CD55 for the treatment of burkitt lymphoma

Abstracts / Immunobiology 217 (2012) 1129–1222

T cell costimulatory receptor. CD46 costimulation of CD4 + T cells drives the activation and differentiation of pro-inflammatory IFNgsecreting cells of the Th1 subset, but in the presence of high levels of the T cell growth factor IL2, CD46 ligation also leads to the development of a subset of anti-inflammatory Th1 T cells, which secrete the immunoregulatory cytokine IL10. We ran microRNA microarrays to study the expression of microRNAs in T cells costimulated by either CD28 or CD46. We found that CD46 costimulation leads to more rapid changes in expression of microRNAs linked to T cell activation, indicating that CD46 gives a more potent costimulatory signal than CD28. We also found that proliferation of T cells, and upregulation of protein markers of activation, including the high affinity alpha chain of the IL2 receptor, are greater after CD46 costimulation compared to CD28. In addition, we ran microRNA expression arrays from the FACS-sorted T cell populations, which arise after CD46 costimulation. We found several microRNAs, which are differentially expressed in the regulatory IL10-secreting Th1 cells compared to the pro-inflammatory IFNg-secreting Th1 T cells. The use of an antisense locked nucleic acid inhibitor of one of these microRNAs, miR-150, led to a decrease in the ratio of IL10 to IFNg secreted from cells after CD46 costimulation, suggesting that miR-150 may positively regulate IL10 secretion after CD46 costimulation. http://dx.doi.org/10.1016/j.imbio.2012.08.057 57 CFHR1 gene variants and their pathophysiological relevance Agustin Tortajada 1,2 , Maria Alba 2,3 , Sheila Pinto 1,2 , Margarita Lopez-Trascasa 2,4 , Pilar Sanchez-Corral 2,3 , Claire L. Harris 5 , Santiago Rodriguez de Cordoba 1,2 1 Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Cientificas, Madrid, Spain 2 Centro de Investigacion Biomedica en Enfermedades Raras, Madrid, Spain 3 Unidad de Investigacion, Hospital Universitario La Paz, Madrid, Spain 4 Servicio de Imnunologia, Hospital Universitario La Paz, Madrid, Spain 5 Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK

Factor H related 1 (FHR-1) is the most abundant FHR protein (50 mg/ml) and that showing the highest sequence similarity with factor H. Analysis of the CFHR1 gene in our population has revealed a relatively long list of genetic variants, including single nucleotide mutations or polymorphisms, two major allotypes and several genomic rearrangements, most of them associated with pathology. Of particular interest are the two major FHR-1*A and FHR-1*B allotypes because of their differential association with AMD and aHUS and a genomic rearrangement resulting in a novel FHR-1 protein with an internal duplication of SCRs1-4, which is associated with C3-GN. To determine whether these genetic variants have functional consequences we have purified the FHR-1 proteins from the plasma of appropriate donors and determined their interaction with C3b/iC3b/C3dg using ELISA and SPR technologies. We have also determined their capacity to compete the factor I-cofactor and convertase decay accelerating activities of factor H and have analysed their interaction with other FHR proteins and heparin. The results of these experiments and the potential mechanisms that justify their association with pathology will be described and discussed. http://dx.doi.org/10.1016/j.imbio.2012.08.058

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58 CFHR5 nephropathy in a family without Cypriot ancestry Nicholas Medjeral-Thomas 1 , Talat H. Malik 1 , Tibor Toth 2 , Terry Cook 1 , Charlie Tomson 2 , Matthew C. Pickering 1 1

Centre for Complement and Inflammation Research, Imperial College, London, UK 2 Southmead Hospital, Bristol, UK C3 glomerulopathy describes glomerular pathology associated with isolated deposition of complement C3. Distinct entities within this classification include dense deposit disease and C3 glomerulonephritis. We have described familial C3 glomerulonephritis in association with two genomic rearrangements within the complement factor H-related (CFHR) locus. In one family the condition segregated with a hybrid CFHR3-1 gene. In another study we described families from Cyprus in which the disease segregated with an internal duplication within the CFHR5 gene (CFHR5 nephropathy). This mutation resulted in an abnormal CFHR5 protein which contained duplication of the first two short consensus repeat domains. Here we demonstrate that this abnormal protein is associated with familial C3 glomerulonephritis in a family without Cypriot ancestry. The mutation is close to but distinct from that which we characterised in Cypriot CFHR5 nephropathy. The demonstration that an identical abnormal CFHR5 protein is associated with familial C3 glomerulonephritis in Cypriots and non-Cypriots provides strong genetic evidence that the change is causative. http://dx.doi.org/10.1016/j.imbio.2012.08.059 59 Characterization of a human bispecific antibody against CD20/CD55 for the treatment of burkitt lymphoma Paolo Macor 1 , Nelly Mezzaroba 1 , Luca De Maso 1 , Chiara Garrovo 2 , Stefania Biffi 2 , Daniele Sblattero 3 , Roberto Marzari 1 , Francesco Tedesco 1 1

