Hypoxia exosome derived CEACAM5 promotes tumor-associated macrophages M2 polarization to accelerate pancreatic neuroendocrine tumors metastasis via MMP9.

Exosomes play significant roles in the communications between tumor cells and tumor microenvironment. However, the specific mechanisms by which exosomes modulate tumor development under hypoxia in pancreatic neuroendocrine tumors (pNETs) are not well understood. This study aims to investigate these mechanisms and made several important discoveries. We found that hypoxic exosomes derived from pNETs cells can activate tumor-associated macrophages (TAM) to the M2 phenotype, in turn, the M2-polarized TAM, facilitate the migration and invasion of pNETs cells. Further investigation revealed that CEACAM5, a protein highly expressed in hypoxic pNETs cells, is enriched in hypoxic pNETs cell-derived exosomes. Hypoxic exosomal CEACAM5 was observed to induce M2 polarization of TAM through activation of the MAPK signaling pathway. Coculturing pNETs cells with TAM or treated with hypoxic exosomes enhanced the metastatic capacity of pNETs cells. In conclusion, these findings suggest that pNETs cells generate CEACAM5-rich exosomes in a hypoxic microenvironment, which in turn polarize TAM promote malignant invasion of pNETs cells. Targeting exosomal CEACAM5 could potentially serve as a diagnostic and therapeutic strategy for pNETs.

Detection and clinical significance of CEACAM5 methylation in colorectal cancer patients.

Colorectal cancer (CRC) is a globally common cancer, and the serum carcinoembryonic antigen (sCEA) is widely applied as a diagnostic and prognostic tumor marker in CRC. This study aimed to elucidate the mechanism of CEA expression and corresponding clinical features to improve prognostic assessments. In CRC cells, hypomethylation of the CEACAM5 promoter enhanced CEA expression in HCT116 and HT29 cells with 5-aza-2'-deoxycytidine (5-Aza-dC) treatment. Our clinical data indicated that 64.7% (101/156) of CRC patients had an sCEA level above the normal range, and 76.2% (77/101) of those patients showed a lower average CpG methylation level of the CEACAM5 promoter. The methylation analysis showed that both CRC cell lines and patient samples shared the same critical methylation CpG regions at -200 to -500 and -1000 to -1400 bp of the CEACAM5 promoter. Patients with hypermethylation of the CEACAM5 promoter showed features of a BRAF mutation, TGFB2 mutation, microsatellite instability-high, and preference for right-sided colorectal cancer and peritoneal seeding presentation that had a similar clinical character to the consensus molecular subtype 1 (CMS1) of colorectal cancer. Additionally, hypermethylation of the CEACAM5 promoter combined with evaluated sCEA demonstrated the worst survival among the patients. Therefore, the methylation status of the CEACAM5 promoter also served as an effective biomarker for assessing disease prognosis. Results indicated that DNA methylation is a major regulatory mechanism for CEA expression in colorectal cancer. Moreover, our data also highlighted that patients in a subgroup who escaped from inactivation by DNA methylation had distinct clinical and pathological features and the worst survival.

New CEACAM-targeting 2A3 single-domain antibody-based chimeric antigen receptor T-cells produce anticancer effects in vitro and in vivo.

Recently, a breakthrough immunotherapeutic strategy of chimeric antigen receptor (CAR) T-cells has been introduced to hematooncology. However, to apply this novel treatment in solid cancers, one must identify suitable molecular targets in the tumors of choice. CEACAM family proteins are involved in the progression of a range of malignancies, including pancreatic and breast cancers, and pose attractive targets for anticancer therapies. In this work, we used a new CEACAM-targeted 2A3 single-domain antibody-based chimeric antigen receptor T-cells to evaluate their antitumor properties in vitro and in animal models. Originally, 2A3 antibody was reported to target CEACAM6 molecule; however, our in vitro co-incubation experiments showed activation and high cytotoxicity of 2A3-CAR T-cells against CEACAM5 and/or CEACAM6 high human cell lines, suggesting cross-reactivity of this antibody. Moreover, 2A3-CAR T-cells tested in vivo in the BxPC-3 xenograft model demonstrated high efficacy against pancreatic cancer xenografts in both early and late intervention treatment regimens. Our results for the first time show an enhanced targeting toward CEACAM5 and CEACAM6 molecules by the new 2A3 sdAb-based CAR T-cells. The results strongly support the further development of 2A3-CAR T-cells as a potential treatment strategy against CEACAM5/6-overexpressing cancers.

