The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro.
ABSTRACT: Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin.
Project description:Natural killer cells (NK cells) play an essential role in the immunological mechanism underlying chronic hepatitis C (CHC). Impairment of NK cell function facilitates persistent infection with hepatitis C virus (HCV) and hepatocellular carcinogenesis. However, the mechanism by which NK cell activity is suppressed in CHC is not completely understood. In this study, we focused on carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1). CEACAM1 is thought to suppress NK cell function. We examined the effect of CEACAM1 on NK cell function in CHC. We investigated the function of CEACAM1 in vitro using Huh7.5.1 cells and the HCV-Japanese fulminant hepatitis (JFH)-1 strain. We analyzed serum CEACAM1 level, NK cell function, and CEACAM1 messenger RNA (mRNA) level in human liver samples. Levels of CEACAM1 on the cell surface, CEACAM1 mRNA levels, and soluble CEACAM1 levels in supernatants were significantly higher in Huh7.5.1 cells infected with JFH-1 (Huh7.5.1/JFH-1 cells) than in Huh7.5.1 cells. Significantly higher NK cell cytotoxicity was observed toward K562 cells after coculture with CEACAM1 knockout Huh7.5.1/JFH-1 cells than after coculture with Huh7.5.1/JFH-1 cells. CEACAM1 expression was induced by the HCV E2 glycoprotein in HCV infection. Significantly higher serum CEACAM1 levels were detected in patients with CHC compared with healthy subjects and patients who achieved sustained virological responses. The expression of CD107a on NK cells from patients with CHC was negatively correlated with serum CEACAM1 levels. Significantly higher levels of CEACAM1 mRNA were detected in HCV-infected livers compared with uninfected livers. Conclusion: CEACAM1 expression was induced in hepatocytes following HCV infection and decreased NK cell cytotoxicity. These results demonstrate a possible role for CEACAM1 in the pathogenesis of CHC and hepatocellular carcinoma progression.
Project description:Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a key molecule in several intracellular and intercellular signaling pathways, with multiple functional and structural roles. CEACAM1 expression in melanoma is often described in the invading part of the tumor and has been associated with increased melanoma cells invasion and migration. We studied CEACAM1 expression in regressing versus non-regressing thin melanomas, knowing that phenomenon of regression represents a valuable model for understanding tumor immunity. In melanoma, through homophilic interactions, CEACAM1 inhibits natural killer cell activity, inhibits effector functions of tumor infiltrating lymphocytes, such as cytotoxicity and interferon-? release. We present a retrospective study including 53 consecutive cases of thin melanoma, 21 with regression and 32 without regression. Comparative analysis of CEACAM1 expression in regressed and non-regressed areas from melanomas with regression and in non-regressed melanomas was performed. We used three different clones of CEACAM1: AA 1-428, extracellular domain, rabbit; AA 1-428, mouse, clone 8B6E2F4; and AA 1-468, full length, mouse, clone 2F6. All three clones had similar reactivity. We identified membrane positivity of tumor cells in non-regressed melanomas and in non-regressed areas in melanomas with regression. Remaining tumor cells in regressed areas were mostly negative for CEACAM1. In non-regressed lesions, there was a stronger positivity of CEACAM1 in the deep invasive front. In thin melanomas, CEACAM1 overexpression is related with invasiveness, suggesting that CEACAM1-positive melanomas are more aggressive. Also, in areas of regression tumor cells lose CEACAM1 expression, probably correlated with the presence of natural killer cells.
