Fc Chimeric Protein Research Articles

Abstract 7259: Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T

Abstract Introduction: In Japan since 2019, CAR-T cell therapy has been used to treat patients with relapsed or refractory B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma and has shown excellent clinical results. Clinical studies of CAR-T cell therapies targeting various antigens, such as HER2, have been conducted, but the therapeutic outcome in sarcoma is still limited. We have previously reported that HER2-targeted CAR-T cells show antitumor effects for synovial sarcoma in vitro. The purpose of this study is to compare HER2-targeted CAR-T cells and NKG2D-based CAR-T cells in antitumor effect and specific responses for synovial sarcoma in vitro. Materials and Methods: Surface expressions of HER2 and NKG2D ligands on three synovial sarcoma cell lines were detected by using recombinant human HER2 Fc chimeric protein and that of NKG2D, followed by staining with PE-conjugated anti-human IgG secondary antibody. We generated 4-1BB-type second generation CAR-T cells targeting NKG2D ligands and examined their antitumor effects and specific responses against synovial sarcoma cells. In addition, we compared the differences between HER2-targeted CAR-T cells and NKG2D-based CAR-T cells by co-culturing with synovial sarcoma cells using short-term assay (WST-8 assay), long-term assay (Real-time Cell Analysis) and CD107a assay. Results: HER2 and NKG2D ligands were found to be expressed by flow cytometry in three synovial sarcoma cell lines. In vitro, NKG2D-based CAR-T cells showed antitumor effects against synovial sarcoma cells, including degranulation and cytokine production. Furthermore, NKG2D-based CAR-T cells showed stronger antitumor effects than HER2-targeted CAR-T cells in both short- and long-term assays and expressed higher levels of CD107a. Conclusions: In this study, NKG2D-based CAR-T cells showed more antitumor effects than HER2-targeted CAR-T cells in vitro. Although the reason for this is not clear, we hypothesize that NKG2D ligands may be induced in synovial sarcoma cells by co-culture with CAR-T cells because HER2 is a cancer-specific antigen, whereas NKG2D ligands are ligands for activating receptors of natural killer cells. Further studies, including in vivo studies, will be conducted to verify the difference in anti-tumor effects between these CAR-T cells. Citation Format: Tomohiro Miyazaki, Chihaya Imai, Naoki Oike, Yudai Murayama, Takashi Ariizumi, Minori Baba, Yasushi Kasahara, Hiroyuki Kawashima. Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7259.

Critical Role of CD6highCD4+ T Cells in Driving Th1/Th17 Cell Immune Responses and Mucosal Inflammation in IBD.

CD6 is a crucial regulator of T cell activation and is implicated in the pathogenesis of multiple autoimmune diseases. ALCAM is the first identified endogenous ligand of CD6. We sought to investigate potential roles of CD6 in regulating intestinal mucosal inflammation in inflammatory bowel disease [IBD]. We analysed the expression of CD6 and ALCAM in the inflamed mucosa of IBD patients using qRT-PCR and immunohistochemistry. Phenotypic properties of CD6low/- and CD6highCD4+ T cells were determined by flow cytometry, qRT-PCR, and ELISA. ALCAM Fc chimeric protein was used to evaluate the role of CD6-ALCAM engagement in regulating IBD CD4+ T cell activation and differentiation. Expression of CD6 and its ligand ALCAM was markedly increased in the inflamed mucosa of IBD patients compared with that in normal controls, and was significantly correlated with disease activity indices of IBD patients. Interestingly, CD6highCD4+ T cells of IBD patients exhibited significantly higher pathogenicity compared with CD6low/-CD4+ T cells, characterized by enhanced T cell activation and preferential Th1 and Th17 cell phenotypes, but a markedly decreased proportion of nTreg [CD25highFoxp3+, CD25highCD127low] cells. Importantly, inclusion of ALCAM Fc chimeric protein significantly facilitated IBD CD4+ T cell, especially CD6highCD4+ T cell, differentiation into Th1/Th17 cells compared with hIgG1 Fc-treated controls. These data indicate that overexpression of CD6 and ALCAM in the inflamed mucosa of IBD patients accelerates intestinal mucosal immune responses via promoting CD4+ T cell proliferation and differentiation into Th1/Th17 cells. Thus, CD6 may serve as a novel therapeutic target for treatment of IBD.