Department of Life Sciences, University of Trieste, Trieste, Italy Optical Imaging Laboratory, CBM, Area Science Park, Trieste, Italy 3 Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy 2

The effect of the antibodies in the treatment of cancer is strongly influenced by the expression of complement regulatory proteins on tumor cells. We have previously developed two minibodies neutralizing CD55 and CD59 that increased the effect of Rituximab in the treatment of a Hu/SCID model of Non-Hodgkin’s lymphoma. Here we have developed two bispecific molecules (CD20/55 or CD20/59) using our anti-CD55 and anti-CD59 miniantibody together with the anti-CD20 (Rituximab) specificity. BsAbs (MB20/55 and MB20/59) were produced using an innovative single vector, purified by affinity chromatography and characterized using western-blot, ELISA and FACS analysis. BsAbs were compared to a minibody (MB20) with the same specificity of Rituximab for CD20, produced and purified in the same conditions of BsAbs. Complement-dependent cytotoxicity assay revealed that MB20/55 or MB20/59 had a killing effect 4 times higher that of MB20, using BJAB or MEC1 as Burkitt Lymphoma and Chronic Lymphocitic Leukemia cell lines respectively. BsAb mixture induced up to 75% of tumor cell cytotoxicity while the cell death caused by MB20 was below do not reach 10%. In vivo pharmacokinetic of BsAbs labeled with Cy5.5 was evaluated by time-domain optical imaging technology in a Hu-SCID model of Burkitt lymphoma. BsAbs distribution profile mimics the data obtained studying the pharmacokinetics

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Abstracts / Immunobiology 217 (2012) 1129–1222

of Rituximab and MB20, in particular the signal: (i) was recorded in the tumor mass developed in the peritoneum, but also in liver, spleen and bone marrow, usually involved in tumor cells dissemination; (ii) was peaked in tumor mass at 3–4 days, and was still seen up to 10 days. To evaluate the therapeutic effect of BsAbs, a group of tumor bearing mice received two ip injection of the BsAb (50 ␮g) 4 and 11 days after tumor cell engraftment. This treatment resulted in a marked reduction in tumor mass size, associated with an increased survival. http://dx.doi.org/10.1016/j.imbio.2012.08.060 60 Chemotherapy of breast cancer patients induces a shift in the glycosylation pattern of plasma complement components C3 and C4 Rudolf Oehler 1 , Christine Schalko 1 , Anna Michlmayr 1 , Rupert Bartsch 2 , Michael Bergmann 1 , Thomas Bachleitner-Hofmann 1 1

Department of Surgery, Medical University of Vienna, Vienna, Austria Department of Internal Medicine-I, Medical University of Vienna, Vienna, Austria 2

Using 2D gel electrophoresis, we recently demonstrated that a combination therapy of mamma-carcinoma patients with epirubicin and docetaxel leads to changes of the plasma proteome. After the initial chemotherapeutic dose, changes in plasma levels of specific isoforms of C3 and C4 were observed which correlated with the final response to chemotherapy. Because many C3 and C4 isoforms showed the same molecular weight but different pI we analysed in the present study whether the observed changes are related to glycosylation. Complement components were analysed in human blood plasma by 1D-WB and 2D-WB analysis. Glycosylation was analysed using specific dyes. Moreover we performed in vitro activation experiments. We could show that the C3 alpha chain is present in a glycosylated as well as in a non-glycosylated form. In contrast, all C4 alpha isoforms were positively stained for glycosylation. Variations in staining intensities suggested that the degree of glycosylation differed between the isoforms. Comparing the glycosylation pattern before and after the initial dose of chemotherapy revealed that the treatment induced a shift in the glycosylation pattern. This shift was not observed after in vitro activation of the complement system. Our data clearly show that complement components C3 and C4 can be glycosylated in vivo. However, the reason of the effect of chemotherapy on this glycosylation remains unclear. http://dx.doi.org/10.1016/j.imbio.2012.08.061 61 Cleavage of intracellular C3 into C3a and C3b by cathepsin L is required for human TH1 induction Kathy M. Liszewski 2 , Martin Kolev 2 , Paula Bertram 1 , Hidekazu Yamamoto 2 , Marilyn K. Leung 1 , John P. Atkinson 1 , Claudia Kemper 2 1 Washington University in Saint Louis, School of Medicine, Division of Rheumatology, Saint Louis, MO, USA 2 MRC Center for Transplantation, Division of Transplantation Immunology and Mucosal Biology, King’s College London, London, UK

