Expression Of Complement Inhibitory Proteins Research Articles

Abstract P044: A genetically engineered mouse Sertoli cell line survives chronic humoral immune factors when grafted into diabetic mice

Abstract Patients with uncontrolled diabetes have an increased risk of developing cancer, therefore controlling their blood glucose and insulin levels is imperative. An effective treatment to accomplish this is transplantation of insulin-producing pancreatic islets. However, these patients must take chronic immunosuppressants to prevent graft rejection, which also increases cancer risk. An alternative to traditional transplantation is to transduce immunoregulatory cells such as Sertoli cells (SC), which survive as grafts without immune suppressants, to express insulin. We have previously transduced a mouse SC tumor line (MSC-1) to express human insulin (MSC1-HI), demonstrated that they survive (at 70-75%) when grafted into diabetic mice after 50 days, and confirmed their sustained expression of human insulin. Since complement is an important part of immune rejection, we hypothesize that one mechanism SC use to survive is inhibition of complement by expression of complement inhibitory proteins (CIP). We confirmed that primary mouse SC (pSC), MSC-1 cells, and MSC1-HI cells survive human complement activation in vitro while controls are killed, indicating that SC must be inhibiting complement, and microarray analyses of pSC and MSC-1 cells identified gene expression of 14 different CIP. We immunostained MSC-1 and MSC1-HI grafts to study deposition of C9, the lytic pore component of the complement membrane attack complex. In MSC1-HI grafts, C9 increased from about 10 positive cells/mm2 graft to roughly 40 positive cells/mm2 graft, and a dramatic increase in C9 deposition was observed on MSC-1 grafts—from about 4 positive cells/mm2 to well over 350 positive cells/mm2. Furthermore, we found that MSC1-HI grafts were significantly larger at day 50 than MSC-1 grafts. The MSC1-HI grafts also increased in size by almost 300% between days 20 and 50 post-transplantation as compared to MSC-1 grafts, which decreased in size by 64%. Thus, it appears that MSC-1 grafts are still rejecting while MSC1-HI grafts are growing, indicating some level of tumorigenesis. Indeed, insulin has been shown to be a growth factor for SC, hence we propose that increased expression of insulin in the MSC-1 cancer cell line is encouraging tumorigenesis in diabetic mice. Taken together, these data indicate that MSC1-HI cells undergo tumorigenesis and can survive and proliferate in spite of complement activation, which may be playing a role in MSC-1 graft reduction. Since complement can be a powerful anti-tumor and transplant rejection mechanism, understanding how these SC tumor cells evade complement killing has significance in both treating cancer and increasing grafted organ survival. As diabetes patients are more susceptible to tumors, further studying the relationship between insulin and tumorigenesis will increase knowledge to allow for more effective management of tumors. Citation Format: Rachel L. Washburn, Taylor Hibler, Jannette M. Dufour. A genetically engineered mouse Sertoli cell line survives chronic humoral immune factors when grafted into diabetic mice. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P044.

Relationship between the expression of complement inhibitory proteins and therapeutic efficacy of antibodies in breast cancer.

Complement regulatory proteins (mCRPs) CD55, CD46 and CD59 have been proposed as key elements in therapeutic resistance against cancer. mCRP-expressing tumor cells, in addition to hindering trastuzumab, pertuzumab and sacituzumab-govitecan therapeutic activity in breast cancer, can regulate biological processes that promote tumor progression. This review describes the structure of mCRPs and analyzes their expression using transcriptomic databases from breast cancer patients, in addition to collecting information on mCRPs interactions and signaling in tumor cells. Given that mCRPs are relevant targets, several strategies that have been explored for their inhibition and regulation in order to increase therapeutic efficacy and prevent cancer resistance and progression are described.

Seeking the Sertoli cell complement inhibitory signature to prolong transplant survival

