Membrane-bound Carcinoembryonic Antigen Research Articles

Impact of Different Selectivity between Soluble and Membrane-bound Forms of Carcinoembryonic Antigen (CEA) on the Target-mediated Disposition of Anti-CEA Monoclonal Antibodies.

Carcinoembryonic antigen (CEA) is a tumor-specific antigen overexpressed in multiple cancers. CEA is expressed as a membrane protein, a part of which is cleaved from the cell membrane and secreted into blood. The soluble form of CEA (sCEA) has been shown to accelerate the clearance of anti-CEA antibody, which limits the antibody distribution in the tumor. To overcome this issue, we developed an anti-CEA monoclonal antibody, 15-1-32, which shows a strong affinity for membrane-bound CEA (mCEA) and relatively weak affinity for sCEA. In this study, we compared the effect of sCEA on the pharmacokinetics of 15-1-32 in mice with that of another anti-CEA monoclonal antibody, labetuzumab, showing less selectivity to mCEA than 15-1-32. As expected, the effect of sCEA on the serum concentration of 15-1-32 was much smaller than that of labetuzumab. The decrease in the area under the curve (AUC) of serum concentration was 22.5% for 15-1-32 when it was coadministered with sCEA, while that of labetuzumab was 79.9%. We also compared the pharmacokinetics of these two antibodies in CEA-positive tumor-bearing mice. The AUCs of 15-1-32 and labetuzumab were decreased in tumor-bearing mice compared with non-tumor-bearing mice to a similar extent (approximately 40% decrease). These results suggested that mCEA also contributes to the clearance of anti-CEA antibodies in CEA-positive tumor-bearing mice. Although the increased selectivity to mCEA minimized the effect of sCEA on the pharmacokinetics of 15-1-32, it may be insufficient to improve the pharmacokinetics in CEA-positive cancer patients. SIGNIFICANCE STATEMENT: Because previous studies reported the rapid clearance of anti-CEA antibodies mediated by soluble CEA, we obtained a monoclonal antibody, 15-1-32, selective to membrane-bound CEA and evaluated the effects of CEA on its pharmacokinetics. Although the effect of soluble CEA on the serum concentration of 15-1-32 was very small, the clearance of 15-1-32 in CEA-positive tumor-bearing mice was still rapid, suggesting membrane-bound CEA also contributes to the clearance of anti-CEA antibodies. These results indicated that increasing selectivity to membrane-bound CEA is not enough to improve the pharmacokinetics of anti-CEA antibody.

In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT

Nanobodies are a novel type of immunoglobulinlike, antigen-binding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and (99m)Tc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEA-transfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with (99m)Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected (99m)Tc-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. This study shows the potency of antigen-binding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis

Background:The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested.Methods:The antibody was modified using EBNA cells cotransfected with β-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes.Results:The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcγRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcγRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcγRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model.Conclusion:The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Targeted killing of colorectal cancer cell lines by a humanised IgG1 monoclonal antibody that binds to membrane-bound carcinoembryonic antigen

The distribution of carcinoembryonic antigen (CEA) in colorectal cancer (CRC) differs from that in normal colorectal tissue, being found on all borders of the cell membrane and hence enabling access to intravenous antibody, making CEA a good target for antibody-based therapy. The distinctive anti-CEA antibody, PR1A3, binds only membrane-bound CEA. Humanised PR1A3 (hPR1A3) was assessed both in vitro cytotoxicity and binding assays with colorectal cancer cell lines expressing varying levels of CEA. Human peripheral blood mononuclear cells (PBMCs) and purified natural killer (NK) cells were used as effectors. The in vitro assays demonstrated hPR1A3 CEA-specific binding and antibody-dependent and CEA-specific killing of human colorectal cancer cell lines by human PBMCs. The effect increased with increasing concentration of antibody and surface CEA, and was lost by using the parent murine IgG1 PR1A3. Killing was also blocked by antibody to the Fc-γIIIA receptor. Purified human NK cells were effective at much lower effector:target ratios than unfractionated PBMCs, indicating that NK cells were the main mediators of hPR1A3-based CEA-specific killing. The results support the development of hPR1A3 for therapy of colorectal cancer.