CEACAM5 and TROP2 define metaplastic and dysplastic transitions in human antral gastric precancerous lesions and tumors.

BACKGROUND: Mucosal gastric atrophy and intestinal metaplasia (IM) increase the risk for the development of gastric cancer (GC) as they represent a field for development of dysplasia and intestinal-type gastric adenocarcinoma. METHODS: We have investigated the expression of two dysplasia markers, CEACAM5 and TROP2, in human antral IM and gastric tumors to assess their potential as molecular markers. RESULTS: In the normal antral mucosa, weak CEACAM5 and TROP2 expression was only observed in the foveolar epithelium, while inflamed antrum exhibited increased expression of both markers. Complete IM exhibited weak CEACAM5 expression at the apical surface, but no basolateral TROP2 expression. On the other hand, incomplete IM demonstrated high levels of both CEACAM5 and TROP2 expression. Notably, incomplete IM with dysplastic morphology (dysplastic incomplete IM) exhibited higher levels of CEACAM5 and TROP2 expression compared to incomplete IM without dysplastic features (simple incomplete IM). In addition, dysplastic incomplete IM showed diminished SOX2 and elevated CDX2 expression compared to simple incomplete IM. CEACAM5 and TROP2 positivity in incomplete IM was similar to that of gastric adenomas and GC. Significant association was found between CEACAM5 and TROP2 positivity and histology of GC. CONCLUSIONS: These findings support the concept that incomplete IM is more likely associated with GC development. Overall, our study provides evidence of the heterogeneity of gastric IM and the distinct expression profiles of CEACAM5 and TROP2 in dysplastic incomplete IM. Our findings support the potential use of CEACAM5 and TROP2 as molecular markers for identifying individuals with a higher risk of GC development in the context of incomplete IM.

A novel human single-domain antibody-drug conjugate targeting CEACAM5 exhibits potent in vitro and in vivo antitumor activity.

Leveraging the specificity of antibody to deliver cytotoxic agent into tumor, antibody-drug conjugates (ADCs) have become one of the hotspots in the development of anticancer therapies. Although significant progress has been achieved, there remain challenges to overcome, including limited penetration into solid tumors and potential immunogenicity. Fully human single-domain antibodies (UdAbs), with their small size and human nature, represent a promising approach for addressing these challenges. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycosylated cell surface protein that rarely expressed in normal adult tissues but overexpressed in diverse cancers, taking part in tumorigenesis, progression, and metastasis. In this study, we investigated the therapeutic potential of UdADC targeting CEACAM5. We performed biopanning in our library and obtained an antibody candidate B9, which bound potently and specifically to CEACAM5 protein (KD = 4.84 nM) and possessed excellent biophysical properties (low aggregation tendency, high homogeneity, and thermal stability). The conjugation of B9 with a potent cytotoxic agent, monomethyl auristatin E (MMAE), exhibited superior antitumor efficacy against CEACAM5-expressing human gastric cancer cell line MKN-45, human pancreatic carcinoma cell line BxPC-3 and human colorectal cancer cell line LS174T with IC50 values of 38.14, 25.60, and 101.4 nM, respectively. In BxPC-3 and MKN-45 xenograft mice, administration of UdADC B9-MMAE (5 mg/kg, i.v.) every 2 days for 4 times markedly inhibited the tumor growth without significant change in body weight. This study may have significant implications for the design of next-generation ADCs.

Identification of a CEACAM5 targeted nanobody for positron emission tomography imaging and near-infrared fluorescence imaging of colorectal cancer.