Enhancement of death receptor 4-mediated apoptosis and cytotoxicity in renal cell carcinoma cells by anisomycin.

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies, and an effective therapy is lacking for metastatic RCC. Anisomycin is known to inhibit protein synthesis and induce ribotoxic stress. The aim of this study was to explore whether anisomycin enhances the cytotoxic effects of mapatumumab, a human agonistic monoclonal antibody specific for death receptor 4 (DR4), in human RCC cells. We examined the cytotoxicity of anisomycin alone and in combination with mapatumumab in human RCC cell lines and primary RCC cell cultures. RCC cells treated with anisomycin showed cytotoxicity in a dose-dependent manner. Anisomyin in combination with mapatumumab showed a synergistic effect not only in two human RCC cell lines but also in five primary RCC cell cultures. The synergy between anisomycin and mapatumumab for cytotoxicity was also observed for apoptosis. Interestingly, anisomycin significantly increased DR4 expression at both the mRNA and the protein level. Furthermore, the combination-induced cytotoxicity was significantly suppressed by a human recombinant DR4:Fc chimeric protein. The combination of anisomycin and mapatumumab also enhanced the activity of caspases 8 and 3, the downstream molecules of death receptors. These findings indicate that anisomycin sensitizes RCC cells to DR4-mediated apoptosis through the induction of DR4, suggesting that combinational treatment with anisomycin and mapatumumab might represent a novel therapeutic strategy for the treatment of RCC.

Soluble vimentin decreases neutrophil adhesion to platelets by inhibiting P‐selectin glycoprotein ligand‐1‐P‐selectin interactions

BackgroundVimentin is an intermediate filament protein mainly thought to be involved in maintaining the cytoskeleton. Recently, there has been increasing evidence of vimentin's role during inflammation, although its precise role is unclear. In a model of murine peritonitis, vimentin‐deficient mice have an increased ability to clear bacteria, suggesting that vimentin somehow inhibits inflammation. This effect may be through inhibiting the interaction between platelets and leukocytes (WBC), since platelets are known to play an important role in the capture and activation of WBC at sites of injury and infection. We and others have described the important role of P‐selectin glycoprotein ligand‐1 (PSGL‐1)‐P‐selectin (P‐sel) interactions on WBC capture by platelets and neutrophil (PMN) transmigration. We hypothesize that soluble vimentin (sVim) decreases PMN‐platelet interactions via interrupting PSGL‐1‐P‐selectin interactions.MethodsAll research was approved by the Institutional Review Board and Institutional Animal Care and Use Committee at Baylor College of Medicine. Recombinant sVim was expressed in E. coli and His‐purified using affinity chromatography. Blood was collected from healthy adult donors and mepacrine‐labelled whole blood or isolated PMN were perfused over fibrinogen, washed platelet monolayers, or P‐selectin/Fc chimeric protein at 2 dyn/cm2 in the presence of varying concentrations of sVim using a microfluidic parallel‐plate flow system (Bioflux). Finally, to determine the role of vimentin on WBC rolling in vivo, mean WBC rolling velocities in both wild type (C57Bl/6) and vimentin−/−mice were measured in cremaster muscle venules (46 ± 0.5 μm) using intravital video microscopy. In vitro data were analyzed as paired t‐test or repeated‐measured analysis of variance, where appropriate, whereas in vivo data were compared using t‐test.ResultsPerfusion of whole blood over fibrinogen resulted in platelet adherence to the fibrinogen coating. WBC adhesion to platelets was reduced in the presence of sVim (50 μg/mL; 29±15% of vehicle control; p<0.05, mean±SEM, n=3). To determine whether this effect was directly related to PMN‐platelet interactions, isolated PMN in the presence of varying concentrations of sVim was perfused over washed platelet monolayers. sVim decreased the number of rolling and adhered PMN on washed platelets in a dose‐dependent fashion (88±22, 35±6, 11±2, 4±2, & 0±0% of vehicle control values for 2.5, 5, 10, 20, & 40 μg/mL, respectively; p<0.05, n=5). To determine whether these observations were due to PSGL‐1‐P‐sel interactions, isolated PMN in the presence varying concentrations of sVim were perfused through P‐sel/Fc chimeric protein (10 μg/mL)‐coated channels. Similar to their effects on PMN‐platelet interactions, sVim significantly decreased PMN adhesion to P‐sel/Fc in a dose‐dependent manner (53±15, 21±3, 16±2, 10±2, 2±1% of vehicle control values for 2.5, 5, 10, 20, & 40 μg/mL, respectively; p<0.05, n=4). In vivo, mean WBC rolling velocities were lower in venules of mice lacking vimentin as compared to wild type mice (42.5±8.8 vs 122.5±23.3 μm/sec; p<0.05, mean±SEM, n=3–4).ConclusionSoluble vimentin decreases neutrophil adhesion to platelets by inhibiting PSGL‐1‐P‐selectin interactions. Further studies need to take place to determine how vimentin disrupts WBC capture and rolling and whether vimentin can be used to modulate inflammation caused by platelet‐leukocyte interactions.Support or Funding InformationNIH grants HL116524, GM0112806, & EY018239 and a Merit Review Grant from the Department of Veterans Affairs

