Expression Of Complement Regulatory Proteins Research Articles

Therapy-induced senescent cancer cells exhibit complement activation and increased complement regulatory protein expression.

Therapy-induced senescence (TIS) is a primary response to chemotherapy, contributing to untoward treatment outcomes such as evasion of immunosurveillance. Despite the established role of the complement system in the immune response to cancer, the role of complement in mediating the immune response against senescent tumor cells remains poorly understood. To explore this relationship, we exposed lung adenocarcinoma (A549), breast adenocarcinoma (MCF7) and pancreatic carcinoma (Panc-1) cell lines to sublethal doses of either etoposide or doxorubicin to trigger TIS. Identification of TIS was based on morphological changes, upregulation of the senescence-associated β-galactosidase, p21Cip1 induction and lamin B1 downregulation. Using immunofluorescence microscopy, quantitative PCR, ELISA of conditioned media and in silico analysis, we investigated complement activation, complement protein expression, C3 levels in the conditioned media of senescent cells and secreted complement proteins as part of the senescence-associated secretory phenotype (SASP), respectively. In cell lines undergoing TIS, complement-related changes included (i) activation of the terminal pathway, evidenced by the deposition of C5b-9 on senescent cells; (ii) an increase in the expression of CD59 and complement factor H and (iii) in A549 cells, an elevation in the expression of C3 with its secretion into the medium. In addition, increased C3 expression was observed in breast cancer samples expressing TIS hallmarks following exposure to neoadjuvant chemotherapy. In conclusion, TIS led to the activation of complement, upregulation of complement regulatory proteins and increased C3 expression. Complement appears to play a role in shaping the cancer microenvironment upon senescence induction.

219 Therapy induced senescence in cancer cell lines affects complement activation and complement regulatory proteins expression

219 Therapy induced senescence in cancer cell lines affects complement activation and complement regulatory proteins expression

Human papillomavirus-mediated expression of complement regulatory proteins in human cervical cancer cells

ObjectivesThis study aimed to evaluate the expression pattern of complement regulatory proteins (CRPs) CD46, CD59, and CD55 in HPV-positive (HPV+) & negative (HPV-) cervical cancer cell lines in search of a reliable differential biomarker. Study DesignWe analysed the expression of CRPs in HPV 16-positive SiHa cell line, HPV 18-positive HeLa cell line, and HPV-negative cell line C33a using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. ResultsWe observed a differential expression profile of CRPs in HPV+ and HPV- cervical cancer cell lines. The mRNA level of CD59 &CD55 showed a higher expression pattern in HPV+ cells when compared to HPV- cancer cells. However, flow cytometry-based experiments revealed that CD46 was preferentially expressed more in HPV 16-positive SiHa cells followed by HPV 18-positive HeLa cells when compared to HPV- C33a cells. Interestingly, confocal microscopy revealed a high level of CD59 expression in Hela cells and SiHa cells but low expression in HPV- C33a cells. In addition, HPV 18-positive HeLa cells expressed more CD55, which was lower in SiHa cells and very weak in C33a cells. ConclusionThe study demonstrates the differential expression of CRPs in both HPV+ and HPV- cervical cancer cells for the first time, and their potential to serve as an early diagnostic marker for cervical carcinogenesis.