We have previously shown that T cell receptor (TCR) activation of human CD4 + T cells leads to local (serum-independent) C3b generation and that the C3b/CD46 interaction is required for TH1 effector function. However, the underlying mechanism of this rapid T cell-derived generation of C3 activation fragments remains

poorly understood and its biological significance unknown. Here we present evidence for the generation of C3a and C3b by a convertase-independent mechanism from intracellular stores of C3. Based on gene array experiments, we observed that the gene signature in CD3/CD46-activated T cells was enriched for genes related to cathepsin L (CSL) activation. CSL secreted by cancer cells has previously been shown to degrade C3 and inhibit complement deposition. However, we found that purified CSL induces rapid and complete in vitro cleavage of C3 into biologically active C3a and C3b only. Further, we determined that resting T cells contain substantial quantities of C3 in vesicles of endoplasmic reticulum origin as well as lysosomally active CSL. Because intracellular and cell-surface C3a and C3b generation is detected within minutes post activation, we suggest that upon TCR stimulation, these compartments merge and move towards the cell surface. Indeed, cell-derived C3b and C3a generation is impaired by a specific CSL inhibitor, an antibody to CSL or an inhibitor of vesicular transport – and these treatments impede TH1 induction. Consistent with our hypothesis, T cells from a C3-deficient patient contained barely detectable C3 and could not mount TH1 responses. Thus, the required rapid local generation of C3 activation fragments for TH1 induction may be (at least initially) convertase-independent and achieved by TCR-induced merging of ‘pre-loaded’ C3- and CSL-containing vesicles. Our data represent a potential paradigm shift in our understanding of C3 activation and may have important implications for the design of next generation complement-targeted therapeutics. http://dx.doi.org/10.1016/j.imbio.2012.08.062 62 Clinical analysis of peri-operative complement activation during renal ischemia-reperfusion injury following transplantation 1 , Daria Sałata 1 , Marta ˛ Wojciech Blogowski 1 , Barbara Dołegowska 2 1 ´ Budkowska , Leszek Domanski 1

Departments of Laboratory Diagnostics and Molecular Medicine, Pomeranian Medical University in Szczecin, Poland 2 Nephrology, Transplantation and Internal Medicine, Pomeranian Medical University in Szczecin, Poland Various previous experimental studies suggest that activation of the complement cascade (CC) may play an important role in the pathogenesis of ischemia reperfusion (I/R) injury during kidney transplantation. Therefore, in this paper we wanted to examine peri-operative complement activation during I/R following human kidney transplantation, and establish whether this is associated with post-transplant outcome of recipients, as well as, may serve as a promising new marker of allograft function. Renal transplant recipients (n = 69) were divided into early, slow and delayed graft function group (EGF, SGF, DGF). Blood samples were collected intraoperatively directly before, and in the 1st, 3rd and 5th minute of allograft reperfusion from the renal vein. Complement C3a, C5a, and C5b-9 levels were measured using ELISA. No significant differences in terms of C3a and C5a levels were observed in analyzed groups of patients. However, during I/R injury significantly higher C5b-9 levels were observed in SGF and DGF patients, comparing to EGF individuals. These were strongly associated with worst early and long-term (1 year) allograft function. Moreover, perioperative C5b-9 levels proved to possess a promising potential for prediction of SDG/DGF occurrence in humans (sensitivity and specificity of 74–87.5%). Our study demonstrated that during human renal transplantation selective activation of the complement cascade occurs, and is more evidently pronounced in SGF/DGF patients. Peri-operative complement levels seem to be associated