Synergistic induction of apoptosis by mapatumumab and anthracyclines in human bladder cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells by engaging the death receptors 4 (DR4) and 5 (DR5). We investigated the effect of chemotherapeutic drugs on DR4-mediated apoptosis in human bladder cancer cells, using a human monoclonal agonistic antibody specific for DR4, mapatumumab. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. Treatment of human bladder cancer T24 cells with mapatumumab in combination with mitomycin C, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with mapatumumab in combination with epirubicin (EPI) had a synergistic cytotoxic effect. Synergy was also obtained in KU7 and RT112 human bladder cancer cells. A synergistic effect was also observed with mapatumumab in combination with pirarubicin. The synergy obtained in cytotoxicity with mapatumumab and EPI was also achieved in apoptosis. EPI markedly increased DR4 expression in the bladder cancer cells at both the mRNA and protein levels. Furthermore, the combination-induced cytotoxicity was significantly suppressed by the DR4:Fc chimeric protein. The combination of EPI and mapatumumab significantly activated the caspase cascade, including caspase-8, -9 and -3, which are the downstream molecules of death receptors. These findings indicate that EPI sensitizes bladder cancer cells to DR4-mediated apoptosis through induction of DR4 and activation of caspases, suggesting that the combination therapy of EPI and mapatumumab may be effective for bladder cancer therapy.

Endothelial Cell–Derived Chemerin Promotes Dendritic Cell Transmigration

ChemR23 is a chemotactic receptor expressed by APCs, such as dendritic cells, macrophages, and NK cells. Chemerin, the ChemR23 ligand, was detected by immunohistochemistry, to be associated with inflamed endothelial cells in autoimmune diseases, such as lupus erythematosus, psoriasis, and rheumatoid arthritis. This study reports that blood and lymphatic murine endothelial cells produce chemerin following retinoic acid stimulation. Conversely, proinflammatory cytokines, such as TNF-α, IFN-γ, and LPS, or calcitriol, are not effective. Retinoic acid-stimulated endothelial cells promoted dendritic cell adhesion under shear stress conditions and transmigration in a ChemR23-dependent manner. Activated endothelial cells upregulated the expression of the atypical chemotactic receptor CCRL2/ACKR5, a nonsignaling receptor able to bind and present chemerin to ChemR23(+) dendritic cells. Accordingly, activated endothelial cells expressed chemerin on the plasma membrane and promoted in a more efficient manner chemerin-dependent transmigration of dendritic cells. Finally, chemerin stimulation of myeloid dendritic cells induced the high-affinity binding of VCAM-1/CD106 Fc chimeric protein and promoted VCAM-1-dependent arrest to immobilized ligands under shear stress conditions. In conclusion, this study reports that retinoic acid-activated endothelial cells can promote myeloid and plasmacytoid dendritic cell transmigration across endothelial cell monolayers through the endogenous production of chemerin, the upregulation of CCRL2, and the activation of dendritic cell β1 integrin affinity.