#5885 HYPOXIA INDUCES COMPLEMENT ACTIVATION ON HUMAN KIDNEY EPITHELIAL CELLS

Abstract Background and Aims Ischemia-reperfusion injury is an inevitable event associated with kidney transplantation (Tx) and has a major impact on short- and long-term graft survival. Proximal tubular epithelial cells are both a source of complement during Tx and a target of complement activation. The normal kidney has a capacity to protect itself from complement activation through cellular expression of complement regulatory proteins. In this study we investigated whether hypoxia and reoxygenation increase C3 deposition and alter complement regulatory protein expression (CD46, CD59 and CD55). Method In vitro, HKC-8 renal proximal tubular epithelial cells were subjected to 24h of hypoxia (1% O2) and then reoxygenated for 4h (O2 = 21%) in the presence of 40% normal human serum. qRT-PCR was used to estimate the level of VEGF-A (as a positive control), C3, CD46, CD59 and CD55 expression. Similar analysis was performed in untreated, normoxic cells. Immunofluorescence staining was used to determine C3 deposition on the cell surface and intensity quantified by ImageJ. Experiments were repeated three times. Results qRT-PCR analysis of HKC-8 cells in hypoxia revealed significant increases in the expression of VEGF-A after 24h (p = 0.0113). C3 expression following hypoxia and reoxygenation increased insignificantly. Increased expression of CD59 with the presence of serum was observed in epithelial cells (p = 0.026). However, no significant change was seen in the expression of CD55 and CD46. Reoxygenation in human serum led to significantly greater deposition of C3 on hypoxic cells compared with normoxic HKC-8 cells (Figure 1, 2). Conclusion Our preliminary data suggests that hypoxia and reoxygenation significantly increases C3 deposition on the proximal tubular cell surface. CD59, as one of the regulators of complement activation is upregulated at mRNA level after hypoxia followed by 4h reperfusion in the presence of serum. These data demonstrate that hypoxia and reoxygenation activate complement system with upregulation of CD59 as a cell defence. However, expression of CD46 and CD55 do not change. Further study is needed to identify the impact of hypoxia on complement regulation in primary epithelial cells and kidney tissues.

Complement and complement regulatory proteins are upregulated in lungs of COVID-19 patients

We explored the pathological changes and the activation of local complement system in COVID-19 pneumonia. Lung paraffin sections of COVID-19 infected patients were analyzed by HE (hematoxylin-eosin) staining. The deposition of complement C3, the deposition of C3b/iC3b/C3d and C5b-9, and the expression of complement regulatory proteins, CD59, CD46 and CD55 were detected by immunohistochemistry. In COVID-19 patients’ lung tissues, fibrin exudation, mixed with erythrocyte, alveolar macrophage and shed pneumocyte are usually observed in the alveoli. The formation of an “alveolar emboli” structure may contribute to thrombosis and consolidation in lung tissue. In addition, we also found that compared to normal tissue, the lung tissues of COVID-19 patients displayed the hyper-activation of complement that is represented by extensive deposition of C3, C3b/iC3b/C3d and C5b-9, and the increased expression level of complement regulatory proteins CD55, and especially CD59 but not CD46. The thrombosis and consolidation in lung tissues may contribute to the pathogenesis of COVID-19. The increased expression of CD55 and CD59 may reflect a feedback of self-protection on the complement hyper-activation. Further, the increased C3 deposition and the strongly activated complement system in lung tissues may suggest the rationale of complement-targeted therapeutics in conquering COVID-19.

Effect of Heme Oxygenase-1 Depletion on Complement Regulatory Proteins Expression in the Rat.

Heme oxygenase has been implicated in the regulation of various immune responses including complement activation. Using a transgenic rat model of HO-1 depletion, the present study assessed the effect of HO-1 absence on the expression of complement regulatory proteins: decay accelerating factor (DAF), CR1-related gene/protein Y (Crry) and CD59, which act to attenuate complement activation. Protein expression was assessed by immunohistochemistry in kidney, liver, lung and spleen tissues. DAF protein was reduced in all tissues retrieved from rats lacking HO-1 (Hmox1-/-) apart from spleen tissue sections. Crry protein was also reduced, but only in Hmox1-/- kidney and liver tissue. C3b staining was augmented in the kidney and spleen from Hmox1-/- rats, suggesting that the decrease of DAF and Crry was sufficient to increase C3b deposition. The observations support an important role of HO-1 as a regulator of the complement system.

