies. There is sound evidence that aPL act as pathogenic autoantibodies being responsible for vascular clots and miscarriages. However, the exact mechanisms involved in the clinical manifestations of the syndrome are still a matter of investigation. In particular, while vascular thrombosis is apparently not associated with inflammation, the pathogenesis of miscarriages can be explained only in part by the aPL-mediated hypercoagulable state and additional non-thrombotic effects, including placental inflammation, have b…

Phenotype characterization of the anti-inflammatory behavior of decidual endothelial cells

Bispecific antibodies targeting tumor-associated antigens and neutralizing complement regulators increase the efficacy of antibody-based immunotherapy in mice.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluate…

C1q is involved in human trophoblast invasion

During the development of human placenta, extravillous trophoblast (EVT) departs from anchoring chorionic villi and invades the maternal decidua. Immunohistochemical analysis of decidua obtained from voluntary abortions showed that C1q was widely distributed in the decidual stroma with intense staining around invading trophoblast, while undetectable in non pregnant uterus. Based on these findings, we hypothesized that C1q may be involved in the migration of EVT. To this end, we investigated the ability of EVT to adhere to solid-phase bound C1q and to migrate using a transwell model system with inserts coated with C1q. Our results showed that EVT strongly adhered to C1q to an extent similar …

Targeting CD34+ cells of the inflamed synovial endothelium by guided nanoparticles for the treatment of rheumatoid arthritis

Abstract Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovi…

P-Selectin Glycoprotein Ligand-1 as a Potential Target for Humoral Immunotherapy of Multiple Myeloma (Supplementry Material)

Monoclonal antibodies (mAbs), successfully adopted in the treatment of several haematological malignancies, have proved almost ineffective in multiple myeloma (MM), because of the lack of an appropriate antigen for targeting and killing MM cells. Here, we demonstrate that PSGL1, the major ligand of P-Selectin, a marker of plasmacytic differentiation expressed at high levels on normal and neoplastic plasma cells, may represent a novel target for mAb-mediated MM immunotherapy. The primary effectors of mAb-induced cell-death, complement-mediated lysis (CDC) and antibody-dependent cellmediated cytotoxicity (ADCC), were investigated using U266B1 and LP1 cell-lines as models. Along with immunolog…

C1q secreted in the tumour microenvironment promotes tumour growth in the absence of complement activation

We have recently shown that locally secreted C1q is involved in trophoblast invasion of decidua during pregnancy (Agostinis et al., J. Immunol. 2010;185;4420–4429). Since this physiologic process resembles to some extent tumor progression, we sought to investigate if C1q plays a similar role in tumor development and progression. Immunohistochemical analysis of several solid tumours including colon, prostate, lung and breast cancer and melanoma revealed the presence of C1q that was localized on the vascular endothelium and also distributed in the stroma in the absence of C4. To investigate the in vivo role of C1q in tumour development, 6/8 week old female WT and C1q−/− C57BL/6 mice received …

Humoral immunotherapy of multiple myeloma: perspectives and perplexities

IMPORTANCE OF THE FIELDS Multiple myeloma (MM) is a hematological malignancy still remaining incurable despite the various therapies available, mainly because of the high fraction of refractory/relapsing cases. Therefore, the development of novel therapeutic approaches is urgently needed to overcome conventional treatment resistance. AREAS COVERED IN THIS REVIEW: In the era of targeted therapies, treatments combining a high specificity for neoplastic cells and the capability to interfere with environmental signals should be regarded as the weapons of choice. Monoclonal antibody (mAb)-based humoral immunotherapy could satisfy both these requirements when applied to MM. Indeed, many of the mo…

Decidual endothelial cells express surface-bound C1q as a molecular bridge between endovascular trophoblast and decidual endothelium.

This study was prompted by the observation that decidual endothelial cells (DECs), unlike endothelial cells (ECs) of blood vessels in normal skin, kidney glomeruli and brain, express surface-bound C1q in physiologic pregnancy. This finding was unexpected, because deposits of C1q are usually observed in pathologic conditions and are associated with complement activation. In the case of DECs, we failed to detect immunoglobulins and C4 co-localized with C1q on the cell surface. Surprisingly, DECs expressed mRNA for the three chains of C1q and secreted detectable level of this component in serum-free medium. The ability to synthesize C1q is acquired by DECs during pregnancy and is not shared by…

Neutralizing human antibodies against CD55 and CD59 targeted to lymphoma cells in vivo potentiate the therapeutic effect of Rituximab

An update on the xenograft and mouse models suitable for investigating new therapeutic compounds for the treatment of B-cell malignancies

B-cell malignancies account for over the 90% of all lymphoid neoplasms. The clonal proliferations of B-cells show a high degree of variation in terms of clinical and presenting features, histopathology, immuophenotype, and genetics. Primary tumor samples are useful for examining the characteristics of a patients own tumor, although both primary leukemic cells and cell lines provide an initial step for screening novel compounds for their activity in some hematological malignancies, they should be followed by models in intact animals. In this review, we try to summarize the animal models generated to study B-cell malignancies, in particular, B-cell lymphoma, B-cell CLL and MM that represent t…

C7 is expressed on endothelial cells as a trap for the assembling terminal complement complex and may exert anti-inflammatory function.