Project description:The prognostic value of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in melanoma was demonstrated more than a decade ago as superior to Breslow score. We have previously shown that intercellular homophilic CEACAM1 interactions protect melanoma cells from lymphocyte-mediated elimination. Here, we study the direct effects of CEACAM1 on melanoma cell biology. By employing tissue microarrays and low-passage primary cultures of metastatic melanoma, we show that CEACAM1 expression gradually increases from nevi to metastatic specimens, with a strong dominance of the CEACAM1-Long tail splice variant. Using experimental systems of CEACAM1 knockdown and overexpression of selective variants or truncation mutants, we prove that only the full-length long tail variant enhances melanoma cell proliferation in vitro and in vivo. This effect is not reversed with a CEACAM1-blocking antibody, suggesting that it is not mediated by intercellular homophilic interactions. Downstream, CEACAM1-Long increases the expression of Sox-2, which we show to be responsible for the CEACAM1-mediated enhanced proliferation. Furthermore, analysis of the CEACAM1 promoter reveals two single-nucleotide polymorphisms (SNPs) that significantly enhance the promoter's activity compared with the consensus nucleotides. Importantly, case-control genetic SNP analysis of 134 patients with melanoma and matched healthy donors show that patients with melanoma do not exhibit the Hardy-Weinberg balance and that homozygous SNP genotype enhances the hazard ratio to develop melanoma by 35%. These observations shed new mechanistic light on the role of CEACAM1 in melanoma, forming the basis for development of novel therapeutic and diagnostic technologies.
Project description:As melanoma cells are immunogenic, they instigate an adaptive immune response and production of anti-tumor T-cells. A central factor in this interaction is CEACAM1 (carcinoembryonic antigen cell adhesion molecule 1), a transmembrane glycoprotein previously shown in our lab to protect melanoma cells from T cell-mediated killing. In this study, we examine the role of transcription factor SOX9 in the regulation of CEACAM1 expression and immune resistance in melanoma cells. Knockdown of endogenous SOX9 results in CEACAM1 up-regulation, while its overexpression leads to the opposite effect. We show that SOX9 controls CEACAM1 expression at a transcriptional level, but in an indirect manner, as regulation of the CEACAM1 promoter remains intact even when all eight potential SOX9-binding sites are abolished. A series of promoter truncations localizes the SOX9-controlled area to the proximal 200bp of the promoter. Point mutations in putative Sp1 and ETS1 binding sites identify these transcription factors as the primary SOX9-controlled mediators. Co-immunoprecipitation studies show that SOX9 and Sp1 physically interact in melanoma cells, while silencing of SOX9 down-regulates ETS1, but not Sp1, in the same cells. Finally, knockdown of SOX9 indeed renders melanoma cells resistant to T cell-mediated killing, in line with the increased CEACAM1 expression. In conclusion, we show that SOX9 regulates CEACAM1 expression in melanoma cells, and thereby their immune resistance. As CEACAM1 is a pivotal protein in melanoma biology and immune crosstalk, further understanding of its regulation can provide new insights and contribute to the development of novel approaches to therapy.
Project description:Widespread metastasis is the leading course of death in many types of cancer, including malignant melanoma. The process of metastasis can be divided into a number of complex cell biological events, collectively termed the "invasion-metastasis cascade." Previous reports have characterized the capability of anchorage-independent growth of cancer cells in vitro as a key characteristic of highly aggressive tumor cells, particularly with respect to metastatic potential. Biological heterogeneity as well as drastic alterations in cell adhesion of disseminated cancer cells support escape mechanisms for metastases to overcome conventional therapies. Here, we show that exclusively the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) splice variant CEACAM1-4L supports an anchorage-independent signature in malignant melanoma. These results highlight important variant-specific modulatory functions of CEACAM1 for metastatic spread in patients suffering malignant melanoma.
Project description:Metastases represent the main cause of death in melanoma patients. Despite the current optimized targeted therapy or immune checkpoint inhibitors the treatment of metastatic melanoma is unsatisfactory. Because of the poor prognosis of advanced melanoma there is an urgent need to identify new biomarkers to differentiate melanoma cells from normal melanocytes, to stratify patients according to their risk, and to identify subgroups of patients that require close follow-up or more aggressive therapy. Furthermore, melanoma progression has been associated with the dysregulation of cell adhesion molecules. We have reviewed the literature and have discussed the important role of the expression of the carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) in the development of melanoma. Thus, novel insights into CEACAM1 may lead to promising strategies in melanoma treatment, in monitoring melanoma patients, in assessing the response to immunotherapy, and in completing the standard immunohistochemical panel used in melanoma examination.
Project description: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.