Targeting C3d in Combination with Daratumumab:a Strategy to Enhance Therapeutic Potential in Multiple Myeloma

The monoclonal antibody (mAb) daratumumab (DARA) targeting CD38 can induce minimal residual disease negative remissions in patients with multiple myeloma (MM). However, majority of patients subsequently relapse or become refractory to treatment (RRMM). Rapid reduction of surface CD38 levels in MM cells following DARA infusion due to trogocytosis by monocytes and granulocytes leads to reduced complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC), effector functions that rely on antigen density. High expression of complement regulatory proteins, CD55 and CD59 on MM cell surface can also impede CDC activity. Therefore, modalities to overcome these limitations and improve clinical outcome for patients with RRMM are needed. Daratumumab is a strong activator of complement. While a subset of cells may be lysed by the membrane attack complex, all cells reacted with daratumumab are opsonized by C3d, the terminal component of C3. C3d remains covalently attached at the cell surface. Thus, the newly acquired membrane bound C3d constitutes a target for additional intervention, particularly when the cell surface CD38 is lost or low following treatment with DARA. Using phage display technology, we generated and characterized recombinant rabbit/human chimeric mAbs that selectively bind C3 fragments including C3d but not full length C3. We selected two high affinity anti-C3d mAbs (clones 5S8 and d301 with kD values 3.35 nM and 0.99 nM, respectively) that showed reactivity against both human and mouse C3d. To deposit C3d on myeloma cell lines (MM1-S, MM1-R, RPMI8226, NCI-H929, KMS-12-PE) without activating CDC, the cells were reacted with DARA in C5 depleted normal human serum. Both 5S8 and d301 bound to the C3d opsonized cells. Based on SPR analysis these two mAbs do not compete for C3d binding suggesting that they recognize distinct epitopes. In normal human serum, the anti-C3d mAbs enhanced killing by CDC of DARA-opsonized target cells compared to DARA alone (P<0.0001). The non-targeting IgG1 isotype control mAb trastuzumab (TRA) did not bind C3d or enhance DARA-mediated killing. To test the combination of DARA with anti-C3d mAbs in vivo, we used Balb/c-SCID mice bearing subcutaneous (s. c.) MM1-R tumors. First, using clones 5S8 and d301, we demonstrated in vivo binding of mouse C3d on MM1-R tumor cells harvested 24 h after a single intraperitoneal (i. p.) injection of DARA. Next, we challenged the mice s. c. with MM1-R cells and randomly divided them into the following treatment groups: 20 mg/kg DARA single agent (n=7), or combination treatments of 10 mg/kg of each of two mAbs: DARA + TRA (n=10), DARA + 5S8 (n=13), DARA + d301 (n=13). The mAbs were given via i. p. injections weekly starting on day 7 post tumor challenge and continued for 6 weeks. Palpable tumors were noticed in mice between days 7 and 14, and tumor growth was monitored by caliper measurements twice weekly. Mice were euthanized when tumors reached pre-defined limits. All the mice that received DARA + TRA showed progressive tumor growth with median survival of 38 days and all mice had to be euthanized by day 50. Doubling the dose of DARA to 20mg/kg slightly increased median survival to 43 days (P = 0.12 vs DARA+TRA). Two of 7 mice that received DARA alone (at 20 mg/kg) survived to day 175. Median survival for mice treated with DARA (10mg/kg) plus an anti-C3d mAb was 162 days (P = 0.006 for comparison to DARA 20mg/kg), and 13 of 26 (50%) mice in this cohort survived tumor-free beyond 200 days. There was no statistically significant difference between groups treated with 5S8 or d301 in combination with DARA (P = 0.23). All the surviving mice showed no evidence of tumor, weight loss or other morbidity indicating that they are essentially tumor-free. In summary, these results demonstrate that combination treatment of DARA with anti-C3d mAbs improves efficacy over single agent DARA leading to long-term survival in the MM.1R xenograft model. The combination of C3d targeting mAbs with DARA could improve clinical efficacy and overcome resistance to single agent therapy.

Bispecific mAb2 Antibodies Targeting CD59 Enhance the Complement-Dependent Cytotoxicity Mediated by Rituximab.