PURPOSE: Here, we aim to identify a CEACAM5-targeted nanobody and demonstrate its application in positron emission tomography (PET) imaging and near-infrared (NIR) fluorescence imaging in colorectal cancer (CRC). METHODS: Immunohistochemistry was applied to verify CEACAM5 expression in CRC and metastatic lymph nodes (mLNs). CEACAM5-targeted nanobodies were obtained by immunization of human CEACAM5 protein in a dromedary, followed by several rounds of phage screenings. Immunofluorescence staining and flow cytometry was carried out to determine the binding affinity of the nanobodies. The nanobodies were radiolabeled by coupling 18F-SFB for PET imaging of CRC subcutaneous xenografts and lymph node metastasis (LNM). IRDye800CW (IR800) were conjugated to form NIR probes for NIR imaging in CRC subcutaneous models. RESULTS: CEACAM5 was overexpressed in either human CRC tissues or mLNs. A CEACAM5 targeted nanobody, Nb41 was successfully generated, with excellent in vitro binding properties. Incorporation of albumin binding domain (ABD) did not affect the affinity of Nb41. In vivo imaging showed that both 18F-FB-Nb41 and 18F-FB-Nb41-ABD showed obvious accumulation in the tumor. Due to the longer retention in the blood, 18F-FB-Nb41-ABD enrichment in tumors was significantly delayed but higher compared to 18F-FB-Nb41. Both 18F-FB-Nb41 and 18F-FB-Nb41-ABD showed prominent LNM enrichment. Similarly, the IR800-conjugated nanobodies Nb41-IR800 and Nb41-ABD-IR800 exhibited superior imaging effects in subcutaneous models, while Nb41-ABD-IR800 exhibited higher fluorescence intensity in the tumor accompanied with a remarkedly delay compared to Nb41-IR800. CONCLUSION: Collectively, we presented the identification and in vivo validation of a CEACAM5-targeted nanobody and a fused nanobody with an ABD, which enabled to the non-invasive visualization of malignancy of CRC using PET imaging and NIR imaging in subcutaneous models as well as LNM models.

Intraoperative molecular imaging of colorectal lung metastases with SGM-101: a feasibility study.

PURPOSE: Metastasectomy is a common treatment option for patients with colorectal lung metastases (CLM). Challenges exist with margin assessment and identification of small nodules, especially during minimally invasive surgery. Intraoperative fluorescence imaging has the potential to overcome these challenges. The aim of this study was to assess feasibility of targeting CLM with the carcinoembryonic antigen (CEA) specific fluorescent tracer SGM-101. METHODS: This was a prospective, open-label feasibility study. The primary outcome was the number of CLM that showed a true positive fluorescence signal with SGM-101. Fluorescence positive signal was defined as a signal-to-background ratio (SBR) ≥ 1.5. A secondary endpoint was the CEA expression in the colorectal lung metastases, assessed with the immunohistochemistry, and scored by the total immunostaining score. RESULTS: Thirteen patients were included in this study. Positive fluorescence signal with in vivo, back table, and closed-field bread loaf imaging was observed in 31%, 45%, and 94% of the tumors respectively. Median SBRs for the three imaging modalities were 1.00 (IQR: 1.00-1.53), 1.45 (IQR: 1.00-1.89), and 4.81 (IQR: 2.70-7.41). All tumor lesions had a maximum total immunostaining score for CEA expression of 12/12. CONCLUSION: This study demonstrated the potential of fluorescence imaging of CLM with SGM-101. CEA expression was observed in all tumors, and closed-field imaging showed excellent CEA specific targeting of the tracer to the tumor nodules. The full potential of SGM-101 for in vivo detection of the tracer can be achieved with improved minimal invasive imaging systems and optimal patient selection. TRIAL REGISTRATION: The study was registered in ClinicalTrial.gov under identifier NCT04737213 at February 2021.

Identification of CEACAM5 as a stemness-related inhibitory immune checkpoint in pancreatic cancer.

