CEACAM1 Expression Research Articles

DOP06 Non-invasive identification of adherent-invasive E. coli in patients with Crohn’s disease

Abstract Background Medications limiting the adhesion of ‘adherent and invasive E. coli’ (AIEC) represent potential strategies to treat Crohn’s disease (CD). However, the ileal AIEC identification is a time-consuming procedure, and the number of AIEC strains which colonise ileal CD mucosa remains unknown. There is an unmet need for non-invasive biomarkers to identify patients colonised by AIEC. We aimed to evaluate non-invasive biomarker of ileal AIEC colonisation in patients with CD. Methods This prospective and multi-centre study included CD patients requiring ileocoloscopy. Saliva, serum, stools and ileal biopsies were collected. Abundance and global invasive ability of ileal or faecal E. coli were performed. Isolated E. coli were characterised as AIEC or non-AIEC on I407 epithelial cells and THP1 macrophages. The ERIC-PCR profiles of ileal E. coli were performed. Ileal E. coli/CEACAM6 interaction was analysed by a yeast aggregation test and T84 assays (CEACAM6 protein expression, adhesion inhibition test with D-mannose). Quantification of serum anti-E. coli and ileal or salivary CEACAM6 was realised by ELISA. Results Overall, 102 CD patients were enrolled in this study and 25.8% of them exhibited ileal AIEC colonisation (AIEC+). The abundance and global invasive ability of ileal mucosa-associated E. coli were higher in AIEC+ CD patients compared with CD patients without AIEC (AIEC−) (p = 0.0065 and p = 0.0007, respectively). There was no difference between faecal abundance and invasive ability of E. coli between AIEC+ and AIEC− patients. The ERIC-PCR profiles of ileal E. coli showed that CD AIEC+ were for 78% of them colonised by not more than 2 clonal AIEC strains. In addition, AIEC were able to interact with CEACAM6 by binding D-mannose residues and to induce CEACAM6 expression in T84 cells (p = 0.0009 and p = 0.0185, vs. non-AIEC; respectively). This was also supported by adhesion inhibition test. Serum anti-E. coli level was higher for CD AIEC+ (vs. CD AIEC-). Ileal CEACAM6 level were positively correlated with abundance of ileal associated E. coli in AIEC+ patients (r = 0.4000; p = 0.0362) and with salivary CEACAM6 level (r = 0.4690; p < 0.0001). The non-invasive biomarker ‘serum anti-E.coli/salivary CEACAM6’ index was higher for CD AIEC+ (p = 0.0174; vs. CD AIEC-). A cut-off value < 1.34 × 10−6 eliminated the presence of ileal AIEC with a high negative predictive value (90% CI95% [69%–95%]). Conclusion Our study reported that identification of faecal AIEC cannot replace identification of AIEC from ileal biopsies, most of AIEC infection are mono or bi-clonal (≤ 2 strains) and that non-invasive biomarker such as ‘serum anti-E.coli/salivary CEACAM6’ index could be helpful to screen CD patients for AIEC infection.

Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6−/− cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

Unaltered Fungal Burden and Lethality in Human CEACAM1-Transgenic Mice During Candida albicans Dissemination and Systemic Infection.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is a receptor for Candida albicans. It is crucial for the immune response of intestinal epithelial cells to this opportunistic pathogen. Moreover, CEACAM1 is of importance for the mucosal colonization by different bacterial pathogens. We therefore studied the influence of the human CEACAM1 receptor in human CEACAM1-transgenic mice on the C. albicans colonization and infection utilizing a colonization/dissemination and a systemic infection mouse model. Our results showed no alterations in the host response between the transgenic mice and the wild-type littermates to the C. albicans infections. Both mouse strains showed comparable C. albicans colonization and mycobiota, similar fungal burdens in various organs, and a similar survival in the systemic infection model. Interestingly, some of the mice treated with anti-bacterial antibiotics (to prepare them for C. albicans colonization via oral infection) also showed a strong reduction in endogenous fungi instead of the normally observed increase in fungal numbers. This was independent of the expression of human CEACAM1. In the systemic infection model, the human CEACAM1 expression was differentially regulated in the kidneys and livers of Candida-infected transgenic mice. Notably, in the kidneys, a total loss of the largest human CEACAM1 isoform was observed. However, the overwhelming immune response induced in the systemic infection model likely covered any CEACAM1-specific effects in the transgenic animals. In vitro studies using bone marrow-derived neutrophils from both mouse strains also revealed no differences in their reaction to C. albicans. In conclusion, in contrast to bacterial pathogens interacting with CEACAM1 on different mucosal surfaces, the human CEACAM1-transgenic mice did not reveal a role of human CEACAM1 in the in vivo candidiasis models used here. Further studies and different approaches will be needed to reveal a putative role of CEACAM1 in the host response to C. albicans.