Inhibition of complement activation via the overexpression of complement-regulatory proteins (CRPs), most notably CD46, CD55 and CD59, is an efficient mechanism of disguise of cancer cells from a host immune system. This phenomenon extends to counteract the potency of therapeutic antibodies that could lyse target cells by eliciting complement cascade. The manifold functions and ubiquitous expression of CRPs preclude their systemic specific inhibition. We selected CD59-specific Fc fragments with a novel antigen binding site (Fcabs) from yeast display libraries using recombinant antigens expressed in bacterial or mammalian cells. To produce a bispecific antibody, we endowed rituximab, a clinically applied anti-CD20 antibody, used for therapy of various lymphoid malignancies, with an anti-CD59 Fcab. This bispecific antibody was able to induce more potent complement-dependent cytotoxicity for CD20 and CD59 expressing Raji cell line measured with lactate dehydrogenase-release assay, but had no effect on the cells with lower levels of the primary CD20 antigen or CD20-negative cells. Such molecules are promising candidates for future therapeutic development as they elicit a higher specific cytotoxicity at a lower concentration and hence cause a lower exhaustion of complement components.

Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti‐TKO pig antibodies in OWMs than humans. Here, we show that long‐term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO‐hTG pigs were transplanted into eight cynomolgus recipients without pre‐screening for low anti‐pig antibody titers. Two recipients of TKO‐hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO‐A) survived for 2 and 61 days, whereas six recipients of TKO‐hTG xenografts with high CRP expression (TKO‐B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+T cell depletion and low anti‐pig antibody titers, which were previously reported important for long‐term survival of αGal knock‐out (GTKO) xenografts, were not always required for long‐term survival of TKO‐hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli.

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX−) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX− sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

Evaluation of Combinatory Effects of Plasmodium Circumsporozoite Protein and Complement Regulatory Protein Expression of Recombinant Baculovirus Vectors.

Baculovirus vectors (BVs) are safely able to transduce foreign genes and express them in mammalian cells. However, the transduction activity of BVs is strongly reduced by the attack of serum complement, which is one of the major obstacles in the use of BVs for in vivo gene transfer. One strategy to overcome this problem is the display of complement regulatory proteins (CRPs) on BV virions. We previously developed CD46-decay accelerating factor (DAF)-CD59 triple fusion type BV showing potent complement resistance. We also developed BVs expressing Plasmodium circumsporozoite protein (CSP) to enhance transduction efficacy in hepatic cells. In this study, we investigated the combination of CSP and CRPs in a BV system to evaluate transduction efficacy along with complement resistance. To accomplish the combination of CSP and CRPs, we generated insect Sf9 cells stably expressing CRPs, to which CSP type BV was infected. The BVs collected from these infected cells were confirmed to possess both CSP and CRPs in virions. We demonstrated that CSP-CD46-DAF-CD59 type BV, containing both CSP and CD46-DAF-CD59, showed a significant increase in transduction efficacy in human hepatoma HepG2 cells under intact serum exposure compared with control type BV or CSP type BV, retaining both advantages of CSP and CD46-DAF-CD59. Collectively, these results demonstrated that the utilization of stably expressing Sf9 cells to introduce the protein products of interest, e.g., CRPs into BVs, would be useful strategy to generate BVs with novel functions such as resistance against serum complement attack.

The complement system in primary Sjögren's syndrome: the expression of certain cascade and regulatory proteins in labial salivary glands - observational study.

