Carcinoembryonic Antigen Cell Adhesion Molecule Research Articles

Abstract 2732: NB203: A Novel therapeutic bispecific antibody targeting CEACAM1/VEGF for tumor immunity and angiogenesis modulation in the tumor microenvironment

Abstract The carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is detected on leukocytes, epithelia and endothelia, and involved in cell adhesion through homophilic or heterophilic binding. In various gastrointestinal cancers, NSCLC (non-small cell lung cancer), and melanoma, CEACAM1 is highly expressed on tumor cells and tumor stroma, with its levels increasing as the disease progresses. Notably, CEACAM1 has been found to dampen T and NK cell functions while promoting tumor angiogenesis and metastasis. In this study, we present NB203, a novel CEACAM1/VEGF bispecific antibody developed using Neologics CEACAM-plus, a bifunctional molecule engineering platform designed to target and modulate the tumor microenvironment (TME). NB203 effectively accumulated in the TME and hindered the homophilic or heterophilic interactions of CEACAM1. It demonstrated a significant enhancement in T cell activities and NK cell-mediated killing of CEACAM1-expressing tumor cells, and the inhibition of tumor cell migration and polyploidy through macrophages. More importantly, NB203 was specifically designed to impede tumor angiogenesis. It exhibited an increased binding capacity to both CEACAM1 and VEGF in pro-angiogenic TME, and undermined intratumoral vessel maturation by blocking VEGF and negatively regulating myeloid cell-dependent tumor angiogenesis. In summary, our preclinical data indicate that NB203 exhibits effective anti-tumor activity by mediating NK and cytotoxic T cell activities, improving the immune-suppressive TME, and suppressing tumor angiogenesis. The use of NB203 holds promise in overcoming resistance to anti-VEGF therapy and substantially improving the safety of therapeutic targeting of angiogenesis in cancer treatment. Citation Format: Jinyu Dong, Binbin Wang, Dong Wang, Jie Ni, Tingting Bu, Haojie Wang, Bishnu Nayak, Baiyang Wang, Xin Dong, Yu Zhang. NB203: A Novel therapeutic bispecific antibody targeting CEACAM1/VEGF for tumor immunity and angiogenesis modulation in the tumor microenvironment [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 2732.

ATOH1, TFAP2B, and CEACAM6 as Immunohistochemical Markers to Distinguish Merkel Cell Carcinoma and Small Cell Lung Cancer.

Merkel cell carcinoma (MCC) and small cell lung cancer (SCLC) can be histologically similar. Immunohistochemistry (IHC) for cytokeratin 20 (CK20) and thyroid transcription factor 1 (TTF-1) are commonly used to differentiate MCC from SCLC; however, these markers have limited sensitivity and specificity. To identify new diagnostic markers, we performed differential gene expression analysis on transcriptome data from MCC and SCLC tumors. Candidate markers included atonal BHLH transcription factor 1 (ATOH1) and transcription factor AP-2β (TFAP2B) for MCC, as well as carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) for SCLC. Immunostaining for CK20, TTF-1, and new candidate markers was performed on 43 MCC and 59 SCLC samples. All three MCC markers were sensitive and specific, with CK20 and ATOH1 staining 43/43 (100%) MCC and 0/59 (0%) SCLC cases and TFAP2B staining 40/43 (93%) MCC and 0/59 (0%) SCLC cases. TTF-1 stained 47/59 (80%) SCLC and 1/43 (2%) MCC cases. CEACAM6 stained 49/59 (83%) SCLC and 0/43 (0%) MCC cases. Combining CEACAM6 and TTF-1 increased SCLC detection sensitivity to 93% and specificity to 98%. These data suggest that ATOH1, TFAP2B, and CEACAM6 should be explored as markers to differentiate MCC and SCLC.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.

CEACAM6 and IFITM3 as potential biomarkers for pelareorep in pancreatic adenocarcinoma (PDAC).