BACKGROUND: Immunotherapy has emerged as a new cancer treatment modality. However, tumour heterogeneity can diminish checkpoint blockade response and shorten patient survival. As a source of tumour heterogeneity, cancer stem cells act as an indispensable reservoir for local recurrence and distant metastasis. Thus, precision immunotherapy targeting tumour heterogeneity requires a comprehensive understanding of cancer stem cell immunology. Our study aimed to identify stemness-related inhibitory immune checkpoints and relevant regulatory pathways in pancreatic cancer. METHODS: Pancreatic cancer-specific datasets in The Cancer Genome Atlas and the Cancer Therapeutics Response Portal were collected for in-depth bioinformatic analysis. Differentially expressed genes between pancreatic cancers with high and low stemness index (mRNAsi) scores were compared to screen out inhibitory immune checkpoints. Survival analysis was used to predict the prognostic value of immune checkpoint plus immune infiltrate in patients with pancreatic cancer. The expression of stemness-related immune checkpoint across immune subtypes of pancreatic cancer was detected and gene set enrichment analysis was performed to figure out the relevant regulatory signallings. RESULTS: The abundance of cancer stemness predicted a low immunotherapy response to pancreatic cancer. The inhibitory immune checkpoint CEACAM5 that was enriched in pancreatic cancers with high mRNAsi scores also exhibited a strong correlation with invasive cell-enriched signature and Msi+ tumour-initiating cell-enriched signature. Levels of CEACAM5 expression were higher in the interferon-γ dominant immune subtype of pancreatic cancers that are characterized by high M1 macrophage infiltration. The patient group with high levels of CEACAM5 expression had a high risk of poor overall survival, even if accompanied by high infiltration of M1 macrophages. Furthermore, prostanoid and long-chain unsaturated fatty acid metabolic processes showed a significant association with cancer stemness and CEACAM5 expression. CONCLUSIONS: Our findings suggest that CEACAM5 is a candidate stemness-related innate immune checkpoint in pancreatic cancer, and is potentially regulated by prostanoid and long-chain unsaturated fatty acid metabolic processes. Immune checkpoint blockade of CEACAM5, which synergizes with inhibition of those regulatory pathways, may improve the efficacy of precision immunotherapy targeting tumour heterogeneity caused by cancer stem cells.

CEACAM5 inhibits the lymphatic metastasis of head and neck squamous cell carcinoma by regulating epithelial-mesenchymal transition via inhibiting MDM2.

Lymph node (LN) metastasis affects both the management and prognosis of head and neck squamous cell carcinoma (HNSCC). Here, we explored the relationship between lymphatic metastasis and CEA family member 5 (CEACAM5), including its possible regulatory role in HNSCC. The levels of CEACAM5 in tissues from patients with HNSCC, with and without LN metastases, were assessed by transcriptome sequencing. The associations between CEACAM5 and the N stage of LN metastasis in HNSCC were predicted through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and a pan-cancer analysis of CEACAM5 expression in 33 common human tumors was conducted. CEACAM5 levels were analyzed in tumor and normal tissue specimens from HNSCC patients and the correlation between CEACAM5 levels and prognosis was evaluated. The influence of CEACAM5 on cell proliferation, invasion, migration, and apoptosis was investigated in HNSCC cell lines, as were the downstream regulatory mechanisms. A mouse model of LN metastasis was constructed. CEACAM5 levels were significantly higher in HNSCC tissue without LN metastasis than in that with LN metastasis. Similar findings were obtained for the clinical specimens. CEACAM5 levels were associated with better clinical prognosis. CEACAM5 was found to inhibit the proliferation and migration and promote the apoptosis of HNSCC cells. A mouse xenograft model showed that CEACAM5 inhibited LN metastasis. In conclusions, CEACAM5 inhibited epithelial-mesenchymal transition (EMT) in HNSCC by reducing murine double minute 2 (MDM2) expression and thereby suppressing LN metastasis. CEACAM5 has potential as both a prognostic marker and a therapeutic target in HNSCC.

Cartilage Oligomeric Matrix Protein, COMP may be a Better Prognostic Marker Than CEACAM5 and Correlates With Colon Cancer Molecular Subtypes, Tumor Aggressiveness and Overall Survival.

BACKGROUND: New tumor biomarkers are needed to improve the management of colon cancer (CC), the second leading cause of cancer-related deaths in the United States. Carcinoembryonic Antigen (CEA), the translated protein of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) gene, is used as a biomarker for CC. Cartilage Oligomeric Matrix Protein (COMP) is overexpressed in CC compared to normal colon tissues. This study aims to evaluate the expression of COMP by disease stage, consensus molecular subtype (CMS), its impact on disease outcomes, and comparison to CEACAM5. MATERIALS AND METHODS: RNA-seq data from 456 CC The Cancer Genome Atlas samples and 41 matching control samples were analyzed for COMP expression and CEACAM5 expression. We stratified tumor samples by stage (I-IV), subtype (CMS1-CMS4), tumor location, and Kirsten RAt Sarcoma (KRAS) mutant status and three quartiles were established based on COMP expression. Kaplan Meier survival outcomes were evaluated. RESULTS: COMP expression was significantly higher in tumor samples, with elevation of expression occurring in stage I and significantly increasing in stage IV. Increased COMP expression occurs in CMS4 with relatively low expression in CMS3. No significant expression difference was attributed to tumor location and KRAS mutant status. Compared to CEACAM5, COMP was a stronger molecular marker across stages and subtypes. CMS4 was associated with the high COMP expression, and higher levels of COMP were associated with poorer overall survival, disease-specific survival, and tumor progression-free intervals. CMS2 and 3 were associated with low expression and better survival. CONCLUSION: COMP is a potential molecular biomarker for CC and may be superior to CEA as an indicator of CC.