1199P - MG1124, a novel CEACAM1-targeted monoclonal antibody, has therapeutic potential as a combination partner of PD-1 inhibitors in NSCLC patients

BackgroundCEACAM1 is a member of the CEA family expressed in cell membranes of T cells and NK cells as well as tumor cells. CEACAM1 overexpressed in tumor cells interacts with CEACAM1 expressed in tumor infiltrating lymphocytes (TIL) and suppresses TIL activity by sending an inhibitory signal to immune cells, which is an immune escape mechanism in cancer. Therefore, we developed MG1124, a monoclonal antibody that specifically binds to CEACAM1 but not to other CEA family members, thereby blocking tumor growth by triggering immune response. MethodsCEACAM1 specific binding of MG1124 was tested in CEACAM1 or other CEA family over-expressing cells. MG1124 was examined by in vitro tumor cell lysis assays using NK92, a human NK cell line. The anti-tumor efficacy of MG1124 alone or in combination with a PD-1 inhibitor was studied in a humanized mouse model engrafted with non-small cell lung cancer (NSCLC) patient-derived tumor xenografts (NSCLC hu-PDX models). ResultsAn anti-CEACAM1 antibody, MG1124 binds to CEACAM1 overexpressing Jurkat cells but not to other CEA family members and blocks ligands interaction in a dose-dependent manner. MG1124 increased NK92-mediated tumor cell lysis against cancer cells with strong CEACAM1 expression, but not to those with no or weak CEACAM1 expression. In a single mouse trial, MG1124 (tumor growth inhibition (TGI) 55%) was efficacious in the high CEACAM1 expressing NSCLC hu-PDX model as monotherapy. In addition, the combination therapy (TGI 42%) with a PD-1 inhibitor displayed enhanced antitumor activity than the anti-CEACAM1 monotherapy (TGI 17%) in a CEACAM1-low expression model as well as in a CEACAM1-high expression model (combination therapy TGI 63% vs monotherapy TGI 46%). Moreover, tumor tissues (n=150) of NSCLC patients showed extensive expression of CEACAM1, suggesting a potential therapeutic use of MG1124 in NSCLC patients. ConclusionsIn this study, the anti-CEACAM1 antibody MG1124 showed antitumor efficacy in human CEACAM1-expressing cancer cell lines and NSCLC hu-PDX xenograft models, and demonstrated potential as a targeted therapy for NSCLC, both as a single agent and in combination with a PD-1 inhibitor. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

P2.13-04 Molecular Nodal Restaging Based on Embryonic Markers Expression Adds No Relevant Clinical Information in Resected Lung Cancer