Abstract Clinical transplantation can treat various illnesses, although without the use of toxic immunosuppressants transplanted tissue is rejected by the immune system. Complement, an enzymatic cascade that leads to cytolysis by insertion of the membrane attack complex (MAC), is a significant component in transplant rejection. Interestingly, Sertoli cells (SC) survive long-term after transplantation across immune barriers without immunosuppression. This study used allotransplant (between same species) and xenotransplant (between different species) models to investigate the role of complement inhibition in SC extended survival. Since SC were resistant to in vitro complement killing, mouse or pig SC were transplanted into mice or rats, respectively. Grafts were collected and analyzed for MAC. MAC was not detected on SC transplants, indicating that SC inhibit complement before MAC deposition, possibly through expression of complement inhibitory proteins (CIP). Bioinformatic analyses of mouse or pig SC identified expression of 13 CIP. After expression of two key CIP, CD46 and CD55, were knocked down, SC were killed by complement in vitro. Taken together, these data indicate that the SC complement inhibitory signature is critical for SC graft survival. Understanding SC complement inhibition is important as it could lead to improved transplant survival with a decreased requirement of harsh immunosuppressants, making this procedure more attainable for patients. Supported by grants from the CH foundation, the South Plains Foundation, and the Mary Lou Clements-Mann Scholarship

Determinants of response to daratumumab in Epstein-Barr virus-positive natural killer and T-cell lymphoma

BackgroundThe potential therapeutic efficacy of daratumumab in natural killer T-cell lymphoma (NKTL) was highlighted when its off-label usage produced sustained remission in a patient with highly refractory disease. This is...

Daratumumab Efficiently Targets NK/T Cell Lymphoma with High CD38 Expression

CD38 expression can be detected on a majority of NK/T cell lymphoma (NKTCL). Based on the clinical data of 94 patients with ENKTL, high CD38 surface expression was an adverse prognostic factor for progression free survival, and these patients had significantly inferior outcomes (Wang, 2015). This corroborates with our own studies showing that relapsed and refractory NKTCL patients almost always exhibit high CD38 expression, thereby proposing that it may be worthwhile to target CD38 as a novel therapeutic option.Daratumumab is a human anti-CD38 monoclonal antibody which has shown significant anti-cancer activity in multiple myeloma. In this study, we investigated the potential cytotoxic effects and mechanisms of actions of Daratumumab in a panel of 10 NKTCL cell lines. Firstly, we identified that cell lines can be stratified into CD38Hi, CD38Mid and CD38Lo as quantified by mRNA expression and CD38 protein surface expression which was qualitatively analysed by FACS and IHC. Daratumumab is able to induce antibody dependent cellular cytotoxicity (ADCC) in a CD38-dependent manner and in a dose-dependent fashion. We also observe a greater induction of antibody dependent cellular phagocytosis (ADCP) activation in correlation with CD38 expression. Furthermore, complement-dependent cytotoxicity (CDC) was significantly triggered by Daratumumab in CD38Hi and CD38Mid cells. However, we found evidence of an additional level of regulation provided by the expression of complement inhibitory proteins(CIPs) CD46, CD55 and CD59 on NKTCL cell lines which can limit and inhibit the induction of CDC regardless of CD38 surface expression levels.All-trans retinoic acid (ATRA) is a vitamin A derivative that binds to retinoic acid receptor alpha leading to the upregulation of CD38 expression. Treatment with ATRA increases CD38 mRNA and surface protein expression in all CD38Hi-Lo NKTCL cell lines. Interestingly CD38Lo cell lines showed the greatest fold increase of CD38 mRNA and surface protein expression. Notably, addition of ATRA to SNK-10, a CD38LoCD55Hi cell line, prior to Daratumumab treatment was able to improve the induction of ADCC but not CDC. This suggests a critical role for CIPS in mediating the induction of CDC in NKTCL.Subsequent investigation for the efficacy of Daratumumab in vivo is currently being performed.We also discovered a synergistic cytotoxic effect in NKTCL CD38Hi cell lines mediated by L-Asparaginase (a component of the current chemotherapeutic regimen in NKTCL) treatment in combination with Daratumumab. Altogether,these results propose a rationale therapeutic option for single or combined Daratumumab treatment in the clinic especially for relapsed and refractory patients. DisclosuresJin:Janssen China R&D: Employment. Yu:Janssen China R&D: Employment. Chen:Janssen China R&D: Employment. Yang:Janssen China R&D: Employment. Chng:Janssen China R&D: Research Funding.