684 Background: Pelareorep (P) is a proprietary formulation of live, replication-competent, naturally occurring Reovirus Type 3 Dearing strain. We previously performed a phase II trial of P with carboplatin (C) and paclitaxel (Pxl) vs C and Pxl alone in metastatic PDAC (NCT01280058). Although P did not improve progression-free survival compared to C and Pxl alone, some exceptional durable responses were seen in the P arm. Low levels of carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) mRNA expression were associated with prolonged PFS in P-treated pts (p=0.05) and there was a 3.3 times higher fold change in Interferon-inducible transmembrane protein 3 (IFITM3) expression in the pts doing poorly versus responders (nominal p<0.05) in P arm. Here we investigate mechanisms of resistance to P infection mediated through CEACAM6 and IFITM3 in a panel of pancreatic ductal adenocarcinoma cell lines. Methods: Prior studies noted CEACAM6 and IFITM3 may restrict cell entry of certain enveloped viruses. CEACAM6 and IFITM3 protein levels were measured in PDAC cell lines using Western blot. Flow cytometry was used to assess P infectivity. We have generated multiple knockdowns of CEACAM6 and IFITM3 in PDAC cell lines to determine changes in P infectivity and other inflammatory signalling pathways. Live cell imaging was used to assess cell viability. Results: Basal expression of CEACAM6 was measured in a panel of 4 PDAC cells (PANC10.5, HPAFII, BxPC3, CFPAC1). Panc10.05 had low basal expression nor could expression be induced by 24hr P infection. All other cell lines showed moderate to high basal expression that was inducible. P infectivity at 24hrs was significantly higher in Panc10.05 cells compared to high CEACAM6 expressing cell lines. There was varying basal expression of IFITM3, however all cell lines showed IFITM3 induction when exposed to P. Of the 3 cell lines that also expressed high levels of CEACAM6 (HPAFII, CFPAC1, BxPC3), low basal expression of IFITM3 was strongly associated with increased sensitivity to P infection and decreased viability at 72hrs, which could be induced by knockdown of IFITM3 in high expressing cell lines. Neither C or Pxl altered levels of CEACAM6 or IFITM3 in any of the four cell lines. Conclusions: High CEACAM6 and IFITM3 play a role in P viral infectivity across multiple pancreatic cell lines, confirming results obtained in translational datasets. Further analysis is ongoing to elucidate mechanism and identify pathways to alter expression of CEACAM6 and IFITM3 and enhance P infectivity in future trials.

Abstract B117: Phase 1 study of CM24 in combination with nivolumab in patients with advanced pancreatic cancer - Survival, potential biomarker and effect on neutrophil extracellular traps (NETs)