AbstractWe describe a novel localization of C7 as a membrane-bound molecule on endothelial cells (ECs). Data obtained by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, Northern blot analysis, and mass spectrometry revealed that membrane-associated C7 (mC7) was indistinguishable from soluble C7 and was associated with vimentin on the cell surface. mC7 interacted with the other late complement components to form membrane-bound TCC (mTCC). Unlike the soluble SC5b-9, mTCC failed to stimulate ECs to express adhesion molecules, to secrete IL-8, and to induce albumin leakage through a monolayer of ECs, and more importantly protected ECs from the proinf…

The soluble terminal complement complex (SC5b-9) up-regulates osteoprotegerin expression and release by endothelial cells: Implications in rheumatoid arthritis

Objective. Complement activation products contribute to a large number of inflammatory diseases, including RA. We have investigated whether osteoprotegerin (OPG) may concur with the soluble terminal complement complex (SC5b-9) to the inflammatory cascade characterizing RA. Methods. Levels of SC5b-9 and OPG in the plasma and SF of patients with active RA were determined by ELISA. The presence of SC5b-9 and OPG in RA synovial lesions was analysed by immunohistochemistry. Cultured endothelial cells were used for in vitro leucocyte/endothelial cell adhesion assays. In addition, endothelial cells were exposed to SC5b-9 in order to evaluate the effects on the production of OPG protein, as well as…

CCL3 and CCL4, the Major Chemokines Produced by CD38+ Chronic Lymphocytic Leukemia Cells, Facilitate Microenvironmental Interactions of Neoplastic Cells Via the CD49d/VCAM Pair.

Abstract CD38, a negative prognostic marker for patients with CLL, has been demonstrated to be a key molecule in the interactions occurring in the context of tumor microenvironment, mediating both survival and migratory signals for CLL cells. By taking advantage of gene expression profiling studies (GEP) comparing 11 CD38pos (CD38>30%) and 15 CD38neg (CD38<10%) CLLs, we identified as over-expressed in CD38pos CLL cells: i) genes for the two C-C chemokines CCL3 and CCL4 (median-log difference, MLD-CCL3= 3.5; MLD-CCL4=4.4); real-time quantitative PCR (RTQ-PCR) of selected cases confirmed GEP results; ii) the gene for CD49d (MLD=4.4); a high correlation between CD38 and CD49d pro…

FRI0030 Anti-TNF-α Antibody Targeted To Inflamed Synovial Tissue for The Treatment of Rheumatoid Arthritis

Background TNF-α neutralizing molecules represent one of the most efficient therapeutic approaches to control inflammation in rheumatoid arthritis (RA). The widespread distribution in the body induces the inhibition of TNF-α in all the tissues, requesting the use of high dose of this expensive drug. Another problem that has not yet been solved in the management of RA patients is how to reduce and possibly avoid the side effects, particularly the increased risk of common and opportunistic infections, which may be associated with long-term administration of these therapeutic drugs. Objectives The aim of the present investigation was to show that a recombinant protein obtained by fusing a syno…

In vivo biodistribution and lifetime analysis of cy5.5-conjugated rituximab in mice bearing lymphoid tumor xenograft using time-domain near-infrared optical imaging.

Rituximab is a chimeric monoclonal antibody directed against human CD20 antigen, which is expressed on B-cell lymphocytes and on the majority of B-cell lymphoid malignancies. Herein we report the conjugate of rituximab with the near-infrared (NIR) fluorophore Cy5.5 (RI-Cy5.5) as a tool for in vitro, in vivo, and ex vivo NIR time-domain (TD) optical imaging. In vitro, RI-Cy5.5 retained biologic activity and led to elevated cell-associated fluorescence on tumor cells. In vivo, TD optical imaging analysis of RI-Cy5.5 injected into lymphoma-bearing mice revealed a slow tumor uptake and a specific long-lasting persistence of the probe within the tumor. Biodistribution studies after intraperiton…

Exploratory study on the effects of biodegradable nanoparticles with drugs on malignant B cells and on a human/mouse model of Burkitt lymphoma.

The aim of this study was to determine if Rituximab coated Biodegradable Nanoparticles (BNPs) loaded with Chlorambucil and Hydroxychloroquine could induce apoptosis of B-Chronic Lymphocytic Leukemia (B-CLL), MEC-1 and BJAB cells in vitro and evaluate their toxic and therapeutic effects on a Human/Mouse Model of Burkitt Lymphoma at an exploratory, proof of concept scale. We found that Rituximab-Chlorambucil-Hydroxychloroquine BNPs induce a decrease in cell viability of malignant B cells in a dose-dependent manner. The mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the B-CLL cells after Annexin V/propidium iodide staining. Additional data revealed that these BN…

Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38(+)CD49d(+) Chronic Lymphocytic Leukemia Cells

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ C…

CD38/CD31, the CCL3 and CCL4 chemokines, and CD49d/vascular cell adhesion molecule-1 are interchained by sequential events sustaining chronic lymphocytic leukemia cell survival.

AbstractCD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Despite evidence that both molecules are involved in interactions occurring between CLL and normal cells in the context of CLL-involved tissues, a functional link is still missing. Using gene expression profiles comparing CD38+CD49d+ versus CD38−CD49d− CLL cells, we showed overexpression of the CCL3 and CCL4 chemokines in cells from the former group. These chemokines were also up-regulated by CD38 signals in CLL; moreover, CCL3 was expressed by CLL cells from bone marrow biopsies (BMB) of CD38+CD49d+ but not CD38−CD49d− cases. High levels of CCR1 and, to a lesser extent, CCR5, the receptors…