Xenogeneic Sertoli cells express complement inhibitory proteins necessary for their survival of hyperacute rejection

Abstract Transplantation is used to treat organ failure associated with various diseases, however there is a severe shortage of organs acceptable for transplant. Utilization of pig organs offers an endless supply of transplantable tissue, but xenografts are rapidly destroyed by hyperacute rejection (HAR). The primary humoral mediator of HAR is the activation of complement (C″) by preformed-antibody binding to xenoantigens, causing a proteolytic cascade that results in cell lysis by membrane attack complex (MAC) insertion. Sertoli cells (SCs) are immunoregulatory testicular cells that survive as xenografts for an extended period without the use of immunosuppressive drugs. The goal of this study was to examine the mechanisms for SC survival of C″. Neonatal porcine SCs (NPSCs) transplanted into rats survived as xenografts while porcine islets (NPIs) did not. Immunohistochemistry of the surviving grafts show deposited C3, but not MAC, suggesting that NPSCs inhibit C″ after activation. An in vitro human serum (HS)-C″ cytotoxicity assay confirmed that NPSCs survive activated human C″. Analysis of NPSC expression of complement inhibitory proteins (CIP) revealed higher levels of membrane cofactor protein (MCP) and decay accelerating factor (DAF) as compared to NPIs, which are killed by C″. Knocking down expression of MCP and DAF using shRNA confirmed their importance in NPSC survival of C″. Less than 10% of these cells survived exposure to the HS-C″ cytotoxicity assay, indicating MCP and DAF are important in NPSC xenograft survival in vitro.

Microglial trogocytosis and the complement system regulate axonal pruning in vivo.

Partial phagocytosis-called trogocytosis-of axons by microglia has been documented in ex vivo preparations but has not been directly observed in vivo. The mechanisms that modulate microglial trogocytosis of axons and its function in neural circuit development remain poorly understood. Here, we directly observe axon trogocytosis by microglia in vivo in the developing Xenopus laevis retinotectal circuit. We show that microglia regulate pruning of retinal ganglion cell axons and are important for proper behavioral response to dark and bright looming stimuli. Using bioinformatics, we identify amphibian regulator of complement activation 3, a homolog of human CD46, as a neuronally expressed synapse-associated complement inhibitory molecule that inhibits trogocytosis and axonal pruning. Using a membrane-bound complement C3 fusion protein, we demonstrate that enhancing complement activity enhances axonal pruning. Our results support the model that microglia remodel axons via trogocytosis and that neurons can control this process through expression of complement inhibitory proteins.

CD55 and CD59 Can Limit the Anti-Tumor Efficacy of Daratumumab in Natural Killer/T-Cell Lymphoma

Complement-dependent cytotoxicity (CDC) is one of the major mechanisms mediating the anti-tumor efficacy of Daratumumab. We have previously demonstrated that a majority of Natural Killer/T- Cell Lymphoma (NKTL) patient samples express CD38 and Daratumumab is highly effective against NKTL cell lines expressing mid-high levels of CD38.In this report we show that subsequent testing in an NKTL mouse xenograft model confirms the potency of Daratumumab in vivo as evidenced by the inhibition in tumour progression and prolongation of mouse survival. When treatment was continued over a month, some tumors began to rapidly enlarge ('Resistant') while the rest remained similar or smaller ('Sensitive') than the tumour volume at the initiation of Daratumumab treatment. An mRNA analysis comparing 'Resistant' and ‘Sensitive’ tumors showed that while both tumours bore similar levels of CD38 expression, resistant tumours displayed an upregulation of complement inhibitory proteins (CIP), CD55 and CD59 but not CD46. This led us to hypothesize that CD59 and CD55 may play a critical role in Daratumumab-mediated CDC in NKTL.FACS analyses demonstrated that the number of membrane molecules of CD55 and CD59 appeared inversely correlated to Daratumumab-mediated CDC. A single CIP knockdown was first performed to delineate the role of each CIP. Silencing CD46 confirmed that it does not have any effect on CDC in NKTL. However, single knockdown of CD55 or CD59 was able to induce cytotoxicity in CDC-resistant cell line CD38midCD55hiCD59lo NKYS, and promote NKS1 CD38hiCD55hiCD59mid to further lysis. Both single and double knockdown of CD55 and CD59 could not enhance Daratumumab-induced CDC in CD38loCD55hiCD59hi HuT78 which recapitulates the importance of CD38 levels. Unexpectedly, the double knockdown did not sensitize CD38hiCD55hiCD59hi KMS12BM either.This led us to conjecture that it may be the ratio of CD38:CIPs which is predictive of response to Daratumumab than CD38 or CIPs alone. All-Trans Retinoic Acid (ATRA) binds the RARE element in CD38 gene leading to upregulation of mRNA and protein expression of CD38. We thus downregulated the expression of CIPs with siRNA followed by amplification of CD38 expression with ATRA in NKTL. This strategy resulted in a significant increase in the CD38:CIP ratio and induced almost a total lysis of NKS1 cells, as well as sensitised HuT78 to a massive amount of Daratumumab-mediated CDC. These experiments suggest that by increasing the CD38: CIP, ratio we can overcome resistance to Daratumumab-mediated CDC.To further statistically study this, a Spearman's rank correlation analyses was performed. The Spearman correlation coefficient shows that the number of surface molecules of CD38 positively correlates to CDC while that of CD55 displays an inverse correlation. CD46 and CD59 do not show any significant correlation. Notably, when correlating the CD38:CIP ratio instead to CDC, the CD38:CD46, CD38:CD55 and CD38:CD59 ratios always show a significant positive correlation coefficient. This suggests that the potential efficacy of Daratumumab can be predicted more accurately based on the ratio of CD38:CIP than any of the molecules alone. Detection of a low CD38:CIP ratio in patient samples could be a biomarker for potentially poorer response to Daratumumab treatment.Daratumumab-resistant NKTL cell lines are being developed in our lab and RNA sequencing comparing sensitive and resistance cells will be subsequently performed in order to gain further insights to mechanisms that may lead to resistance. Preliminary analyses on CD38 and CIP expression so far has shown that CD38 protein and mRNA expression are prominently downregulated in resistant cell lines while the level of CIPs remain similar or increased. The total outcomes of these studies will contribute valuable insights to clinical trials that currently involve Daratumumab treatment. DisclosuresZhou:Janssen R&D: Employment. Yang:Janssen R&D: Employment. Chng:Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding.

CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma

AbstractThe anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow–localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).

Ofatumumab Exhibits Enhanced In Vitro and In Vivo Activity Compared to Rituximab in Preclinical Models of Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell lymphoma considered to be incurable with current treatments, including first-line rituximab in combination with multiagent chemotherapy and for those eligible, high-dose chemotherapy and stem cell support or rituximab maintenance. On the other hand, achieving a complete remission by high-sensitive flow cytometry is associated with prolonged duration of remission, stressing the need to develop and/or incorporate novel agents into the management of MCL. To this end, we examined the activity of ofatumumab, an anti-CD20 monoclonal antibody with distinct binding and immunologic properties compared to rituximab, in MCL preclinical models. MCL cells were labeled with (51)Cr before incubation with rituximab or ofatumumab (10 μg/mL) plus human serum or effector cells. (51)Cr-release was measured and the percentage of lysis was calculated. Surface CD20, CD55, and CD59 were measured by Imagestream analysis. SCID mice inoculated subcutaneously with Z138 cells were assigned to control versus four doses of ofatumumab or rituximab (10 mg/kg/dose). Ofatumumab exhibited enhanced in vitro complement-dependent cytotoxicity activity compared with rituximab in MCL cell lines, despite a high degree of in vitro resistance to rituximab associated with low CD20 levels and/or high expression of complement inhibitory proteins. Ofatumumab also delayed tumor progression and prolonged survival in a murine model of MCL. Our results demonstrate that ofatumumab is more effective than rituximab in MCL preclinical models, including in the presence of rituximab resistance, and support the clinical investigation of ofatumumab in combination with standard systemic chemotherapy in MCL (NCT01527149).

Anti-Leukemic Activity of Daratumumab in Acute Myeloid Leukemia Cells and Patient-Derived Xenografts