Abstract Background: The monoclonal antibody CM24 blocks the activity of carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), which is known to have several important roles in cancer progression, immune evasion, and metastasis. The homophilic interaction of CEACAM1 on tumor cells and tumor infiltrating lymphocytes (TILs) silences the anti-tumor immunity. Additionally, CEACAM1 is part of the neutrophil extracellular trap (NET) complex. NETs are regarded as an important component in the tumor microenvironment, which contribute to metastasis and immune evasion. Previously we have demonstrated direct binding of CM24 to NETs and inhibition of NET-induced cancer cell migration and tumor metastasis by the antibody. Here, we present the results from the dose escalation part of a Phase 1/2 study of CM24 and nivolumab (NCT 04731467) and the findings of exploratory studies that were conducted as part of this trial. Methods: In the dose escalation part of the study, 11 pancreatic ductal adenocarcinoma (PDAC) patients who received 2 prior lines of therapy were administered CM24 at 10, 15 and 20mg/kg q2w and nivolumab at 480mg q4w. Levels of CEACAM1 and myeloperoxidase (MPO) were measured in serum by ELISA. Pretreatment biopsies from 8 evaluable PDAC patients were analyzed, along with pretreatment and on treatment serum samples. Quantitative multiplex IF and immunohistochemistry (IHC) were used to evaluate CEACAM1, PDL1, TIM3 and CEACAM1-positive TILs in patient biopsies. Results: Nine PDAC patients were evaluable for exploratory efficacy assessment with a median OS of 4.5 (95% CI 2.0-11.1) months, including 1 confirmed PR and 2 pts with SD for a disease control rate of 33%. Percentage of CEACAM1+TILs in patient biopsies ranged between 0% and 10%, and a cutoff rate to define low (0-5%) vs. high (> 5%) expression was used to stratify the patient survival data. High CEACAM1+TILs (median of 10%) were measured in 3/8 PDAC patients with median survival of 11.1 months (range: 9.6-14.6 months) compared to 5/8 patients with low CEACAM1+TILs (median 2%) who exhibited shorter survival (range: 2-4.5 months), suggesting positive association (p=0.017). IHC analysis of CEACAM1 further revealed CEACAM1-positive NETs in the biopsies of all PDAC patients. In serum, enhanced levels of the NET biomarker MPO were detected in PDAC patients (n=10) as compared to healthy volunteers (n=30), demonstrating a 3-fold difference in the mean value (p<0.01). Interestingly, MPO levels decreased by an average of 40% (p<0.05) across all dose levels within 2 weeks of CM24 therapy. Additional findings will be presented at the conference. Conclusions: CM24 in combination with nivolumab shows promising initial activity results and a favorable safety profile in PDAC patients who have progressed after second-line therapy. The positive association between the level of CEACAM1+TILs and OS as well as CM24 effects on NETs, advocate for the role of CM24 in controlling immune evasion. These 2 potential biomarkers are being investigated in the ongoing randomized Phase 2 study. Citation Format: Erkut Borazanci, Shubham Pant, Ruth Perets, Talia Golan, Mohammed Najeeb Al Hallak, Michael Cecchini, Tomer Maierson, Hava Ben David, Michael Schickler, Hadas Reuveni. Phase 1 study of CM24 in combination with nivolumab in patients with advanced pancreatic cancer - Survival, potential biomarker and effect on neutrophil extracellular traps (NETs) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B117.

Abstract B125: Neutralizing acidosis with L-DOS47 urease immunoconjugate enhances responses to anti-PD1 checkpoint blockade in a preclinical orthotopic model of pancreatic adenocarcinoma

Abstract Background: Acidosis in the tumor microenvironment is an important immunosuppressive mechanism that leads to tumor growth. We have shown that neutralization of tumor pH using sodium bicarbonate improves responses to immune checkpoint blockade (ICB) in pre-clinical models, but phase I/IIa clinical trials failed due to poor patient compliance. Thus, we aimed to test clinically translatable agents to neutralize tumor acidity and improve cancer outcomes in response to ICB. We tested L-DOS47, a targeted urease immunoconjugate designed to directly neutralize tumor acidity that has been well-tolerated in phase I/IIa trials. The nanobody component of L-DOS47 binds carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6), a cell surface protein highly expressed in gastrointestinal and lung cancers; the urease moiety cleaves endogenous urea into two NH4+ and one CO2 thereby raising local pH. Materials and Methods: Immunohistochemistry screening for CEACAM6 expression and L-DOS47 binding was performed on tumor microarrays from pancreatic ductal adenocarcinoma (PDAC) cases (n=10 plus 2 normal tumor adjacent cases). We established a preclinical in vivo PDAC model by orthotopically inoculating human CEACAM6-expressing KPC961 into immunocompetent mice. Pharmacodynamic studies were performed using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure tumor extracellular pH (pHe) at baseline (pre-L-DOS47) and at various time points post-L-DOS47 (4, 18, 24, 48, 72 and 96 hours). Mean tumor pHe and histograms of pHe distributions were compared among groups. Additional biological replicates were conducted to assess therapeutic efficacy. For these experiments, mice were randomized, after measurable tumors are established, into groups with equal tumor volume averages to initiate twice-weekly therapies with 300 µg anti-PD1 or 90 µg/kg L-DOS47 monotherapies, 300 µg anti-PD1 + 90 µg/kg L-DOS47 compared to a control group receiving no therapy. Tumor volumes were measured weekly by ultrasound imaging up to 4 weeks. Results: Immunohistochemistry screening of patient tumor microarrays confirmed high CEACAM6 expression and L-DOS47 binding to PDAC tissues. CEST-MRI results showed that L-DOS47 increased tumor pHe from 4h to 96h post-injection; however, pHe increases were only evident in tumors with baseline pHe ≤ 6.60. Efficacy studies in the PDAC model showed that the combination of anti-PD1 + L-DOS47 significantly reduced tumor growth when compared with control, L-DOS47 and anti-PD1 groups at week 3 (p<.0001; <.0001 and 0.0368, respectively) and week 4 (p<.0001; <.0001 and 0.0142, respectively). Conclusion: These studies provide strong evidence that L-DOS47, by neutralizing acidic tumor pH, significantly improves responses to anti-PD1 immunotherapy. Citation Format: Bruna V Jardim-Perassi, Pietro Irrera, Dominique Abrahams, Christopher J Whelan, Matthew S Beatty, Samantha R Byrne, Andrew A Odeja, Dario L Longo, Young Ou, Kim Gaspar, Martin Koebel, Shari A Pilon-Thomas, Christof Boehler, Gabrielle M Siegers, Robert J Gillies, Arig A Ibrahim-Hashim. Neutralizing acidosis with L-DOS47 urease immunoconjugate enhances responses to anti-PD1 checkpoint blockade in a preclinical orthotopic model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B125.