The relapse rate in non-small cell lung cancer (NSCLC) is high, even in localised disease, suggesting that the current approach to pathological staging is insufficiently sensitive to detect occult micrometastases present in resected lymph nodes. Therefore, we aimed to determine the prognostic value of the expression of embryonic molecular markers in histologically-negative lymph nodes of completely-resected NSCLC. 76 NSCLC patients undergoing radical resection were included. Primary tumours and 347 lymph nodes were studied. The molecular markers finally were selected based on testing of 27 normal lung and 129 lung tumour samples as well as 25 lymph nodes obtained from non-neoplastic diseases. CEACAM5, FGFR2b, and PTPN11 expression levels were evaluated through mRNA analysis using real-time RT-qPCR assay. Statistical analyses included the Kruskal-Wallis test, Kaplan Meier curves, and log-rank tests. CEACAM5 expression levels were scored as high in 90 lymph nodes (26%). The molecular-positive lymph nodes lead to the restaging of 37 (62%) pN0 patients as molecular N1 or N2 and 5 (31%) pN1 cases were reclassified as molecular-positive N2. Surprisingly, molecular-positive patients (42, 55%) associated with a better OS (overall survival, p=0,04) than molecular-negative patients (34, 45%). FGFR2b overexpression was observed in 41 (12%) lymph nodes leading to the restaging of 17 patients (22%). Again a trend was observed towards a better DFS (disease-free survival) in the restaged patients (p=0,09). PTPN11 expression levels were high in 109 (31%) lymph nodes and led to the restaging of 41 (54%) patients who did not correlate with clinical outcome (p=0,61). The combination of CEACAM5-FGFR2b restaged the same number of patients than CEACAM5 only. Accordingly, high expression levels of CEACAM5 or FGFR2b in the primary tumour were related to better DFS (p<0,06; p<0,02, respectively); PTPN11 did not correlate with prognosis (p=0,37). Molecular nodal restaging based on expression levels of CEACAM5 and/or FGFR2b, does not add relevant clinical information to pathological staging of NSCLC likely related to the better prognosis of their overexpression in primary tumors.

Enhanced E. coli LF82 Translocation through the Follicle-associated Epithelium in Crohn’s Disease is Dependent on Long Polar Fimbriae and CEACAM6 expression, and Increases Paracellular Permeability

Background and AimsPatients with Crohn’s disease [CD] harbour an increased number of adherent-invasive E. coli [AIEC]. The strain LF82, identified in the ileal mucosa of CD patients, has been extensively studied for pathogenic mechanisms. However, understanding of the interaction of LF82 with the intestinal mucosa of CD patients is lacking.MethodsHere, we investigated the importance of long polar fimbriae [LPF] type 1 pili and the carcinoembryonic antigen-related cell-adhesion molecule 6 [CEACAM6] for translocation of LF82 in an in vitro model of follicle-associated epithelium [FAE], and in the FAE and villus epithelium [VE] of patients with CD and controls, using Ussing chambers.ResultsSignificantly greater LF82 passage occurred in the FAE model compared with in the VE Caco-2cl1 mono-culture. Moreover, bacterial translocation was inhibited by either LPF disruption or pre-incubation with anti-CEACAM6 antibody. Tissue mounted in Ussing chambers showed significantly higher LF82 passage in FAE from patients with CD compared with control FAE, that was diminished in LF82 lacking LPF and by blocking host CEACAM6. Interestingly, addition of LF82 to the CD FAE tissues significantly increased paracellular permeability [of 51Chromium-EDTA] compared with baseline, and the increase was inhibited by anti-CEACAM6. Immunofluorescence and immunoblots showed higher expression of CEACAM6 in FAE of patients with CD compared with in FAE from controls.ConclusionsThese data suggest that the FAE of CD patients is a site of vulnerability for invasion by LF82 via a mechanism that requires both bacterial LPF and host CEACAM6. Further, LF82 has the ability to increase paracellular passage through the FAE of patients with CD. These data can help define novel therapeutic targets in CD for the prevention of clinical recurrence.

OR13 Increased expression of ceacam1 in human liver transplantation is cytoprotective during ischemia-reperfusion injury

OR13 Increased expression of ceacam1 in human liver transplantation is cytoprotective during ischemia-reperfusion injury

Tumor intrinsic immunity related proteins may be novel tumor suppressors in some types of cancer

Immune checkpoint blockade therapy (ICBT) can unleash T-cell responses against cancer. However, only a small fraction of patients exhibited responses to ICBT. The role of immune checkpoints in cancer cells is not well understood. In this study, we analyzed T-cell coinhibitory/costimulatory genes across more than 1100 samples of the Cancer Cell Line Encyclopedia (CCLE). Nearly 90% of such genes were not expressed or had low expression across the CCLE cancer cell lines. Cell line screening showed the enrichment of cancer cells deprived of the expression of CD27, CEACAM1, CTLA4, LRIG1, PDCD1LG2, or TNFRSF18, suggesting their role as tumor suppressor. The metagene expression signature derived from these six genes - Immu6Metagene was associated with prolonged survival phenotypes. A common set of five oncogenic pathways were significantly inhibited in different types of tumors of the cancer patients with good survival outcome and high Immu6Metagene signature expression. These pathways were TGF-β signaling, angiogenesis, EMT, hypoxia and mitotic process. Our study showed that oncoimmunology related molecules especially the six genes of the Immu6Metagene signature may play the tumor suppressor role in certain cancers. Therefore, the ICBT targeting them should be considered in such context to improve the efficacy.