Daratumumab is a human antibody that binds to CD38 on the cell surface and induces cell killing by multiple mechanisms including complement mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and apoptosis. In pre-clinical and clinical studies, daratumumab has been shown to effectively kill multiple myeloma (MM) cells and to enhance the potency of other treatments against MM. The purpose of the study was to investigate in vitro and in vivo efficacy of daratumumab against 9 acute myeloid leukemia (AML) cell lines and patient-derived samples.First, we evaluated the expression of CD38, complement inhibitory proteins (CIP) CD46, CD55, CD59, and FcgR1 (CD64) on AML cell lines (n=9), AML patient cells (n=10) and healthy donor bone marrow using flow cytometry. CD38 enumeration showed a substantial variation between cell lines (12,827±19, 320 molecules/cell) and between AML patients (11,560±8, 175 molecules/cell), while CD38 expression was more consistent in bone marrow (BM) from healthy donors (1,176±355 molecules/cell). The daratumumab-induced apoptosis observed in cell lines (MOLM-13, MOLM-16, MV-4-11, NB4) in vitro was not correlated with CD38 expression levels. Daratumumab induced minimal ADCC (5-20%) and low levels of (2-5%) CDC mediated cell killing in 6 AML cell lines tested. We did not observe a direct correlation between CD38 expression and ADCC, CDC, nor between CDC and CIP expression. Interestingly, treatment of two human Acute Promyelocytic Leukemia (M3) cell lines HL-60 and NB-4 with all-trans retinoic acid (ATRA) induced a 10-30-fold increase in CD38 expression, suggesting that ATRA could be used in combination with daratumumab.While we, and others, have shown that pre-incubation of primary AML cells with anti-CD38 antibodies inhibits engraftment in NSG mice, we aimed at evaluating the anti-leukemic activity of daratumumab in a therapeutic xenograft model using 3 different AML patients. NSG mice (10/group/patient) were transplanted with T cell-depleted AML cells and BM aspirates were collected 4-6 weeks later to assess leukemia burden in each mouse prior to treatment. Animals were untreated (Ctrl) or received daratumumab (10 mg/kg), or IgG1 isotype once a week for five weeks. We assessed AML burden (% huCD45+ CD33+) in BM, spleen (SPL) and peripheral blood (PB) within 5 days after the last treatment. First, we evaluated an AML (#3406, FLT3-ITD, see figure) with high expression of CD38 (13,445 molecules/cell) and low CD64 (489/cell) was evaluated. Daratumumab significantly reduced leukemia burden in SPL and PB, but had no effect in BM. The same daratumumab-induced reduction in peripheral blasts and lack of effect in BM was observed in 2 other AML patient xenografts (#7577, M1 IDH mutant/FLT3-ITD with 6,529 CD38 molecules/cell; #8096, M2 with 335 CD38 molecules/cell). Interestingly, we observed that daratumumab treatment led to a drastic reduction in CD38 surface expression in AML blasts including in BM, indicating that daratumumab efficiently targeted CD38 in bone marrow blasts. Our results suggest that the bone marrow microenvironment can impair the anti-leukemic activity of daratumumab observed in other tissues. Ongoing xenograft studies are testing whether induction with chemotherapy (Ara-C+doxorubicin), or with other agents disrupting the bone marrow microenvironment, can enhance the anti-leukemic activity of daratumumab. [Display omitted] DisclosuresDos Santos:Janssen R&D: Research Funding. Xiaochuan:Janssen R&D: Research Funding. Doshi:Janssen R&D: Employment. Sasser:Janssen R&D: Employment. Danet-Desnoyers:Janssen R&D: Research Funding.

Spontaneous abortion is associated with elevated systemic C5a and reduced mRNA of complement inhibitory proteins in placenta.

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto-maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion.

Crosstalk between TGF-β1 and complement activation augments epithelial injury in pulmonary fibrosis.

The epithelial complement inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated complement activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in complement activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of complement component 3a and 5a (C3a and C5a), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C5a (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C5a-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C5a down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C5a receptor (C3aR/C5aR) expression, pharmacologic C3aR/C5aR inhibition protected against C3a-/C5a-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to complement activation that down-regulates CIPs and induces TGF-β1 expression

Complement inhibitory proteins expression in placentas of thrombophilic women

Factors controlling complement activation appear to exert a protective effect on pregnancy. This is particularly important in women with thrombophilia. The aim of this study was to determine the transcript and protein levels of complement decay-accelerating factor (DAF) and membrane cofactor protein (MCP) in the placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western blot. We observed a higher transcript (p < 0.05) and protein (p < 0.001) levels of DAF and MCP in the placentas of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of proteins that control activation of complement control proteins is only seemingly contradictory to the changes observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.

Complement inhibitory proteins expression in placentas of thrombophilic women

Factors controlling complement activation appear to exert a protective effect on pregnancy. This is particularly important in women with thrombophilia. The aim of this study was to determine the transcript and protein levels of complement decay-accelerating factor (DAF) and membrane cofactor protein (MCP) in the placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women. Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia. The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western blot. We observed a higher transcript (p &lt; 0.05) and protein (p &lt; 0.001) levels of DAF and MCP in the placentas of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of proteins that control activation of complement control proteins is only seemingly contradictory to the changes observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.