Abstract 1867: Pre-clinical characterization of anti-CEACAM1 antibody

Abstract Targeting checkpoint molecules cytotoxic T lymphocyte antigen-4 (CTLA-4) or programmed death receptor-1 (PD-1) expressed on immune cells has shown promising results in the treatment of cancer patients. Nevertheless only a small portion of population benefited from current ICI treatments, so there should be other mechanisms for immune evasion potentially. The carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is an activation-induced inhibitory molecule on T and NK cells. In a wide range of cancers, it’s also expressed on tumor cells and other immune cells including macrophages and neutrophils. Homophilic interaction between CEACAM1 expressed on tumor cells and immune cells dampens NK and T cell function, mediates tumor migration, and promotes tumor angiogenesis, which implies that this homophilic interaction is an important mechanism for tumor progression. Here, we describe the identification and characterization of a novel humanized anti-human CEACAM1 therapeutic antibody developed via Neologics’ novel target validation and antibody screening Tier-A platform. This anti-CEACAM1 antibody specifically binds to recombinant human CEACAM1 with nM level affinity and significantly enhances T cell activities and NK killing on CEACAM1 expressing tumor cells, inhibits tumor cell migration in vitro as well. In humanized mouse models, this antibody evidently reduces tumor growth alone or combined with other immune checkpoint blockers. Taken together, we have demonstrated this anti-CEACAM1 antibody specifically enhance NK cell activity, modulate T cell immune activation and inhibit tumor migration supporting further clinical investigation for various indications. Citation Format: Jinyu Dong, Yu Zhang, Baiyang Wang, Tingting Pu, Liegang Shao, Binbin Wang, Dong Wang, Jie Ni, Sunan Li, Xin Dong. Pre-clinical characterization of anti-CEACAM1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1867.

Glycan Conformation in the Heavily Glycosylated Protein, CEACAM1.

Glycans attached to glycoproteins can contribute to stability, mediate interactions with other proteins, and initiate signal transduction. Glycan conformation, which is critical to these processes, is highly variable and often depicted as sampling a multitude of conformers. These conformers can be generated by molecular dynamics simulations, and more inclusively by accelerated molecular dynamics, as well as other extended sampling methods. However, experimental assessments of the contribution that various conformers make to a native ensemble are rare. Here, we use long-range pseudo-contact shifts (PCSs) of NMR resonances from an isotopically labeled glycoprotein to identify preferred conformations of its glycans. The N-terminal domain from human Carcinoembryonic Antigen Cell Adhesion Molecule 1, hCEACAM1-Ig1, was used as the model glycoprotein in this study. It has been engineered to include a lanthanide-ion-binding loop that generates PCSs, as well as a homogeneous set of three 13C-labeled N-glycans. Analysis of the PCSs indicates that preferred glycan conformers have extensive contacts with the protein surface. Factors leading to this preference appear to include interactions between N-acetyl methyls of GlcNAc residues and hydrophobic surface pockets on the protein surface.