Abstract LB-075: Increased T cell- activation resulting from the combination of the anti-CEACAM6 function-blocking antibody BAY 1834942 with checkpoint inhibitors targeting either PD-1/PD-L1 or TIM-3

Abstract CEACAM6 (CD66c) is a novel immune checkpoint regulator suppressing the activity of effector T cells against tumors. CEACAM6 is expressed on tumor cells of multiple malignancies e.g.adenocarcinomas of the lung, colon, pancreas and stomach. In these tumor types higher CEACAM6 expression is associated with advanced stages and a poor prognosis. In immunohistochemistry analyses on primary tumor tissue slides and tissue microarrays the tumor cell expression of CEACAM6 was found to be independent from that of programmed death ligand 1 (PD-L1). BAY 1834942 is a humanized monoclonal antibody selectively blocking CEACAM6-mediated suppression of human T cells. Because there is no rodent orthologue of CEACAM6, BAY 1834942 was fully characterized in in vitro studies as reported earlier (AACR 2018, abstract nr. 1771). Benchmarking and combination studies showed that CEACAM6-mediated inhibition of T cell activation is apparently non-redundant with the programmed death-receptor 1 (PD-1)/ PD-L1 axis. Combination experiments were performed in co-cultures of PD-1, T cell immunoglobulin and mucin domain 3 (TIM3) and CEACAM1 (CD66a) positive virus antigen-specific T cells and virus peptide-loaded CEACAM6-expressing tumor cells (HCC2935, HCT116-C6 cells). When BAY 1834942 was combined with antibody inhibitors of either PD-1 or PD-L1, we consistently observed enhanced secretion of proinflammatory cytokines by T cells in the presence of PD-L1 positive tumor cells (HCC2935). Unexpectedly, combination of BAY 1834942 with an anti-TIM3 antibody resulted in an even more pronounced, synergistic increase of cytokine secretion. The combined effect of CEACAM6 and TIM-3 blockade was confirmed using survivin peptide-specific T cells as alternative T cell source. In contrast, combination of BAY 1834942 with an anti-CEACAM1 function blocking antibody was not superior to anti-CEACAM1 treatment alone, which is in line with our hypothesis that CEACAM1 is the main T cell receptor for CEACAM6 in our functional assays. In summary, BAY 1834942 is a novel immune checkpoint inhibitor with monotherapy potential for the treatment of patients with CEACAM6-expressing cancers. The data shown here provide a rationale for examining its combination potential with immune checkpoint inhibitors targeting either PD-1, PD-L1 or TIM-3. BAY 1834942 is currently under investigation in Ph1 clinical trials (NCT03596372). Citation Format: Joerg Willuda, Hans-Henning Boehm, Jessica Pinkert, Mark Trautwein, Wolf-Dietrich Doecke, Oliver von Ahsen, Karl Ziegelbauer, Rienk Offringa, Bertolt Kreft, Philipp Beckhove. Increased T cell- activation resulting from the combination of the anti-CEACAM6 function-blocking antibody BAY 1834942 with checkpoint inhibitors targeting either PD-1/PD-L1 or TIM-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-075.