IntroductionThe complement cascade and regulatory proteins are involved in the pathogenesis of the Sjögren’s syndrome and other autoimmune diseases. The complement activation via the alternative pathway was recognized as a major pathogenic mechanism in autoimmune conditions. The aim of this study was to assess expression of complement cascade components and regulatory proteins in minor salivary glands in patients with primary Sjögren’s syndrome (pSS).Materials and methodsThe expression of C1q and C5b-9 – membrane attack complex and regulatory proteins such as: membrane cofactor protein (MCP), decay-accelerating factor (DAF) and protectin were examined using immunochemistry method in specimens from biopsy of minor salivary glands in pSS patients. The biopsy material was obtained from 20 pSS patients, 5 patients with non-specific sialadenitis and from 5 patients with suspicion of dryness syndrome without sialadenitis confirmation.ResultsNone of the examined samples showed the expression of C1q or the effector C5b-9. Membrane cofactor protein expression was lower in pSS group than in both non-specific sialadenitis and noninflamed salivary glands. The inflammatory cells in pSS samples partially expressed MCP. There were differences in the sites and intensity of membrane protectin expression exclusively on the luminal surfaces in pSS; on the luminal and, partially, antiluminal surface in non-specific inflammation, and on the entire cell surface in unaffected salivary glands. There were no DAF expression in salivary gland tissue in biopsy specimens in all studied subjects.ConclusionsThe study demonstrated the absence of complement-cascade proteins (C1q, MAC) in the salivary glands of pSS patients, which may indicated a lack of local complement activation via the classical pathway and the observed gland tissue damage being due to a mechanism other than MAC-induced cytolysis. The differences in the expression of complement regulatory proteins between pSS, non-specific sialadenitis, and normal salivary glands may indicate that alternative functions of these regulatory proteins may be of greater significance in pSS. Low MCP expression in pSS in comparison with non-specific sialadenitis and normal salivary glands, may suggest altered modulation of cell-mediated immunity in pSS. The differences in the location and intensity of protectin (CD59) expression indicates a possibility of reducing the proinflammatory effect of protectin in pSS.

Thrombotic risk in paroxysmal nocturnal hemoglobinuria-like (PNH-like) phenotype.

The complement system is an essential component of the innate immune defence that, if overly activated, may damage organs and tissues. For this reason, there is a fine complement regulatory system. The complement modulation system includes two proteins with important regulatory activity, CD55 or decay accelerating factor (DAF) and CD59 or membrane inhibitor of reactive lysis (MIRL).The paroxysmal nocturnal hemoglobinuria (PNH) is a clonal and non-neoplastic disease characterized by intravascular haemolysis, occurrence of thrombosis and bone marrow failure.In clinical practice, in opposition to PNH, a variety of pathological conditions have been observed with an acquired and non-genetic deficiency of the regulatory proteins CD55 and CD59. This abnormal, non-clonal, reduced expression of complement regulatory proteins configures what we may define as PNH-like phenotype.Similarly to PNH, even in the PNH-like phenotype diseases there has been a greater exposure to the mediated complement cellular lysis and, a likely increased risk of thromboembolic events.Therefore, the knowledge of the potential roles of the complement system becomes necessary for a deeper understanding of several pathological conditions and for an improved clinical management of the patients.

Loss of complement regulatory proteins on red blood cells in mild malarial anaemia and in Plasmodium falciparum induced blood-stage infection

BackgroundAnaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection.MethodsThe expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated.ResultsCRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM.ConclusionsRemoval of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia.

Targeting CD37 in B-Cell Malignancies Using the Novel Therapeutic Duohexabody-CD37 Results in Efficient Killing of Tumor B-Cells Ex Vivo Via Complement-Dependent Cytotoxicity, Even in Relapsed and/or Refractory Patient Samples