Integrated multiple analytes and semi-mechanistic population pharmacokinetic model of tusamitamab ravtansine, a DM4 anti-CEACAM5 antibody-drug conjugate.

Tusamitamab ravtansine (SAR408701) is an antibody-drug conjugate (ADC), combining a humanized monoclonal antibody (IgG1) targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and a potent cytotoxic maytansinoid derivative, DM4, inhibiting microtubule assembly. SAR408701 is currently in clinical development for the treatment of advanced solid tumors expressing CEACAM5. It is administered intravenously as a conjugated antibody with an average Drug Antibody Ratio (DAR) of 3.8. During SAR408701 clinical development, four entities were measured in plasma: conjugated antibody (SAR408701), naked antibody (NAB), DM4 and its methylated metabolite (MeDM4), both being active. Average DAR and proportions of individual DAR species were also assessed in a subset of patients. An integrated and semi-mechanistic population pharmacokinetic model describing the time-course of all entities in plasma and DAR measurements has been developed. All DAR moieties were assumed to share the same drug disposition parameters, excepted for clearance which differed for DAR0 (i.e. NAB entity). The conversion of higher DAR to lower DAR resulted in a DAR-dependent ADC deconjugation and was represented as an irreversible first-order process. Each conjugated antibody was assumed to contribute to DM4 formation. All data were fitted simultaneously and the model developed was successful in describing the pharmacokinetic profile of each entity. Such a structural model could be translated to other ADCs and gives insight of mechanistic processes governing ADC disposition. This framework will further be expanded to evaluate covariates impact on SAR408701 pharmacokinetics and its derivatives, and thus can help identifying sources of pharmacokinetic variability and potential efficacy and safety pharmacokinetic drivers.

Site-Specific N-Linked Glycosylation Analysis of Human Carcinoembryonic Antigen by Sheathless Capillary Electrophoresis-Tandem Mass Spectrometry.

With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X].

Assessment of the Efficacy and Clinical Utility of Different Circulating Tumor Cell (CTC) Detection Assays in Patients with Chemotherapy-Naïve Advanced or Metastatic Non-Small Cell Lung Cancer (NSCLC).

We herein investigated the detection frequency and clinical relevance of circulating tumor cells (CTCs) in chemotherapy-naïve stage IIIB/IV non-small cell lung cancer (NSCLC), by using the CellSearch and real-time CEACAM5mRNA assays. Blood samples from 43 patients were obtained at different time points during first-line chemotherapy. CellSearch revealed the detection of ≥1 CTCs in 41.9%, 40.9%, and 16.7% of patients at baseline, post-1st, and post-2nd treatment cycle, respectively, and of ≥5 CTCs in 11.6%, 9.1%, and 5.6%, respectively. CEACAM5mRNA+ CTCs were detected in 29.3% and 16% of patients pre- and post-treatment, respectively. The positivity concordance between the two assays was 2.2%. CTC-detection by CellSearch (≥5 CTCs: p = 0.004), CEACAM5mRNA (p = 0.010), or by any assay (p = 0.000) was associated with disease progression. Reduced survival was demonstrated for patients harboring ≥5 CTCs (progression-free survival; PFS: p = 0.000; overall survival; OS: p = 0.009), CEACAM5mRNA+ CTCs (PFS: p = 0.043; OS: p = 0.039), and CTCs by any assay (PFS: p = 0.005; OS: p = 0.006, respectively). CTC-detection by any assay independently predicted for increased risk of relapse (hazard ratio; HR: 3.496; p = 0.001) and death (HR: 2.866; p = 0.008). CellSearch-positivity either pre-, post-1st, or post-2nd cycle, was predictive for shorter PFS (p = 0.036) compared to negativity in all time points. Persistent CEACAM5mRNA-positivity pre- and post-treatment was associated with reduced PFS (p = 0.036) and OS (p = 0.026). In conclusion, CTC detection and monitoring using the CellSearch and CEACAM5mRNA assays provides valuable and complementary clinical information for chemo-naïve advanced or metastatic NSCLC.