Abstract 3947: Mechanisms of action of a neoantigen-targeting antibody NEO-201

Abstract NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that reacts to tumor-associated antigens (TAA) derived from pooled allogeneic colon tumor tissue extracts. NEO-201 recognizes tumor-associated variants of CEACAM family members. This mAb is remarkably tumor-specific in its staining profile and reacts to a wide range of human carcinoma cell lines by flow cytometry and tumor tissues by immunohistochemistry. NEO-201 exhibited both ADCC and complement-dependent cellular cytotoxicity (CDC) activity against human carcinoma cell in vitro and counteracted the growth of human pancreatic xenograft tumors in vivo. We investigated an additional mechanism of action of NEO-201 and to further confirm the rationale for clinical testing. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-surface protein expressed by immune cells and tumor cells, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function. Binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing. This study was designed determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore antitumor functionality to NK cells. In vitro assays using human tumor cell lines were conducted to identify CEACAM family members bound by NEO-201. Functional assays were conducted to assess the ability of NEO-201 to potentiate the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16 and the ability to mediate ADCC. NEO-201 was found to react with distinct variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed differing levels of the native forms of CEACAM5/6 vs. the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment augmented the cytolytic activity of NK-92 cells against NEO-201+ tumor cells in proportion to their level of CEACAM5 expression (average increase of 2-fold), but not against NEO-201+ cells that only expressed CEACAM6. This study demonstrates that NEO-201 is capable of an additional function. This antibody can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cytotoxicity. Experiments are in progress to determine the involvement of NK cell CEACAM1 and/or other checkpoint pathways in this mechanism of action. These results suggest that NEO-201 may potentially reverse CEACAM1-dependent immunosuppression of NK cells in patients whose tumors express the NEO-201-reactive variant of CEACAM5. Citation Format: Philip Martin Arlen, Massimo Fantini, Justin David, Christina M. Annunziata, Kwong Y. Tsang, Pia Morelli. Mechanisms of action of a neoantigen-targeting antibody NEO-201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3947.

Co-expression of carcinoembryonic antigen-related cell adhesion molecule 6 and 8 inhibits proliferation and invasiveness of breast carcinoma cells.

The carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 and CEACAM8 form heterodimers and exert their effects. Therefore, we examined the effects of CEACAM6 and CEACAM8 co-expression in breast cancer. We first studied CEACAM6/8 expression using immunohistochemistry in 109 patients with breast cancer. We then established MCF-7 cells that were stably transfected with CEACAM8 and studied cell proliferation, invasion and adhesion. The number of CEACAM6 and CEACAM8 double-positive breast carcinoma cells significantly increased in patients with low histopathological grade and stage. Proximity ligation assay (PLA) confirmed high CEACAM6/8 expression in MCF-7 cells. CEACAM6/8 expression promoted the adhesion of MCF-7 cells to endothelial cell monolayers but inhibited their invasion and proliferation. Furthermore, CEACAM6 status in carcinoma cells was significantly higher in bone than in lung metastases. CEACAM6/8 expression is associated with the inhibition of vascular invasion and cell proliferation. CEACAM6 expression was also considered to be involved in bone metastases of breast cancer. This is the first study to demonstrate the possible role of CEACAM6/8 heterodimer and CEACAM6 expression in breast cancer patients.

Preclinical characterization and efficacy of 4R9, a novel immune checkpoint blockade targeting CEACAM1 for cancer therapy.

e14155 Background: CEACAM1 is one of the several immune checkpoint receptors expressed on T cells that mediate suppression of inflammatory T cell response. It is known that CEACAM1-CEACAM1 homophilic interaction induces downregulation of ZAP70 phosphorylation in response to T cell receptor (TCR) stimulation. Also, CEACAM1 is highly expressed on non-small cell lung cancer (NSCLC) and its expression correlated with cancer progression and poor prognosis. We developed a fully human monoclonal antibody 4R9, human CEACAM1-targeting antibody. Methods: T cell activation of 4R9 was determined by NFAT-luciferase reporter assay with CEACAM1 overexpressing Jurkat stable cells. In vitro efficacy of 4R9 was examined using NK-mediated tumor cell killing assay. The anti-tumor efficacy of 4R9 alone or in combination was studied in vivo in a humanized mouse model engrafted with NSCLC patient-derived tumor xenografts. Results: Anti-CEACAM1 antibody 4R9 binds to CEACAM1 overexpressed in HEK293 or Jurkat cells but not to other CEA family members. 4R9 blocks CEACAM1-CEACAM1 homophilic interaction by binding to N domain of CEACAM1. CEACAM1-CEACAM1 homophilic interaction induced downregulation of ZAP70 phosphorylation in response to TCR stimulation in CEACAM1 overexpressing Jurkat stable cell line, which was rescued by 4R9 resulting in augmentation of NFAT activity and IL-2 expression. NK cell-mediated tumor lysis was increased by 4R9 in a CEACAM1 expression-dependent manner. Out of 49 NSCLC tumor tissues, 20 cases exhibited dominant expression of CEACAM1 over PD-L1 with 6 cases showing &gt; 50% of CEACAM1 positivity. In a single mouse trial using NSCLC PDX-huNSG mouse model, 4R9 (20 mpk, 2qW) suppressed tumor progression more than 30% as monotherapy (10/19) as well as in combination (16/22) with pembrolizumab (5 mpk, 2qW). Moreover, PDX of adenocarcinoma origin with more than 50% of CEACAM1 expression were more efficiently prohibited for progression with 4R9, suggesting potential therapeutic use of 4R9 in patients with NSCLC. Conclusions: Anti-CEACAM1 antibody blocked CEACAM1-mediated negative regulation and restored T/NK cell activities. Different expression patterns of CEACAM1 compared with PD-L1 and efficacies in a single mouse trial support the rationale for developing anti-CEAEAM1 antibody as cancer therapeutics.

CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

BackgroundSystemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1β. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1β in murine neutrophils through its ITIM receptor.ResultsWe now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1−/− mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G−) express IL-6 in the early response (< 2 h) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment perhaps due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and β-actin in response to LPS. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and β-actin from RP105.ConclusionThis novel pathway and regulation of IL-6 signaling by CEACAM1 defines a novel role for monocytes in the fever response of mice to LPS.

Expression of Chosen Carcinoembryonic-Related Cell Adhesion Molecules in Pancreatic Intraepithelial Neoplasia (PanIN) Associated with Chronic Pancreatitis and Pancreatic Ductal Adenocarcinoma (PDAC)

Aims: Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are members of the glycosylphosphatidylinositol (GPI)-linked immunoglobulin (Ig) superfamily and take part in regulation of cell adhesion, tumor suppression and angiogenesis. Overexpression of CEACAM 1, 5 and 6 is widely described in several gastrointestinal epithelial tumors. The aim of study was to evaluate the expression of CEACAM 1, CEACAM 5 and CEACAM 6 in the most common precursor lesions of pancreatic ductal adenocarcinoma -pancreatic intraepithelial neoplasia (PanIN).Methods and results: The study group consisted of 32 patients treated for chronic pancreatitis and 38 patients with pancreatic ductal adenocarcinoma who also had pancreatic intraepithelial neoplasia. The expression of CEACAM was performed by immunohistochemical method and evaluated using 3-point scale: 0 - lack of positive reaction in pancreatic intraepithelial neoplasia, 1 (weak and moderate) - reaction present in 1-30% epithelial cells in PanIN and 2 (strong) - reaction present in >30% epithelial cells in PanIN. Expression of CEACAM 1, 5 and 6 increased with increasing degree of advancement of PanIN. Differences in expression of CEACAM 1, 5 and 6 between normal pancreatic ducts and different degrees of PanIN were statistically significant (p<0.001). We observed relationship between CEACAM1 expression and localization of PanIN in different parts of the pancreas.Conclusions: CEACAM 1, CEACAM 5 and CEACAM 6 expression appears to be an early event in pancreatic carcinogenesis. Moreover, expression of CEACAM 1, 5 and 6 may represent a useful biomarker that may aid in the identification of precancerous lesions in the pancreas.

Prognostic implication of CEACAM1 expression in squamous cell carcinoma of the larynx: Pilot study.

CEACAM1, a valuable biomarker for several cancers, have remained unexplored up to the present in laryngeal squamous cell carcinoma (LSCC). We aimed to examine CEACAM1 expression and evaluate its combinational clinical significance for the diagnosis or prognosis and treatment decision making in LSCC. CEACAM1 expression was assessed by immunohistochemistry in 54 LSCCs and evaluate its correlation with clinical and histopathological features. CEACAM subtype 1 (CEACAM1) expression was positive in 50% of the cases. No significant difference was observed in relation to age, gender, tumor size, and tumor stage. CEACAM1 expression correlated with tumor grade, development of local recurrence, node and distant metastasis. Kaplan-Meier survival curves showed that CEACAM1 staining was inversely correlated with both overall and disease-specific 5-year survival. Our study is the first to demonstrate that CEACAM1 expression is associated with an adverse prognosis in LSCC. CEACAM1 is a valuable biomarker and a promising therapeutic target in LSCC.