Old and New Blood Markers in Human Colorectal Cancer.

Cancer is a predominant cause of mortality all over the world. Lung, prostate, and colorectal cancer are the more frequent in men while breast and colorectal have a high incidence in women. Major progress aside, some cancers are still frequent and one major issue is improvements in detection methods. Imaging techniques have a major role, but inflammatory, tumoral markers and calculated scores may contribute to the assessment of prognosis. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and carcinoembryonic antigen cell adhesion molecule (CEACAM) have been used for decades and do not have a clear use for diagnosis or prognosis yet. The CEACAM family includes 12 human members, and some of them have a cluster differentiation (CD). CD66 may be an interesting indicator of disease severity. Beside interleukin-6 (IL-6), the high level of which is observed in patients with a high mortality rate, other cytokines IL-17A, IL-22, and transforming growth factor -β (TGF-β) are expressed at the tumor level. The detection of circulating tumor cells has been improved but is still of undetermined value. Circulating tumor DNA (ctDNA) was recently studied in CRC stage II patients and may be helpful for chemotherapy management.

241 (PB021) - Development of a novel antibody-drug conjugate targeting both CEACAM5 and CEACAM6

Background: Carcinoembryonic antigen cell adhesion molecule (CEACAM) family members 5 and 6 are tumor-associated antigens frequently upregulated in epithelial cancers, including pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC), where they contribute to invasion, metastasis, survival, resistance to chemotherapy, and immune escape. CT109 is a novel antibody with dual specificity for both CEACAM5 and 6, mediated by binding to a shared glycoepitope. CT109 exhibits a high affinity (in the single nM range) to the target.

Abstract CT136: Interim safety and efficacy results from a phase 1b study of CM24 in combination with nivolumab in adults with advanced solid tumors

Abstract Objectives: The monoclonal antibody CM24 binds to and blocks carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), which is known to have several important roles in cancer progression. A Phase 1/2 study (NCT 04731467) includes in its Phase 1 part, a dose escalation of CM24 administered with nivolumab for the treatment of refractory cancer patients. The primary objectives of this part are to evaluate safety, tolerability, PK and determine the recommended Phase 2 dose (RP2D) of CM24. In the Phase 2 part of this study, patients with NSCLC relapsed from or refractory to first-line immuno-oncology will be treated with CM24 and nivolumab, and patients with advanced/metastatic pancreatic adenocarcinoma relapsed from or refractory to first-line therapy will be treated with CM24, nivolumab, and chemotherapy, the objective will be to evaluate safety and preliminary efficacy of the combination treatment. Methods: In the dose escalation part of the study, enrolled patients were administered with CM24 at 10, 15 and 20mg/kg q2w and nivolumab 480mg q4w. Safety was assessed according to CTCAE v5 and preliminary anti-tumor activity was assessed by the investigators according to RECISTv1.1 using CT/MRI. CM24 and CEACAM1 measurements in serum, biopsy specimens and TILs, as well as tumor and TILs PD-L1 levels were determined. Results: As of data cutoff date of January 31, 2022, a total of 13 patients were in the dose escalation phase, of which 10 are evaluable for dose limiting toxicity (DLT) determination, including 7 with pancreatic cancer (PDAC), 2 with colorectal cancer (CRC) and one with papillary thyroid cancer (PTC). All patients but one had received 2 prior regimens for metastatic disease. Six Grade 3 adverse events (AEs) (unrelated to CM24 or nivolumab), were observed, each in a single patient, including diarrhea, hypokalemia, abdominal pain, small bowel obstruction, atrial flutter, and GI bleed. No Grade 4 AEs or deaths were reported. For the 10 evaluable patients, best overall response included one confirmed PR (PDAC patient, 6-month duration of response) two SD (one PDAC and one PTC patient, both with duration of 3.5 months), and two patients are awaiting follow up MRI/CT scans. As of the cutoff date, 8/10 of the patients that completed the DLT period are on treatment or in follow up (range 2 to 9.5 months). Pharmacokinetic analysis of CM24 shows exposure is dose-related and approximately dose-proportional. Dose proportionality is shown by CLss and dose normalized Cmax and AUC. Receptor occupancy analyses in peripheral blood reached a mean of 100% occupancy of CEACAM1 by CM24 at the higher dose levels. Conclusion: The Phase 2 portions of the study will be initiated at the conclusion of this dose escalation part. Citation Format: Erkut Borazanci, Mohammed Najeeb Al Hallak, Joseph P. Eder, Talia Golan, Shubham Pant, Ruth Perets, Gal Markel, Michael Schickler, Hadas Reuveni, Lixian Jin, Bertrand Liang, Alexander I Spira. Interim safety and efficacy results from a phase 1b study of CM24 in combination with nivolumab in adults with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT136.