Pretargeted Anti–Carcinoembryonic-Antigen Radioimmunotherapy for Medullary Thyroid Carcinoma

significant survival advantage of patients with progressive medullary thyroid carcinoma treated with pretargeted anti–carcinoembryonic-antigen radioimmunotherapy over an untreated control group matched on calcitonin doubling time (median, 110 months v 61 months; P .030). This survival advantage doubled for biologic responders relative to these controls (median, 159 months v 61 months; P .010), and was still evident when biologic responders were compared with biologic nonresponders (median, 159 months v 109 months; P .035). In delineating clinical risk, Chatal et al relied on calcitonin doubling times, with cut-offs set at 2 years and 5 years. In clinical practice, however, a risk classification based on calcitonin doubling times may not really be helpful because of the need for protracted expectant observation. Anti–carcinoembryonic-antigen radioimmunotherapy is targeted on membrane-bound carcinoembryonic antigen. There is some evidence to suggest that serum levels of carcinoembryonic antigen from patients with medullary thyroid carcinoma may correspond to the expression of carcinoembryonic antigen in the cancer cells. 2,3 In addition, the duration of response to pretargeted anti–carcinoembryonic-antigen radioimmunotherapy was previously shown to correlate with the duration of the blood carcinoembryonic antigen response. 4 The suitability of serum levels of carcinoembryonic antigen for risk stratification of patients with medullary thyroid carcinoma before initial operation has emerged only recently. 5 Preoperative serum levels of carcinoembryonic antigens greater than 30 ng/mL almost always conflicted with surgical curability and were associated with an approximately 70% rate of involvement of the central and lateral lymph node compartment on the side of the primary tumor. This rate of involvement increased to approximately 90% when the preoperative serum level of carcinoembryonic antigen exceeded 100 ng/mL. In this setting, involvement of the lateral lymph node compartment on the side opposite to the primary tumor, and distant metastases were seen in three quarters of the patients at the time of operation. 5 These data suggest that pretherapeutic serum levels of carcinoembryonic antigen, which are available within a few days, may predict the chance of patients with medullary thyroid carcinoma to benefit from anti– carcinoembryonic-antigen radioimmunotherapy. We wonder if Chatal et al have the data to break down their patients with medullary thyroid carcinoma by pretherapeutic serum level of carcinoembryonic antigen and response to pretargeted anti– carcinoembryonic-antigen radioimmunotherapy. This piece of information would further elucidate the effectiveness of this novel therapeutic modality in select subgroups of patients with medullary thyroid carcinoma.

Effective production of carcinoembryonic antigen by conversion of the membrane-bound into a recombinant secretory protein by site-specific mutagenesis

Carcinoembryonic antigen (CEA), the most widely used human tumor marker, is a heavily glycosylated protein over-expressed by a wide range of tumors. It has been indicated that CEA might be a useful target for human anti-tumor immunotherapy. CEA assay for research as well as clinical trials demands a continuous source of CEA protein preparations. In a multi-purpose research program to provide a reliable source for large production of CEA, we converted the membrane-bound carcinoembryonic antigen into a secretory protein by site-specific mutagenesis. We made the secretory CEA protein by introducing a new stop codon at 99 bp upstream of the original stop codon in CEA cDNA by PCR-based mutagenesis. The glycosylation of recombinant CEA proteins, especially those destined for administration to human trials is crucially important. To produce CEA with the same glycosylation pattern and immunogenicity as the native CEA expressed by human tumors in vivo, the truncated CEA cDNA which does not encode the last C-terminal 33-amino acid hydrophobic domain was transfected into HT29, a human colon carcinoma cell line by the calcium phosphate method. Stable transfectants were selected and pooled. CEA secretion from the cells was verified by analysis of the transfectant culture supernatant for CEA protein. As determined by ELISA, 16 microg/L of recombinant CEA was secreted per 106 transfectants within 48 hrs, an increase over 40 times relative to the untransfected cells. The size of the recombinant CEA secreted by HT29 transfectants in our experiment is identical to that of reference CEA secreted from tumors and is fully antigenic. It seems that the C-terminal truncation does not affect CEA glycosylation in HT29 cells. It is predicted that human cancer immunotherapy using recombinant CEA expressed in this system would be more effective than the commercial protein which is usually prepared from bacterial or other heterologous expression systems.