Expression of CEACAM1 or CEACAM5 in AZ-521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori.

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c-Src and c-Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ-521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA-dependent fashion. In contrast, we discovered that AZ-521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin-β1 or carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ-521 cells readily express integrin-β1 , but overexpression of integrin-β1 constructs did not restore the T4SS defect. We further show that AZ-521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ-521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ-521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori-infected AGS cells. Our results suggest the existence of an integrin-β1 - and CEACAM1- or CEACAM5-dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.

MiR-335-5p has an important role in the progression of gastric cancer by down-regulation of CEACAM5

BackgroundDue to the lack of suitable biomarker to early diagnosis of gastric cancer patients for effective treatment, nowadays there are many experiment acts to use novel biomarker such as miRNAs to early diagnosis of GC. MethodsPeripheral blood specimens were collected before treatment from 113 patients with gastric cancer. The CEACAM5 and miR-335 expression were evaluated by quantitative RT-PCR. ResultCEACAM5 mRNA expression was significantly lower in patients with AGC than patients with EGC (P < .05) and miR-335 was significantly up-regulated in AGC compare to EGC (P < .05). Moreover, CEACAM5 and miR-335 expression level correlated significantly with LNM, distant metastasis, and TNM stage (P > .05). ConclusionOur findings demonstrate a possible relationship between the deregulation of miR-335 and CEACAM5 with GC progression, also miR-335 and CEACAM5 have the potential to recognize the AGC than EGC.

Abstract 1771: BAY 1834942 is an immunotherapeutic antibody blocking the novel immune checkpoint regulator CEACAM6 (CD66c)

Abstract CEACAM6 (CD66c) was previously shown to act as a novel immune checkpoint regulator suppressing the activity of effector T cells against tumors (Witzens-Harig et al., Blood 2013). CEACAM6 is a GPI-linked protein that is strongly expressed at the tumor cell surface in multiple cancer indications such as non-small cell lung adenocarcinoma (NSCLC), colorectal carcinoma (CRC), gastric adenocarcinoma and pancreatic cancer. In general, elevated CEACAM6 expression is associated with advanced tumor stages and poor prognosis. In vitro experiments showed that engagement of T-cells with CEACAM6, either expressed on tumor cells or presented on beads, resulted in suppression of TCR-mediated T-cell activation and ZAP70 phosphorylation. Based on these findings, we hypothesized that antibodies targeting CEACAM6 may be employed to enhance T-cell responses against CEACAM6-expressing cancers. Here we report the generation and characterization of BAY 1834942, a humanized monoclonal antibody selectively blocking the inhibitory impact of CEACAM6 on human T cells. There is no rodent ortholog of CEACAM6 precluding in vivo efficacy studies. In tumor cell / T cell co-culture systems, BAY 1834942 increased secretion of T-cell cytokines and effector molecules (e.g. IFNγ, TNFα, IL-2, granzyme B) and resulted in improved tumor cell killing. The effects of BAY 1834942 were dose-dependent, only observed in the context of CEACAM6-expressing tumor cells and could be reproduced in experiments using tumor cell lines and T-cell preparations from different sources, including T cells derived from tumor infiltrating lymphocytes from pancreatic cancer. BAY 1834942 is cross-reactive with the cynomolgus CEACAM6 ortholog and was well-tolerated in monkey toxicology studies. In summary, BAY 1834942 is a novel checkpoint inhibitor with potential for the treatment of patients with CEACAM6 expressing cancers, both as single agent and in combination with other checkpoint inhibitors. First-in-man trials are expected to commence in 2018. Citation Format: Joerg Willuda, Mark Trautwein, Jessica Pinkert, Wolf-Dietrich Doecke, Hans-Henning Boehm, Florian Wessel, Yingzi Ge, Eva Maria Gutierrez, Joerg Weiske, Christoph Freiberg, Uwe Gritzan, Julian Glueck, Dieter Zopf, Sven Golfier, Oliver von Ahsen, Ruprecht Zierz, Sabine Wittemer-Rump, Heiner Apeler, Ziegelbauer Karl, Rienk Offringa, Bertolt Kreft, Beckhove Philipp. BAY 1834942 is an immunotherapeutic antibody blocking the novel immune checkpoint regulator CEACAM6 (CD66c) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1771.