Project description:Bv8 (prokineticin 2) expressed by Gr1(+)CD11b(+) myeloid cells is critical for VEGF-independent tumor angiogenesis. Although granulocyte colony-stimulating factor (G-CSF) has been shown to be a key inducer of Bv8 expression, the basis for Bv8 production in driving tumor angiogenesis is undefined. Because the cell adhesion molecule CEACAM1, which is highly expressed on Gr1(+)CD11b(+) myeloid cells, is known to regulate G-CSF receptor (G-CSFR) signaling, we hypothesized that CEACAM1 would regulate Bv8 production in these cells. In support of this hypothesis, we found that Bv8 expression was elevated in Gr1(+)CD11b(+) cells from Ceacam1-deficient mice implanted with B16 melanoma, increasing the infiltration of Gr1(+)CD11b(+) myeloid cells in melanoma tumors and enhancing their growth and angiogenesis. Furthermore, treatment with anti-Gr1 or anti-Bv8 or anti-G-CSF monoclonal antibody reduced myeloid cell infiltration, tumor growth, and angiogenesis to levels observed in tumor-bearing wild-type (WT) mice. Reconstitution of CEACAM1-deficient mice with WT bone marrow cells restored tumor infiltration of Gr1(+)CD11b(+) cells along with tumor growth and angiogenesis to WT levels. Treatment of tumor-bearing WT mice with anti-CEACAM1 antibody limited tumor outgrowth and angiogenesis, albeit to a lesser extent. Tumor growth in Ceacam1-deficient mice was not affected significantly in Rag(-/-) background, indicating that CEACAM1 expression in T and B lymphocytes had a negligible role in this pathway. Together, our findings show that CEACAM1 negatively regulates Gr1(+)CD11b(+) myeloid cell-dependent tumor angiogenesis by inhibiting the G-CSF-Bv8 signaling pathway.
Project description:Vasculogenic mimicry (VM) promotes tumor migration, metastasis, and invasion in various types of cancer, but the relationship between VM and these phenotypes remains undefined. In this study, we examined carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) as a novel target of VM. We found that ectopic expression of CEACAM1 in HT1080 human fibrosarcoma cells suppressed the formation of a VM-like network. Further, cell migration and proliferation were abated by the introduction of CEACAM1 into HT1080 cells. Conversely, knockout (KO) of the CEACAM1 gene in SK-MEL-28 melanoma cells, which normally express high levels of CEACAM1, inhibited formation of a VM-like network, which was covered on reintroduction of CEACAM1. These results suggest that CEACAM1 differentially regulates formation of the VM-like network between cancer cell types and implicate CEACAM1 as a novel therapeutic target in malignant cancer.
Project description:Reprogramming of NK cells with a chimeric antigen receptor (CAR) proved an effective strategy to increase NK cell reactivity and recognition specificity toward tumor cells. To enhance the cytotoxicity of NK cells against CD138-positive multiple myeloma (MM) cells, we generated genetically modified NK-92MI cells carrying a CAR that consists of an anti-CD138 single-chain variable fragment (scFv) fused to the CD3? chain as a signaling moiety. The genetic modification through a lentiviral vector did not affect the intrinsic cytolytic activity of NK-92MI toward human erythroleukemic cell line K562 cells or CD138-negative targets. However, these retargeted NK-92MI (NK-92MI-scFv) displayed markedly enhanced cytotoxicity against CD138-positive human MM cell lines (RPMI8226, U266 and NCI-H929) and primary MM cells at various effector-to-target ratios (E:T) as compared to the empty vector-transfected NK-92MI (NK-92MI-mock). In line with the enhanced cytotoxicity of NK-92MI-scFv, significant elevations in the secretion of granzyme B, interferon-? and proportion of CD107a expression were also found in NK-92MI-scFv in response to CD138-positive targets compared with NK-92MI-mock. Most importantly, the enhancement in the cytotoxicity of NK-92MI-scFv did not attenuate with 10Gy-irradiation that sufficiently blocked cell proliferation. Moreover, the irradiated NK-92MI-scFv exerted definitely intensified anti-tumor activity toward CD138-positive MM cells than NK-92MI-mock in the xenograft NOD-SCID mouse model. This study provides the rationale and feasibility for adoptive immunotherapy with CD138-specific CAR-modified NK cells in CD138-positive plasmacytic malignancies, which potentially further improves remission quality and prolongs the remission duration of patients with MM after upfront chemotherapy.