CD37 is a tetraspanin molecule expressed on mature B-cells, but absent on normal stem cells and plasma cells. Due to this cellular distribution, which is highly similar to CD20, CD37 has gained attention as a target for B-cell lymphoma, especially for rituximab-resistant and relapsed patients.The CD37-targeting antibody-based products that are currently being evaluated in the clinic are generally poor inducers of complement-dependent cytotoxicity (CDC) - a rapid and powerful Fc-mediated effector function. DuoHexaBody-CD37 is a bispecific IgG1 antibody targeting two non-overlapping epitopes in the large extracellular loop of CD37, with a hexamerization-enhancing mutation (E430G) in the IgG Fc domain. The E430G mutation increases the intrinsic capacity of IgG1 molecules to form hexameric antibody clusters upon binding to membrane-bound antigens, thereby facilitating binding of the hexavalent complement component C1q and subsequent CDC activity (Diebolder et al., Science 2014; de Jong et al., PLoS Biology 2016). The combination of dual epitope targeting and enhanced hexamerization of DuoHexaBody-CD37 upon binding to the cell surface significantly increases complement activation and CDC efficacy in CD37-positive B-cells.To assess its clinical relevance and potential, we tested the capacity of DuoHexaBody-CD37 to induce ex vivo CDC activity using a broad range of B-cell lymphoma and leukemia patient samples (Diffuse Large B-Cell Lymphoma, Follicular Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Chronic Lymphocytic Leukemia, n=40) derived from both newly diagnosed (ND) (n=23) and relapsed and/or refractory (RR) patients (n=17), most of whom had received prior treatment with rituximab. The CDC activity of DuoHexaBody-CD37 was compared to the CDC activity of the CD20 antibodies rituximab, ofatumumab and obinutuzumab. Our results revealed that, independent of the B-cell malignancy subtype, DuoHexaBody-CD37 induced potent CDC activity with a median lysis of 82% (n=40, both RR and ND) at 10 µg/ml, and significantly outperformed all three tested CD20 antibodies (18%, 24% and 51% median lysis for rituximab, obinutuzumab and ofatumumab, respectively). Furthermore, CDC induced by DuoHexaBody-CD37 was shown to be less sensitive to the expression of complement regulatory proteins (CD46, CD55, CD59), compared to the tested CD20 antibodies, although the few patient samples that were resistant to DuoHexaBody-CD37 in the ex vivo CDC assays expressed significantly higher CD59 levels than the responsive samples.Importantly, while CDC induced by the tested CD20 antibodies was lower in samples derived from RR patients than from ND patients, DuoHexaBody-CD37 was equally effective in inducing CDC in samples derived from ND and RR patients that received prior treatment with rituximab. In a direct comparison to the strong CDC inducer ofatumumab, DuoHexaBody-CD37 induced significantly more potent CDC in samples from 17 RR CLL and B-cell lymphoma patients with a median lysis of 82% for DuoHexaBody-CD37 versus 29% for ofatumumab (p<0.001) (Fig. 1).In conclusion, our results indicate great therapeutic potential for DuoHexaBody-CD37 in B cell malignancies, based on its strong capacity to induce CDC, even in ex vivo samples from patients that had relapsed from or were refractory to rituximab-containing treatments.Figure1 Complement-dependent cytotoxicity induced by DuoHexaBody-CD37, rituximab, ofatumumab or obinutuzumab at 10 µg/mL in frozen CLL or lymphoma B-cells of relapsed and/ or refractory patients that received prior treatment with rituximab (n=17). Statistical analysis was performed using a non-parametric t-test (Wilcoxon). ***p < 0.001 and ****p < 0.0001. Data is shown as mean ± SD. [Display omitted] DisclosuresVan Der Horst:Genmab: Research Funding. Oostindie:Genmab: Employment, Equity Ownership. Overdijk:Genmab: Employment, Equity Ownership. Chamuleau:Gilead: Research Funding; Genmab: Research Funding; celgene: Research Funding; BMS: Research Funding. Breij:Genmab: Employment, Equity Ownership. Mutis:Celgene: Research Funding; Novartis: Research Funding; Gilead: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding.

Loss of complement regulatory proteins on uninfected erythrocytes in vivax and falciparum malaria anemia.