E-Cadherin – Fc Chimeric Protein-Based Biomaterial: Breaking the Barriers in Stem Cell Technology and Regenerative Medicine

Chimeric proteins have been used for years for various purposes ranging from biomaterials to candidate drug molecules, and from bench to bulk. Regenerative medicine needs various kinds of proteins for providing essential factors for maintaining starting cells, like induced pluripotent stem cells (iPSC), and renewal, proliferation, targeted differentiation of these cells, and as extracellular matrix for the experimental cells. However, there are several challenges associated with making functional chimeric proteins for effective application as biomaterial in this field. Fc-chimeric protein technology could be an effective solution to overcome many of them. These tailored proteins are recently becoming superior choice of biomaterials in stem cell technology and regenerative medicine due to their specific advantageous biophysical and biochemical properties over other chimeric forms of same proteins. Recent advances in recombinant protein-related science and technology also expedited the popularity of this kind of engineered protein. Over the last decade our lab has been pioneering this field, and we and others have been successfully applied Fc-chimeric proteins to overcome many critical issues in stem cell technologies targeting regenerative medicine and tissue engineering. Fc-chimeric protein-based biomaterials, specifically, E-cad-Fc have been preferentially applied for coating of cell culture plates for establishing xenogeneic-agent free monolayer stem cell culture and their maintenance, enhanced directed differentiation of stem cells to specific lineages, and non-enzymatic on-site one-step purification of target cells. Here the technology, recent discoveries, and future direction related with the E-cad-Fc-chimeric protein in connection with regenerative medicine are described.

The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells

Emerging evidence has pointed to biological roles of melanoma-associated antigens (MAGEs) in cancer development, progression and resistance to treatment. However, the mechanisms involved remain to be fully elucidated. In this report, we show that one of the MAGE proteins, MAGE-D2, suppresses the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 2 (TRAIL-R2) and plays an important role in protecting melanoma cells from apoptosis induced by TRAIL. MAGE-D2 was commonly expressed at increased levels in melanoma cells compared with melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis. This was associated with enhanced formation of TRAIL death-inducing signaling complex and up-regulation of TRAIL-R2, and was blocked by a recombinant TRAIL-R2/Fc chimeric protein or siRNA knockdown of TRAIL-R2. Regulation of TRAIL-R2 by MAGE-D2 appeared to be mediated by p53, in that knockdown MAGE-D2 did not up-regulate TRAIL-R2 in p53-null or mutant p53 melanoma cells. In addition, inhibition of MAGE-D2 did not result in up-regulation of TRAIL-R2 in wild-type p53 cell lines with p53 inhibited by short hairpin RNA. Indeed, knockdown of MAGE-D2 led to up-regulation of p53 due to a transcriptional increase. The regulatory effect of MAGE-D2 on TRAIL-R2 expression and TRAIL-induced apoptosis was recapitulated in studies on fresh melanoma isolates. Taken together, these results identify the expression of MAGE-D2 as an important mechanism that inhibit TRAIL-induced apoptosis and suggest that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma.