CEACAM-1 Induced CSF3-receptor Downregulation in Bone Marrow Associated With Refractory Neutropenia in Advanced Cirrhosis.

Background and AimsCirrhosis patients exhibit cytopenia, and, at times refractory neutropenia to granulocyte colony-stimulating factor (G-CSF), which acts through the CSF3-receptor (CSF3R), and changes in CSF3R can affect the response. We conducted this study to assess the CSF3R status and its relevance in cirrhotic patients.MethodsCirrhotic patients (n=127) and controls (n=26) with clinically indicated bone marrow (BM) examination were studied. BM assessment was done by qRT-PCR and immunohistochemistry (IHC) for CSF3R. Circulating G-CSF, CSF3R, and carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) were measured. BM hematopoietic precursor cells and their alterations were examined by flow cytometry. The findings were validated in liver cirrhosis patients who received G-CSF for severe neutropenia.ResultsThe mean age was 48.6±13.4 years, and 80.3% were men. Circulatory CSF3R reduction was noted with the advancement of cirrhosis, and confirmed by qRT-PCR and IHC in BM. CSF3R decline was related to decreased hematopoietic stem cells (HSCs) and downregulation of CSF3R in the remaining HSCs. Cocultures confirmed that CEACAM1 led to CSF3R downregulation in BM cells by possible lysosomal degradation. Baseline low peripheral blood-(PB)-CSF3R also predisposed development of infections on follow-up. Decreased CSF3R was also associated with nonresponse to exogenous G-CSF treatment of neutropenia.ConclusionsAdvanced liver cirrhosis was associated with low CSF3R and high CEACAM1 levels in the BM and circulation, making patients prone to infection and inadequate response to exogenous G-CSF.

SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects.

SUMMARYDevelopmental factors may regulate the expression of immune modulatory proteins in cancer, linking embryonic development and cancer cell immune evasion. This is particularly relevant in melanoma because immune checkpoint inhibitors are commonly used in the clinic. SRY-box transcription factor 10 (SOX10) mediates neural crest development and is required for melanoma cell growth. In this study, we investigate immune-related targets of SOX10 and observe positive regulation of herpesvirus entry mediator (HVEM) and carcinoembryonic-antigen cell-adhesion molecule 1 (CEACAM1). Sox10 knockout reduces tumor growth in vivo, and this effect is exacerbated in immune-competent models. Modulation of CEACAM1 expression but not HVEM elicits modest effects on tumor growth. Importantly, Sox10 knockout effects on tumor growth are dependent, in part, on CD8+ T cells. Extending this analysis to samples from patients with cutaneous melanoma, we observe a negative correlation with SOX10 and immune-related pathways. These data demonstrate a role for SOX10 in regulating immune checkpoint protein expression and anti-tumor immunity in melanoma.