Anemia is a major complication of malaria, driven largely by loss of uninfected RBCs during infection. RBC clearance through loss of complement regulatory proteins (CRPs) is a significant contributor to anemia in Plasmodium falciparum infection, but its role in Plasmodium vivax infection is unknown. CRP loss increases RBC susceptibility to macrophage clearance, a process that is also regulated by CD47. We compared CRPs and CD47 expression on infected and uninfected RBCs in adult patients with vivax and falciparum malaria and different anemia severities from Papua, Indonesia. Complement activation and parasite-specific complement-fixing antibodies were measured by ELISA. Levels of CR1 and CD55 were reduced in severe anemia in both falciparum and vivax malaria. Loss of CRPs and CD47 was restricted to uninfected RBCs, with infected RBCs having higher expression. There was no association among complement-fixing antibodies, complement activation, and CRP loss. Our findings demonstrate that CRP loss is a pan-species, age-independent mechanism of malarial anemia. Higher levels of CRP and CD47 expression on infected RBCs suggest that parasites are protected from complement-mediated destruction and macrophage clearance. Lack of associations between protective antibodies and CRP loss highlight that complement pathogenic and protective pathways are distinct mechanisms during infection.

Activation of the complement system in an osteosarcoma cell line promotes angiogenesis through enhanced production of growth factors

There is increasing evidence that the complement system is activated in various cancer tissues. Besides being involved in innate immunity against pathogens, the complement system also participates in inflammation and the modulation of tumor microenvironment. Recent studies suggest that complement activation promotes tumor progression in various ways. Among some cancer cell lines, we found that human bone osteosarcoma epithelial cells (U2-OS) can activate the alternative pathway of the complement system by pooled normal human serum. Interestingly, U2-OS cells showed less expression of complement regulatory proteins, compared to other cancer cell lines. Furthermore, the activated complement system enhanced the production of growth factors, which promoted angiogenesis of human endothelial cells. Our results demonstrated a direct linkage between the complement system and angiogenesis using the in vitro model, which suggest the complement system and related mechanisms might be potential targets for cancer treatment.

The endometria of women with endometriosis exhibit dysfunctional expression of complement regulatory proteins during the mid secretory phase

The control of complement activation within embryo-endometrium environment is critical for embryo survival. Cell evasion from complement attack requires interaction of complement regulatory proteins (CRPs) with cell adhesion αvβ3 integrin. We aim to compare the expression of CRPs in endometria of women with and without endometriosis and to examine the molecular interaction of decay accelerating factor (DAF) with αvβ3 integrin. Endometrial expression of Membrane cofactor protein (CD46), Decay accelerating factor (DAF), Membrane attack complex inhibitory factor (CD59) and β3 integrin subunit were determined through menstrual cycle by immunohistochemistry. DAF protein quantity was determined by Western blot and mRNA levels measured in epithelial cells isolated by laser capture microdissection (LCM). Using in vitro assay, we examined DAF and β3 integrin expression through paracrine regulation between endometrial compartments. To determine whether β3 integrin and DAF interacts in vivo, endometrial samples were subjected to immunoprecipitation and colocalization using dual immunofluorescence technique. DAF and β3 integrin expression were significantly low in samples from women with endometriosis during mid secretory phase. This observation was supported by decreased DAF protein quantity; faint DAF and β3 integrin interaction and reduced mRNA levels in cells dissected by LCM. Moreover epithelial DAF and β3 integrin expression through paracrine regulation by progesterone from stromal compartment was disrupted in endometriosis. Endometria from women with endometriosis exhibits aberrant expression of complement proteins. The abnormal DAF expression potentially compromises embryo survival, contributing to understand the implantation failure in women with endometriosis.

Complement regulatory proteins in kidneys of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

The complement system activation is involved in the development of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). The study aimed to investigate the expression of complement regulatory proteins (CRPs) CD46, CD55 and CD59 in kidneys of 51 AVV patients. The expression of CD46, CD55 and CD59 in kidneys was detected by immunohistochemistry and double immunofluorescence staining. The immunohistochemical examination revealed that expression of the three CRPs could be detected in the glomeruli and tubules of both AAV patients and normal controls. The expression levels of the three CRPs in glomeruli of patients with AAV were significantly lower than those of normal controls. The scores of CD46 and CD55 expression in the tubules of AAV patients were significantly lower than those of normal controls, while there was no significant difference between the scores of CD59 expression in tubules of AAV patients and those of normal controls. Among AAV patients, the expression level of CD46 in glomeruli correlated inversely with the proportion of normal glomeruli, while it correlated with tubular atrophy in renal interstitium (r=-0·305, P=0·026; r=0·330, P=0·023, respectively). The expression levels of CD55 and CD59 in glomeruli correlated with the proportion of total crescents (r=0·384, P=0·006; r=0·351, P=0·011, respectively). Double immunofluorescence staining indicated that all three CRPs were expressed on endothelial cells, podocytes and mesangial cells in glomeruli. The expression levels of the three CRPs were dysregulated in kidneys of patients with AAV. The expression levels of CD46, CD55 and CD59 were associated with the severity of renal injury of AAV patients.