Delineation of apoptotic genes for synergistic apoptosis of lexatumumab and anthracyclines in human renal cell carcinoma cells by polymerase chain reaction array

Lexatumumab, a human agonistic monoclonal antibody against tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor-2 (TRAIL-R2), is a promising molecular-targeted therapeutic agent. Our past study indicated that low concentrations of doxorubicin sensitized renal cell carcinoma (RCC) cells to lexatumumab-mediated apoptosis. The present study was designed to examine the cellular and molecular effects of lexatumumab and anthracyclines in RCC cells. The treatment of human RCC cells with lexatumumab in combination with anthracyclines, epirubicin, and pirarubicin had a synergistic cytotoxicity. A marked synergistic apoptosis was induced by lexatumumab in combination with epirubicin or pirarubicin. Epirubicin and pirarubicin significantly increased the TRAIL-R2 expression at both the mRNA and the protein levels. The combination-induced cytotoxicity was significantly suppressed by the human recombinant DR5:Fc chimeric protein. To further explore the molecular mechanisms in this synergistic cytotoxicity with lexatumumab and anthracyclines, the changes in 84 apoptosis-related genes were evaluated by a quantitative polymerase chain reaction (PCR) array. Among these genes, 18 (CD40LG, FASLG, LTA, TNSF7, FAS, BAG3, BAK1, BAX, BID, BIK, BCL10, caspase-1, caspase-5, caspase-6, caspase-10, TNF receptor-associated factor 1, PYCARD, and CIDEA) were significantly upregulated and eight (TNF receptor-associated factor 4, TNFRSF11B, TNF, BCL2, BCL2L1, BNIP3L, caspase-9, and DAPK1) were downregulated at mRNA levels in RCC cells cotreated with lexatumumab and epirubicin. Furthermore, the upregulation of mRNA levels of PYCARD and CIDEA was confirmed using real-time reverse transcriptase-PCR analysis. The present study demonstrates that anthracylines sensitize RCC cells to lexatumumab-mediated apoptosis by inducing TRAIL-R2 expression, and the utility of PCR array to elucidate the mechanism of synergistic apoptosis.

Feto-maternal immunologic interactions mediated by inducible costimulator (ICOS) signaling

OBJECTIVE: ICOS is a costimulatory receptor expressed on effector/memory T cells. The ICOS pathway is a potent regulator of immune cell cytokine production and functional differentiation. Although expression of a specific ICOS ligand, B7-H2, has been reported in extravillous trophoblast cells, the significance of ICOS:B7-H2 interactions during pregnancy are not well-described. The present study aimed to determine the expression pattern and function of ICOS in decidual T cells. DESIGN: In vitro study of ICOS expression and function using human peripheral and decidual T cells. MATERIALS AND METHODS: T cells were isolated using immunobeads from enzymatically- digested term decidual (d) tissues from uncomplicated cesarean sections. Peripheral (p) T cells were collected via venipuncture from healthy donors. ICOS expression on pT and dT cell subsets was examined using flow cytometry. T cells activated with an anti-CD3 mAb were then stimulated with a B7-H2/Fc chimeric protein and cytokine secretion was measured using ELISA. Co-culture of a trophoblast cell line (JEG3) and peripheral or decidual T cells was conducted to examine intercellular communications via ICOS:B7H2. RESULTS: Surface expression of ICOS was intense on dT cells but weak on pT cells. Subset evaluation revealed higher expression on CD45RO+ effector/memory cells when compared to CD45RA+ naïve cells. Among CD4+ T cells, strong expression was detected on Foxp3high regulatory T cells. ICOS ligation prompted T cells to secrete IFN-γ and IL-10 but not IL-2. In co-culture assays, antiCD3-activated T cells increased their production of IFN-γ and IL-10 in response to B7-H2 on JEG3 cells; this effect was abrogated in the presence of aB7-H2 neutralizing antibody. CONCLUSION: Our findings suggest that the ICOS pathway might play an important role in the regulation of cytokine production by dT cells. This may help to regulate immune reactivity and host defense against pathogens at the fetomaternal interface.