T-cell bispecific antibodies in node-positive breast cancer: novel therapeutic avenue for MHC class I loss variants.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) represent a prognostic factor for survival in primary breast cancer (BC). Nonetheless, neoepitope load and TILs cytolytic activity are modest in BC, compromising the efficacy of immune-activating antibodies, which do not yet compete against immunogenic chemotherapy. PATIENTS AND METHODS: We analyzed by functional flow cytometry the immune dynamics of primary and metastatic axillary nodes [metastatic lymph nodes (mLN)] in early BC (EBC) after exposure to T-cell bispecific antibodies (TCB) bridging CD3ε and human epidermal growth factor receptor 2 (HER2) or Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 (CEACAM5), before and after chemotherapy. Human leukocyte antigen (HLA) class I loss was assessed by whole exome sequencing and immunohistochemistry. One hundred primary BC, 64 surrounding 'healthy tissue' and 24 mLN-related parameters were analyzed. RESULTS: HLA loss of heterozygosity was observed in EBC, at a clonal and subclonal level and was associated with regulatory T cells and T-cell immunoglobulin and mucin-domain-3 expression restraining the immuno-stimulatory effects of neoadjuvant chemotherapy. TCB bridging CD3ε and HER2 or CEACAM5 could bypass major histocompatibility complex (MHC) class I loss, partially rescuing T-cell functions in mLN. CONCLUSION: TCB should be developed in BC to circumvent low MHC/peptide complexes.

KRT19 and CEACAM5 mRNA-marked circulated tumor cells indicate unfavorable prognosis of breast cancer patients.

AIM: To investigate the clinical and prognostic significance of circulated tumor cells (CTC) marked by cytokeratin 19 coding gene KRT19 mRNA and carcinoembryonic antigen coding gene CEACAM5 mRNA in preoperative peripheral blood of breast cancer patients and provide molecular markers for breast cancer metastasis risk. METHODS: The mRNA levels of KRT19 and CEACAM5 in preoperative peripheral blood of breast cancer patients without (n = 603) and with (n = 76) distant metastases at the time of initial diagnosis were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The relationship between CTCKRT19, CTCCEACAM5 and clinicopathological features, local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), or overall survival (OS) was statistically analyzed. RESULTS: In different pathological stages of breast cancer, the rates of CTCKRT19-pos and CTCCEACAM5-pos increased with the increase of the stages (P = 0.077 and P = 0.004). Preoperative CTCKRT19-pos in breast cancer patients was closely related to the lymph node metastasis statues (P < 0.0001), and had no significant correlation with other clinicopathological features. There was no significant correlation between CTCCEACAM5 and the clinicopathological features. Patients with high levels of CTC double-marked by KRT19 and CEACAM5 mRNA had shorter DMFS (P < 0.0001) and OS (P = 0.016) for patients with breast cancer. The 7-year DMFS rates for the low-, intermediate-, and high-risk groups were 90.7%, 67.5%, and 59.1%, respectively (P < 0.0001). The prognosis of patients with decreased KRT19 and CEACAM5 mRNA after treatment is better than that of patients who have not decreased, and the combination of the two indicators is better than the single one for predicting PFS (P = 0.002 compare with P = 0.036 or P = 0.047). CONCLUSION: Double-marked CTC by KRT19 and CEACAM5 mRNA is a prognostic index of breast cancer patients before surgery and after chemotherapy. Single-marked CTC by KRT19 mRNA indicates lymph node statues of preoperative patients. Therefore, the RT-qPCR-based molecular diagnosis of CTC could be used for prognostic prediction of breast cancer patients and guiding clinical treatment.

An efficient constitutive expression system for Anti-CEACAM5 nanobody production in the yeast Pichia pastoris.