Abstract 4592: Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy

Abstract Background: Resistance to anti-cancer therapies results in relapsed/refractory disease of Glioblastoma (GBM) and Ewing’s Sarcoma. Up-regulation of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59 can enable solid tumors to confer resistance to antibody-mediated immunotherapy by preventing complement and antibody-dependent cytotoxicity. mCRPs’ inhibitory role in monoclonal antibody treatments for liquid tumors have been reported, but their role and regulation in solid tumors has not been explored. In the context of refractory tumors, others have reported that vascular endothelial growth factor-A (VEGF-A) can induce mCRP expression in endothelial cells. Notably, p53 mutational status induces VEGF-A and its receptor (VEGFR2) in breast cancer cell lines. To investigate potential links among p53 status, VEGF-A, and mCRP, we screened wildtype (wt-p53) and mutant p53 solid tumor cell lines for mCRP expression and VEGF-A secretion. Our data suggest that p53 mutational status is associated with expression of CD55 and VEGF-A secretion. These studies provide foundation for potentially recognizing mCRPs as immune biomarkers in solid tumors, ultimately, resulting in development of novel immunotherapies for improved clinical outcomes. Methods: Pediatric Ewing’s sarcoma (CHLA9 and CHLA10) and adult GBM (GBM10 and GBM43) cell lines differing in p53 status were selected for in vitro studies. GBM43 originates from a primary GBM, while GBM10 is from a recurrent GBM patient. Ewing’s Sarcoma cell lines, CHLA9 and CHLA10, were generated from the same patient at primary diagnosis and at relapse respectively. Western blot, and sequencing confirmed the expression and p53 mutational status. mCRP expression was evaluated using RT-PCR and flow cytometry. Milliplex platform assessed VEGF-A expression in cell supernatants. Results: Whole genome sequencing data confirmed p53 mutations in all cell lines. CHLA9 and GBM10 harbor wt-p53. CHLA10 cells have p53 deletion and GBM43 cells have a F270C p53 mutation in both alleles CD55 transcripts were undetected in wt-p53 lines (CHLA9 and GBM10), but CD55 transcripts were increased in mutant/deleted p53 lines (CHLA10 and GBM43). Flow cytometry data show increased CD55 expression in mutant p53 glioblastoma (GBM43) versus wt-p53 (GBM10) cells (p&amp;lt;0.001). Transcript and flow cytometric analysis of CD46 and CD59 in Ewing’s sarcoma and GBM cell lines are in progress. Mutant p53 Ewing’s sarcoma and GBM cell lines secreted more VEGF-A compared to wt-p53 cell lines (p&amp;lt;0.05 and p&amp;lt;0.001, respectively). Conclusion: These findings highlight the importance of further investigating role of VEGF-A in regulating mCRPs in wt-p53 versus mutant p53 solid tumor cell lines. Elucidating mechanisms for mCRP regulation is critical for immune biomarker development and in facilitating the use of antibody-based therapeutic approaches for solid tumors. Citation Format: Pankita Hemant Pandya, M. R. Saadatzadeh, Jixin Ding, Barbara Bailey, Sydney Ross, Khadijeh Bijangi-Vishehsaraei, Mary E. Murray, Karen E. Pollok, Jamie L. Renbarger. Complement regulatory protein expression in solid tumors: implications for resistance to antibody-mediated immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4592. doi:10.1158/1538-7445.AM2017-4592