Cutting Edge: FcR-Like 6 Is an MHC Class II Receptor

Receptors for the Fc portion of Ig have been extensively characterized and are known to regulate humoral responses, but members of the closely related FcR-like (FCRL) family have not been found to bind Ig, and to date, no ligand has been identified for any FCRL. Using a cell-based GFP reporter system and a recombinant Fc chimeric protein, we show that human FCRL6, a receptor selectively expressed by cytotoxic T and NK cells, directly binds HLA-DR, an MHC class II molecule. Given the similarity among constant regions of Ig and MHC molecules, these findings suggest that representatives of the FcR and FCRL multigene families may have independently evolved to engage two ancestral elements fundamental to adaptive immunity. This discovery may offer new insight into the interaction between cytotoxic lymphocytes and APCs and may have important implications for better understanding HLA disease susceptibility and pathogenesis.

Von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina.

In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. In this study, we seek other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor alphas (HIF-alphas) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, we generated retina-specific conditional-knockout mice for VHL (Vhl(alpha)(-CreKO) mice). These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1alpha gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina.

Abstract 2424: Synergistic induction of apoptosis in human solid cancer cells by Lexatumumab and cisplatin in a caspase-dependent manner

Abstract Introduction and Objectives: Mapatumumab and Lexatumumab are human agonistic antibodies against TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 (TRAIL-R1) and TRAIL-R2, respectively, which induce apoptosis specifically in cancer cells. Here, we investigated whether chemotherapeutic agents enhance Mapatumumab- or Lexatumumab-mediated apoptosis against various solid cancer cells. Methods: Cytotoxicity and synergy was assessed by MTT and isobolograpic analyses, respectively. TRAIL-R2 expression was detected by real-time RT-PCR, flow cytometry, and Western blotting. Apoptosis was assessed by DNA Ladder and caspase assays. Results: Treatment of ACHN human renal cell carcinoma (RCC) cells with cisplatin combined with Mapatumumab did not overcome resistance to these agents. However, treatment with cisplatin in combination with Lexatumumab had a synergistic cytotoxicity. Synergy was also achieved in six primary RCC cell cultures. Lexatumumab and cisplatin also synergistically enhanced apoptosis. Pretreatment with cisplatin followed by Lexatumumab resulted in high cytotoxicity compare with the reverse sequence. Cisplatin significantly increased TRAIL-R2 expression at both mRNA and protein levels. Furthermore, the combination of Lexatumumab and cisplatin significantly enhanced caspase-8 activity, Bid cleavage, up-regulation of Bax, cytochrome c release, and caspase-9, caspase-6, and caspase-3 activities. Importantly, the activation of capase-8 was significantly abrogated by the specific inhibitors of caspase-9, caspase-6, and caspase-3. Furthermore, combination-induced cytotoxicity was significantly suppressed by DR5: Fc chimeric protein and the specific inhibitors of caspase-8, caspase-9, caspase-6, and caspase-3. Similar effect was observed in prostate cancer, bladder cancer, lung cancer, and cervical cancer cells. Conclusions: Cisplatin sensitizes solid cancer cells to Lexatumumab-induced apoptosis by potentiation of the extrinsic and intrinsic apoptotic pathways that lead to enhancement of caspase activation, particularly caspase-8. These results suggest that combination of cisplatin plus Lexatumumab is promising against solid cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2424.

Enhancement of Lexatumumab-Induced Apoptosis in Human Solid Cancer Cells by Cisplatin in Caspase-Dependent Manner