Preclinical activity of ofatumumab (OFA) in mantle cell lymphoma (MCL) in vitro, ex vivo, and in vivo models.

e18537 Background: MCL is characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. OFA is a fully human anti-CD20 mAb targeting a novel membrane-proximal epitope on CD20. To characterize the activity of ofatumumab in MCL, we conducted pre-clinical studies in cell lines, primary tumor cells derived from MCL patients and a MCL bearing severe combined immunodeficiency (SCID) mouse model. Methods: Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in 51Cr labeled Mino, Jeko, Rec-1 and Z-138 cells comparing RTX or OFA. Primary tumor cells were exposed ex vivo to OFA or RTX with human serum, differences in cell viability were determined by Cell Titer Glo assay. Expression of CD20 and complement inhibitory proteins (CIPs) CD55 and CD59 was determined by Imagestream analysis and Western blot. SCID mice were inoculated SQ with 10x106 Z-138 cells. Once tumors were established, mice were assigned to observation versus 4 doses of either OFA or RTX, and anti-tumor activity was measured by changes in tumor volume. Results: OFA induced higher CDC than RTX in all MCL cell lines tested (Mino: 65.9% vs. 0.5% ; Jeko 43.9% vs. 13.3% ; Rec-1 25.4% vs. 4.7% ; Z-138: 56.4% vs. 0.65%). No differences in ADCC were noted between OFA and RTX. In primary tumor cells, OFA and RTX demonstrated similar activity. CD20 levels were similar in all MCL cell lines tested. Of interest, CIP expression in MCL cell lines was higher when compared to other NHL cell lines, explaining differences observed between OFA and RTX. In vivo OFA was more effective in slowing tumor progression than RTX. Conclusions: Our data suggest OFA is more potent than RTX against MCL pre-clinical models. In addition and as expected, OFA exhibits potent CDC despite high expression of CIP. Our results support the evaluation of ofatumumab in future prospective clinical trials for patients with MCL.

Activity of Rituximab and Ofatumumab Against Mantle Cell Lymphoma (MCL) in Vitro in MCL Cell Lines and Ex Vivo in Tumor Cells Derived From Patient Tumor Samples

Abstract 4972Mantle cell lymphoma (MCL) is an aggressive form of non-Hodgkin lymphoma (NHL) that frequently presents with advanced stage disease. The addition of rituximab, a monoclonal anti-CD20 antibody, to high dose chemotherapy regimens often followed by stem cell transplant has improved outcomes, but survival still remains low at 3–5 years. Novel agents are needed to improve outcomes in MCL. Ofatumumab is a fully human anti-CD20 monoclonal antibody directed against a novel epitope on the CD20 antigen. Ofatumumab has been shown to be more potent than rituximab against B-NHL cells in pre-clinical investigations. Ofatumumab is FDA approved for the treatment of CLL that is fludarabine and alemtuzumab refractory or with bulky disease resistant to fludarabine and is being investigated in clinical trials in NHL. In order to characterize the activity of ofatumumab against MCL, we performed pre-clinical investigations into the activity of ofatumumab against MCL cell lines and primary MCL tumor cells derived from patient tumor samples (n=2). Antibody-dependant cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in the MCL cell lines Mino, Jeko, Rec-1 and Z-138 to demonstrate sensitivity to rituximab and ofatumumab. Lymphoma cells were labeled with 51Cr prior to incubation with rituximab or ofatumumab at 10ug/mL plus human serum or effector cells (efector:target ratio of 20:1). 51Cr-release was measured and the percentage of lysis was calculated. Patient tumor cells were isolated from tumor biopsy samples by MACS sorting (negative selection). Patient tumor cells were incubated with ofatumumab or rituximab at 10ug/mL in the presence of human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Means were compared using a t-test. Expression of CD20 and the complement inhibitory proteins (CIPs) CD55 and CD59 in MCL cell lines were determined by flow cytometry and compared to the rituximab-sensitive cell line Raji and the rituximab-resistant cell line Raji 4RH. Surface density of CD20, CD55 and CD59 were determined by Imagestream analysis. Western blot was performed to measure total CD20 protein expression. Ofatumumab induced significantly higher levels of cell lysis compared to rituximab in CDC assays of all MCL cell lines tested (Mino: 65.9% vs 0.5%; JeKo 43.9% vs 13.3%; REC-1 25.4% vs 4.7%; Z-138: 56.4% vs 0.65%; all p-values <0.05). The ADCC assays showed a similar degree of lysis with ofatumumab when compared to rituximab in all cell lines tested. In primary tumor cells, ofatumumab and rituximab demonstrated similar levels of decreased cell viability following 48 hours of antibody exposure. MCL cell lines demonstrated similar expression of surface and total CD20 when compared to the rituximab-sensitive B-NHL Raji cell line. CIP expression was increased in all MCL cell lines compared to Raji cells and was similar to the rituximab-resistant Raji 4RH cell line. Our data suggest ofatumumab is more potent than rituximab against MCL cells in vitro and retains CDC activity despite high expression levels of CIPs. This increased activity was not seen in patient tumor samples; however we were limited by the number of available patient samples. In vivo experiments investigating the activity of ofatumumab in a SCID mouse MCL xenograft model and investigations into the activity of ofatumumab in MCL cells in combination with cytotoxic agents and novel small molecule inhibitors are ongoing. Disclosures:Czuczman:Genmab: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding. Hernandez-Ilizaliturri:Genmab: Research Funding.