Abstract 3821: The neoantigen-targeting antibody NEO-201 enhances NK cell-dependent killing of tumor cells through blockade of the inhibitory CEACAM5/CEACAM1 immune checkpoint pathway

Abstract Background: Immunotherapy using checkpoint blockade antibodies that target effector cell inhibitory receptors, like PD-1 and CTLA-4, have elicited some dramatic and durable responses in several tumor types. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-surface protein expressed by immune cells and tumor cells, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function; binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing. NEO-201 is a novel humanized IgG1 monoclonal antibody (mAb) that was derived from an immunogenic preparation of tumor-associated antigens (TAAs) from pooled allogeneic colon tumor tissue extracts. It reacts against a wide variety of human carcinoma cell lines and tumor tissues, but is largely non-reactive against normal tissues. NEO-201 binds to members of the CEACAM family, and can activate innate immune mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to kill tumor cells. This investigation was designed determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore antitumor functionality to NK cells. Methodology: In vitro assays using human tumor cell lines were conducted to identify CEACAM family members bound by NEO-201. Functional assays were conducted to assess the ability of NEO-201 to potentiate the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16 and the ability to mediate ADCC. Results: NEO-201 was found to react with distinct variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed differing levels of the native forms of CEACAM5/6 vs. the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment augmented the cytolytic activity of NK-92 cells against NEO-201+ tumor cells in proportion to their level of CEACAM5 expression (average increase of 2-fold), but not against NEO-201+ cells that only expressed CEACAM6. Conclusions: This study demonstrates that NEO-201 is reactive against a tumor-associated variant of CEACAM5/6, and provides evidence that this antibody can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cytotoxicity. Experiments are in progress to determine the involvement of NK cell CEACAM1 and/or other checkpoint pathways in this mechanism of action. These results suggest that NEO-201 may potentially reverse CEACAM1-dependent immunosuppression of NK cells in patients whose tumors express the NEO-201-reactive variant of CEACAM5. Citation Format: Justin M. David, Massimo Fantini, Christina M. Annunziata, Philip M. Arlen, Kwong Y. Tsang. The neoantigen-targeting antibody NEO-201 enhances NK cell-dependent killing of tumor cells through blockade of the inhibitory CEACAM5/CEACAM1 immune checkpoint pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3821.

Regulation of CEACAM1 Protein Expression by the Transcription Factor ETS-1 in BRAF-Mutant Human Metastatic Melanoma Cells

BRAF becomes constitutively activated in 50% to 70% of melanoma cases. CEACAM1 has a dual role in melanoma, including facilitation of cell proliferation and suppression of infiltrating lymphocytes, which are consistent with its value as a marker for poor prognosis in melanoma patients. Here we show that BRAFV600E melanoma cells treated with BRAF and MEK inhibitors (MAPKi) downregulate CEACAM1 mRNA and protein expression in a dose- and exposure time–dependent manners. Indeed, there is a significant correlation between the presence of BRAFV600E and CEACAM1 expression in melanoma specimens obtained from 45 patients. Vemurafenib-resistant cell systems reactivate the MAPK pathway and restore basal CEACAM1 mRNA and protein levels. These combined results suggest transcriptional regulation. Indeed, luciferase reporting assays show that CEACAM1 promoter (CEACAM1p) activity is significantly reduced by MAPKi. Importantly, we show that the MAPK-driven CEACAM1p activity is mediated by ETS1, a major transcription factor and downstream effector of the MAPK pathway. Phosphorylation mutant ETS1T38A shows a dominant negative effect over CEACAM1 expression. The data are consistent with independent RNAseq data from serial biopsies of melanoma patients treated with BRAF inhibitors, which demonstrate similar CEACAM1 downregulation. Finally, we show that CEACAM1 downregulation by MAPKi renders the cells more sensitive to T-cell activation. These results provide a new view on a potential immunological mechanism of action of MAPKi in melanoma, as well as on the aggressive phenotype observed in drug-resistant cells.