Identification of KLF6/PSGs and NPY-Related USF2/CEACAM Transcriptional Regulatory Networks via Spinal Cord Bulk and Single-Cell RNA-Seq Analysis.

Background To further understand the development of the spinal cord, an exploration of the patterns and transcriptional features of spinal cord development in newborn mice at the cellular transcriptome level was carried out. Methods The mouse single-cell sequencing (scRNA-seq) dataset was downloaded from the GSE108788 dataset. Single-cell RNA-Seq (scRNA-Seq) was conducted on cervical and lumbar spinal V2a interneurons from 2 P0 neonates. Single-cell analysis using the Seurat package was completed, and marker mRNAs were identified for each cluster. Then, pseudotemporal analysis was used to analyze the transcription changes of marker mRNAs in different clusters over time. Finally, the functions of these marker mRNAs were assessed by enrichment analysis and protein-protein interaction (PPI) networks. A transcriptional regulatory network was then constructed using the TRRUST dataset. Results A total of 949 cells were screened. Single-cell analysis was conducted based on marker mRNAs of each cluster, which revealed the heterogeneity of neonatal mouse spinal cord neuronal cells. Functional analysis of pseudotemporal trajectory-related marker mRNAs suggested that pregnancy-specific glycoproteins (PSGs) and carcinoembryonic antigen cell adhesion molecules (CEACAMs) were the core mRNAs in cluster 3. GSVA analysis then demonstrated that the different clusters had differences in pathway activity. By constructing a transcriptional regulatory network, USF2 was identified to be a transcriptional regulator of CEACAM1 and CEACAM5, while KLF6 was identified to be a transcriptional regulator of PSG3 and PSG5. This conclusion was then validated using the Genotype-Tissue Expression (GTEx) spinal cord transcriptome dataset. Conclusions This study completed an integrated analysis of a single-cell dataset with the utilization of marker mRNAs. USF2/CEACAM1&5 and KLF6/PSG3&5 transcriptional regulatory networks were identified by spinal cord single-cell analysis.

Fusobacterium nucleatum CbpF Mediates Inhibition of T Cell Function Through CEACAM1 Activation.

F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4+ T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.

Nano-Strategies Targeting the Integrin αvβ3 Network for Cancer Therapy

Integrin αvβ3, a cell surface receptor, participates in signaling transduction pathways in cancer cell proliferation and metastasis. Several ligands bind to integrin αvβ3 to regulate proliferation and metastasis in cancer cells. Crosstalk between the integrin and other signal transduction pathways also plays an important role in modulating cancer proliferation. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) activates the downstream integrin FAK to stimulate biological activities including cancer proliferation and metastasis. Blockage of signals related to integrin αvβ3 was shown to be a promising target for cancer therapies. 3,3′,5,5′-tetraiodothyroacetic acid (tetrac) completely binds to the integrin with the thyroid hormone to suppress cancer proliferation. The (E)-stilbene analog, resveratrol, also binds to integrin αvβ3 to inhibit cancer growth. Recently, nanotechnologies have been used in the biomedical field for detection and therapeutic purposes. In the current review, we show and evaluate the potentiation of the nanomaterial carrier RGD peptide, derivatives of PLGA-tetrac (NDAT), and nanoresveratrol targeting integrin αvβ3 in cancer therapies.