Nanobodies offer multiple advantages over conventional antibodies in terms of size, stability, solubility, immunogenicity, and production costs, with improved tumor uptake and blood clearance. Additionally, the recombinant expression of nanobodies is robust in various expression systems, such as Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris. P. pastoris is the most widely used microorganism for nanobody production, but all or almost all expression vectors developed for this system are based on the regulated promoter of the alcohol oxidase 1 gene (AOX1) that requires methanol for full induction. In this study, a constitutive anti-CEACAM5 nanobody expression system was constructed under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) promoter. The effects of different carbon sources and pH on nanobody expression were evaluated in shaking flask cultures. After 96 h of constitutive expression in shaking flask, a yield of 51.71 mg/L was obtained. In addition, this constitutive expression system produced nanobodies at equivalent yield and affinity to that produced by methanol-induced expression. The results of this study indicated that the use of a constitutive expression system is a promising alternative for the production of nanobodies applied for cancer diagnosis and therapy.

Moraxella catarrhalis induces an immune response in the murine lung that is independent of human CEACAM5 expression and long-term smoke exposure.

In patients with chronic obstructive pulmonary disease (COPD), Moraxella catarrhalis infection of the lower airways is associated with chronic colonization and inflammation during stable disease and acute exacerbations. Chronic smoke exposure induces chronic inflammation and impairs mucociliary clearance, thus contributing to bacterial colonization of the lower airways in COPD patients. The human-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, expressed in human airways, has been shown to contribute to epithelial colonization of CEACAM-binding pathogens. To investigate the impact of CEACAM5 expression on pulmonary M. catarrhalis colonization, we infected mice transgenic for human CEACAM5 (hCEACAM5) and wild type mice intratracheally with M. catarrhalis with or without preceding smoke exposure and analyzed bacterial colonization and local and systemic inflammation. Our results show that airway infection with M. catarrhalis accelerated acute local but not systemic inflammation, albeit independent of hCEACAM5 expression. Long-term smoke exposure alone or prior to M. catarrhalis infection did not contribute to increased local or systemic inflammation. No difference was found in pulmonary clearance of M. catarrhalis in hCEACAM5-transgenic mice compared with wild-type mice. Smoke exposure neither altered time nor extent of persistence of M. catarrhalis in the lungs of both genotypes. In conclusion, M. catarrhalis induced a local acute immune response in murine airways. Neither hCEACAM5 expression nor chronic smoke exposure nor a combination of both was sufficient as prerequisites for the establishment of chronic M. catarrhalis colonization. Our results demonstrate the difficulties in mirroring conditions of chronic airways colonization of M. catarrhalis in a murine model.

Preclinical evaluation of a novel CEA-targeting near-infrared fluorescent tracer delineating colorectal and pancreatic tumors.

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA-targeted near-infrared fluorescent (NIRF) tracer, based on a disulfide-stabilized single-chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell-based plate assays and orthotopic colorectal (HT-29, well differentiated) and pancreatic (BXPC-3, poorly differentiated) xenogeneic human-mouse models. NIRF signals were visualized using the clinically compatible FLARE™ imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor-to-background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent-guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.

Improving the therapeutic index in cancer therapy by using antibody-drug conjugates designed with a moderately cytotoxic drug.

The antibody-drug conjugate (ADC), IMMU-130, of the moderately cytotoxic topoisomerase I inhibitor, SN-38, and the CEACAM5-targeted humanized antibody (mAb), labetuzumab, was evaluated in model systems of human colon carcinoma and in phase I clinical trials of heavily pretreated patients with metastatic colorectal cancer. The conjugate, designed with a near-homogeneous drug substitution of 7-8 SN-38/mAb and with a linker that released 50% of the drug in ∼20 h, showed significant antitumor effects compared to a nontargeted ADC in human tumor xenografts, which could be augmented in combination with bevacizumab. The advantage of fractionated dosing was demonstrated, with potential implications for the clinical dosing schedule. Biodistribution comparing IMMU-130 with labetuzumab showed that the conjugate cleared somewhat faster from the blood, but this did not affect tumor uptake and retention. The use of an ultrastable linker in the conjugate design abrogated antitumor effects. A tolerability study in rabbits showed a high safety margin, with no-observed-adverse-effect level (NOAEL) corresponding to a cumulative human-equivalent protein dose of 40-60 mg/kg. The preclinical findings appear to be corroborated in two phase I clinical trials, with high tolerability and evidence of antitumor activity, including objective responses. The impact of the ADC design on the utility of IMMU-130, tailored to a poorly internalizing target, is discussed.

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue.

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.