This study was designed to evaluate the apoptotic effect of mapatumumab or lexatumumab, human agonistic antibodies that target the tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2), in combination with chemotherapeutic agents, against human solid cancer cells. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. Treatment of ACHN human renal cell carcinoma cells with cisplatin combined with mapatumumab did not overcome resistance to these agents. However, treatment with cisplatin in combination with lexatumumab had a synergistic cytotoxicity. Synergy was also achieved in six primary renal cell carcinoma cell cultures. Lexatumumab and cisplatin also synergistically enhanced apoptosis. Pretreatment with cisplatin followed by lexatumumab resulted in high cytotoxicity compared with the reverse sequence. Cisplatin significantly increased TRAIL-R2 expression at both the mRNA and the protein levels. Furthermore, the combination of lexatumumab and cisplatin significantly enhanced caspase-8 activity, Bid cleavage, up-regulation of Bax, cytochrome c release, and caspase-9, caspase-6, and caspase-3 activities. Importantly, the activation of caspase-8 was significantly abrogated by the specific inhibitors of caspase-9, caspase-6, and caspase-3. Furthermore, combination-induced cytotoxicity was significantly suppressed by the DR5:Fc chimeric protein and the specific inhibitors of caspase-8, caspase-9, caspase-6, and caspase-3. A similar effect was observed in prostate cancer, bladder cancer, lung cancer, and cervical cancer cells. Cisplatin sensitizes solid cancer cells to lexatumumab-induced apoptosis by potentiation of the extrinsic and intrinsic apoptotic pathways that lead to amplification of caspase activation, particularly caspase-8, suggesting the combination treatment of solid cancers with cisplatin and lexatumumab might overcome their resistance.

Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.

Bacterial Fimbriae Stimulate Proinflammatory Activation in the Endothelium through Distinct TLRs

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.

Low concentrations of doxorubicin sensitizes human solid cancer cells to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐receptor (R) 2‐mediated apoptosis by inducing TRAIL‐R2 expression

There is accumulating evidence suggesting that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2 is a promising molecular target for cancer therapy. Therefore, we investigated the effect of chemotherapeutic agents on TRAIL-R2-mediated apoptosis and cytotoxicity in various human solid cancer cells. Treatment of the ACHN human renal cell carcinoma (RCC) cell line with agonistic TRAIL-R2 antibody (lexatumumab) in combination with 5-fluorouracil, vinblastine, paclitaxel, or docetaxel did not overcome resistance to these agents. However, treatment with lexatumumab in combination with doxorubicin had a synergistic cytotoxicity. Synergy was also achieved in two other human RCC cell lines, Caki-1 and Caki-2, and in eight primary RCC cell cultures. Sequential treatment with doxorubicin followed by lexatumumab induced significantly more cytotoxicity than reverse treatment or simultaneous treatment. Low concentrations of doxorubicin (0.1 and 1 microg/mL) significantly increased TRAIL-R2 expression at both the mRNA and protein levels. Furthermore, the combination of doxorubicin and lexatumumab significantly enhanced caspase 8 activity, Bid cleavage, Bcl-xL decrease, release of cytochrome c, and caspase 9 and caspase 3 activity, and induced synergistic apoptosis. The activation of caspases and apoptosis induced with lexatumumab and doxorubicin was blocked by the human recombinant DR5:Fc chimeric protein. In addition, synergistic cytotoxicity was also observed in human prostate, bladder, and lung cancer cells, but was inhibited by the DR5:Fc chimeric protein. These findings suggest that doxorubicin sensitizes solid cancer cells to TRAIL-R2-mediated apoptosis by inducing TRAIL-R2 expression, and that the combination treatment with lexatumumab and doxorubicin might be a promising targeted therapy for cancers, including RCC, prostate, bladder, and lung cancers.

Specific and high-affinity binding of tetramerized PD-L1 extracellular domain to PD-1-expressing cells: possible application to enhance T cell function