Molecular targets for diagnostic and intraoperative imaging of pancreatic ductal adenocarcinoma after neoadjuvant FOLFIRINOX treatment

Neoadjuvant systemic treatment is increasingly being integrated in the standard treatment of pancreatic ductal adenocarcinoma (PDAC) patients to improve oncological outcomes. Current available imaging techniques remain unreliable in assessing response to therapies, as they cannot distinguish between (vital) tumor tissue and therapy induced fibrosis (TIF). Consequently, resections with tumor positive margins and subsequent early post-operative recurrences occur and patients eligible for potential radical resection could be missed. To optimize patient selection and monitor results of neoadjuvant treatment, PDAC-specific diagnostic and intraoperative molecular imaging methods are required. This study aims to evaluate molecular imaging targets for PDAC after neoadjuvant FOLFIRINOX treatment. Expression of integrin αvβ6, carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5), mesothelin, prostate-specific membrane antigen (PSMA), urokinase-type plasminogen activator receptor, fibroblast activating receptor, integrin α5 subunit and epidermal growth factor receptor was evaluated using immunohistochemistry. Immunoreactivity was determined using the semiquantitative H-score. Resection specimens from patients after neoadjuvant FOLFIRINOX treatment containing PDAC (n = 32), tumor associated pancreatitis (TAP) and TIF (n = 15), normal pancreas parenchyma (NPP) (n = 32) and tumor positive (n = 24) and negative (n = 56) lymph nodes were included. Integrin αvβ6, CEACAM5, mesothelin and PSMA stainings showed significantly higher expression in PDAC compared to TAP and NPP. No expression of αvβ6, CEACAM5 and mesothelin was observed in TIF. Integrin αvβ6 and CEACAM5 allow for accurate metastatic lymph node detection. Targeting integrin αvβ6, CEA, mesothelin and PSMA has the potential to distinguish vital PDAC from fibrotic tissue after neoadjuvant FOLFIRINOX treatment. Integrin αvβ6 and CEACAM5 detect primary tumors and tumor positive lymph nodes.

BRAF inhibitor resistance of melanoma cells triggers increased susceptibility to natural killer cell-mediated lysis

BackgroundTargeted therapies and immunotherapies are first-line treatments for patients with advanced melanoma. Serine–threonine protein kinase B-RAF (BRAF) and mitogen-activated protein kinase (MEK) inhibition leads to a 70% response rate in...

Cell-dependent regulation of vasculogenic mimicry by carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1)

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.

Abstract LB-C15: EpCAM-high liver cancer stem cells resist natural killer cell-mediated cytotoxicity by upregulating CEACAM1

Abstract Natural killer (NK) cells are innate lymphoid cells with potent cytolytic functions that defend the host against microbial infections and tumours. Here, we investigated the cytotoxic effects of NK cells on epithelial cell adhesion molecule (EpCAM)-expressing liver cancer stem cells (CSCs). The frequency of recurrence after curative resection was higher in patients with peritumoural EpCAM expression than patients with pantumoural or negative EpCAM expression. Co-culture experiments demonstrated that EpCAMhigh Huh-7 cells resisted NK cell-mediated cytotoxicity. Silencing carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), which is overexpressed on the surface of EpCAMhigh Huh-7 cells and other hepatoma cells or primary hepatocytes, restored cytotoxicity of NK cells against EpCAMhigh Huh-7 cells. Neutralization of CEACAM1 on the NK cells also enhanced the killing of Huh-7 cells, suggesting CEACAM1 expressed on the NK cells transmits inhibitory signals into the NK cells. EpCAMhigh Hepa1-6 cells formed larger tumours with higher CEACAM1 expression levels in vivo. Neutralization of CEACAM1 in Hepa1-6 xenograft mouse model resulted in more activated phenotype of tumour-infiltrating NK cells and delayed growth of the tumour. Finally, CEACAM1 expression positively correlated with EpCAM expression in human HCC tissues and the serum CEACAM1 levels were also significantly higher in patients with EpCAM+ HCC. Moreover, analyses with TCGA dataset revealed that EpCAM expression shows positive correlation with CEACAM1 expression in various types of cancers. In conclusion, our data demonstrated that EpCAMhigh liver CSCs resist NK cell-mediated cytotoxicity by upregulating CEACAM1 expression. Citation Format: Pil Soo Sung, Seung Kew Yoon. EpCAM-high liver cancer stem cells resist natural killer cell-mediated cytotoxicity by upregulating CEACAM1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-C15. doi:10.1158/1535-7163.TARG-19-LB-C15