Upregulated expression of complement inhibitory proteins on bladder cancer cells and anti‐MUC1 antibody immune selection

Membrane complement inhibitors (CD46, CD55 and CD59) are upregulated in some human cancers indicating that they play a role in immune evasion. We investigated complement inhibitor expression in bladder cancer and examined the hypothesis that selective pressure of an antibody response (anti-MUC1) results in the upregulated expression of complement inhibitors on tumor cells. Paired samples of tumor and normal tissue from 22 bladder cancer patients were analyzed for expression of MUC1, CD46, CD55 and CD59, and matched serum samples analyzed for anti-MUC1 IgM and IgG levels. Relationships between anti-MUC1 antibody levels and complement inhibitor expression were investigated. MUC1 mRNA was upregulated in 86% of tumor samples. CD46 was upregulated in 77%, CD55 in 55% and CD59 in 59% of tumors. Low titer anti-MUC1 IgM was detected in normal human sera, but was elevated in 41% of the bladder cancer patients. Anti-MUC1 IgG was virtually absent from normal sera, but present in 32% of the cancer patients. There was a direct relationship between anti-MUC1 antibody titer and expression level of complement inhibitors. Analysis of the correlation of each antibody with the expression of each complement inhibitor by Spearman's rank test revealed a strong correlation between both anti-MUC1 IgM and IgG levels and increased expression of CD46 and CD55, and combined anti-MUC1 IgM/IgG levels correlated with increased expression of all 3 complement inhibitors. In conclusion, the data demonstrate upregulated complement inhibitor expression and the presence of an anti-MUC1 antibody response in bladder cancer patients and support the hypothesis of antibody-mediated immune selection.

Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni

Schistosomes are believed to evade complement-mediated damage by expression of complement inhibitory proteins. Our previous results [Deng, J., Gold, D., LoVerde, P.T., Fishelson, Z., 2003. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect. Immun. 71, 6402–6410.] have demonstrated that paramyosin (Pmy) of the blood fluke S. mansoni binds to the human complement proteins C8 and C9, inhibits complement activation at the terminal stage and protects the parasite from complement-mediated damage. In order to locate the Pmy binding site to C8 and C9, various fragments of Pmy cDNA were PCR-cloned into a pET28a bacterial expression vector. Recombinant His-tagged Pmy fragments were expressed in BL21 Escherichia coli and purified over a nickel–nitrilotriacetic acid column. Binding assays by Western blotting with monoclonal anti-His antibody demonstrated that PmyCC (Pmy amino acids 744Asp– 866Met) was the only Pmy fragment that bound to human C8 and C9. Functional analyses demonstrated that PmyCC inhibited hemolysis of rabbit erythrocytes and of antibody-sensitized sheep erythrocytes by human complement. Importantly, PmyCC inhibited in vitro killing of trypsin-sensitized schistosomula of S. mansoni by human complement. In the presence of PmyCC, Zn 2+-induced C9 polymerization was inhibited. Most of the immunodominant B-cell antigenic epitopes of Pmy are present in the PmyCC region, as antibodies collected from mice immunized with recombinant Pmy bound primarily to PmyCC. Taken together, this study has mapped the complement regulatory domain in Pmy, capable of binding to C8 and C9 and preventing polyC9 formation, to its C-terminal region.

Cyclooxygenase-2 Induction and Prostacyclin Release by Protease-activated Receptors in Endothelial Cells Require Cooperation between Mitogen-activated Protein Kinase and NF-κB Pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.