The negative co-stimulatory receptor, programmed cell death 1 (PD-1), is induced on activated T cells and delivers inhibitory signals upon engagement with its ligands PD-L1 and PD-L2, which are expressed on various somatic cells and certain cancers. Accumulating evidence suggests that interfering with the PD-1-PD-L1 interaction may result in the restoration of defective T cell functions in cancer and chronic viral infection. Herein, we established procedures to produce large amounts of renatured recombinant extracellular domain proteins of mouse PD-1 (mPD-1) and PD-L1. While monomeric mPD-1 and mouse PD-L1 (mPD-L1) only marginally interacted with the cells expressing their counterpart proteins, their tetramerization markedly enhanced the affinity with the K(d) of mPD-L1 tetramer being nearly 100-fold lower than that of the corresponding monomer. The affinity of mPD-L1 tetramer was even higher than a high-affinity anti-PD-1 mAb, and it efficiently inhibited the binding of mPD-L1/Fc-chimeric protein to mPD-1(+) cells. Functionally, mPD-L1 tetramer significantly enhanced the proliferative responses as well as the cytotoxic activity of T cells against specific target cells in vitro. The results suggest that oligomeric PD-L1 extracellular domains may provide a potential means to restore T cell functions in cancer and viral infection in humans.

Tunicamycin Sensitizes Human Melanoma Cells to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis by Up-regulation of TRAIL-R2 via the Unfolded Protein Response

We have reported previously low expression of death receptors for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in fresh isolates and tissue sections of melanoma. This seemed to correlate with relative resistance of freshly isolated melanoma cells to TRAIL-induced apoptosis. We show in this study that the endoplasmic reticulum (ER) stress inducer, tunicamycin, selectively up-regulated the cell surface expression of TRAIL-R2, but not other members of the TNF receptor family, and enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. Tunicamycin-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was associated with increased activation of the caspase cascade and reduction in mitochondrial membrane potential and was inhibited by a recombinant TRAIL-R2/Fc chimeric protein. Up-regulation of TRAIL-R2 on the melanoma cell surface was associated with increased transcription of TRAIL-R2 and its total protein levels. Two signaling pathways of the ER stress-induced unfolded protein response mediated by inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha) and activation of transcription factor 6 (ATF6), respectively, seemed to be involved. In one melanoma line, there was clear evidence of activation of the IRE1alpha pathway, and small interfering RNA (siRNA) knockdown of IRE1alpha substantially reduced the up-regulation of TRAIL-R2. Similarly, there was evidence for the activation of the ATF6 pathway, and siRNA knockdown of ATF6 had a delayed effect on TRAIL-R2 expression in one but not another melanoma cell line. Moreover, the transcription factor CCAAT/enhancer-binding protein homologous protein seemed to be involved in the up-regulation of TRAIL-R2 by tunicamycin, but its role varied between different melanoma lines. Taken together, our results suggest that agents that induce ER stress may enhance TRAIL-R2 expression and increase the therapeutic response to TRAIL in melanoma.

Establishment of a Functionally Active Collagen-Binding Vascular Endothelial Growth Factor Fusion Protein In Situ

Tissue regeneration requires both growth factor and extracellular matrix such as collagen, serving as a scaffold for cell growth. We established FNCBD-VEGF121, consisting of the fibronectin collagen-binding domain (FNCBD) and vascular endothelial growth factor (VEGF) 121, and investigated its properties. FNCBD-VEGF121 specifically bound to gelatin and type I, II, III, IV, and V collagen. This collagen-bound FNCBD-VEGF121 captured soluble VEGF receptor 2 (VEGFR-2)/Fc chimeric protein. Cell growth-promoting activity of FNCBD-VEGF121 was almost identical to that of VEGF121. The VEGF fusion protein significantly enhanced the expression of VEGFR-2 (71.6+/-0.8%) on endothelial progenitor cells (EPCs) derived from umbilical cord blood. Expectably, the collagen-bound VEGF fusion protein not only promoted the growth of endothelial cells (ECs) but also induced the expression of VEGFR-2 (63.7+/-0.8%) on non-adherent cells expanded from bone marrow CD34+ cells. Moreover, the VEGF fusion protein enhanced sprout formation of ECs in a matrigel model. In vivo experiments revealed that FNCBD-VEGF121 had local effects but not systemic effect on EPC mobilization. These results suggest that FNCBD-VEGF121 stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates both ECs and EPCs in situ, supplying a vascular regeneration niche.