Cleavage of carcinoembryonic antigen induces metastatic potential in colorectal carcinoma

Carcinoembryonic antigen (CEA), a widely used tumor marker, is attached by a glycosylphosphatidylinositol (GPI) anchor motif to the cell membrane. Recent study suggested that membrane-bound CEA might be cleaved by glycosylphosphatidylinositol-phospholipase D (GPI-PLD). We studied the effect of GPI-PLD on the cleavage of CEA to elucidate the implication for metastatic potential in colorectal carcinoma cells. CEA amount of conditioned medium was changed by suramin and phenanthroline (activator and inhibitor of GPI-PLD) only in SW620 and SW837 which expressed both CEA and GPI-PLD mRNA. Suramin treatment also augmented migratory activity and decreased cell surface CEA expression in SW620 and SW837. Furthermore, GPI-PLD knockdown cells using GPI-PLD-specific siRNA in SW620 and SW837 showed decreased CEA secretion from cell membrane and the migration activity, increased membrane-bound CEA amount. Splenic injection of SW620 and SW837 induced marked hepatic metastases in nude mice. These results suggest that membrane-bound CEA is cleaved by GPI-PLD and that this cleavage enhances the metastatic potential in colorectal carcinoma cells.

Morphometric analysis of liver macrophages in patients with colorectal liver metastasis.

Kupffer cells, residential liver macrophages, have binding sites for carcinoembryonic antigen (CEA), but their role in metastatic liver tumor progression has not been well addressed clinically. Liver macrophages were analyzed morphometrically and their relationship with CEA and tumor microvessel density (MVD) was examined in 71 patients who underwent macroscopic curative hepatectomy for metastatic liver tumors from colorectal cancer. In paraffin-embedded sections, MVD was evaluated by CD34-positive cell counts, liver macrophages visualized using anti PG-M1 (CD68) antibody were analyzed, and membrane-bound CEA was assessed by immunoreactivity of tumor cells for CEA. The area of liver macrophages in peritumoral regions (43.0 +/- 11.6 microm2) was significantly larger than that in non-tumor regions (25.2 +/- 24.2 microm2) (P < 0.0001). The liver macrophage density in peritumoral regions was 154 +/- 49 per field, and was significantly higher than in non-tumor regions (74 +/- 24 per field) (P < 0.001). The density and the area of liver macrophages had no correlation with serum CEA levels and the degree of tumor CEA expression. A weak positive correlation was observed between MVD and liver macrophage density (P = 0.0104, Spearman rank correlation coefficient = 0.31). Cox multivariate regression analysis showed that MVD greater than 50 (P = 0.0233, hazard ratio = 2.463), and the area of liver macrophages larger than 40.9 microm2 (P = 0.0485, hazard ratio = 2.127), were significant independent prognostic factors. The present morphometric analysis suggests that the liver macrophages are accumulated and activated in peritumoral regions in patients with colorectal liver metastasis and this accumulation and activation of liver macrophages, with which soluble or membrane-bound CEA might not be associated, are related with patients' poor prognosis.

A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA.

Chimeric T cell receptors with specificity for tumor-associated antigens are successfully used to target T cells to tumor cells. The efficacy of this approach, however, is reduced by soluble antigen that is frequently present in high serum concentrations. To overcome this situation, we constructed an anti-CEA chimeric receptor whose extracellular moiety is composed of a humanized single chain antibody fragment (scFv) derived from the anti-CEA mAb BW431/26 and the CH2/CH3 constant domains of human IgG. The intracellular moiety consists of the gamma-signaling chain of the human Fc epsilon RI receptor constituting a completely humanized chimeric receptor. After transfection, the humBW431/26 scFv-CH2CH3-gamma receptor is expressed as a homodimer on the surface of MD45 T cells. Co-incubation with CEA+ tumor cells specifically activates grafted MD45 T cells indicated by IL-2 secretion and cytolytic activity against CEA+ tumor cells. Notably, the efficacy of receptor-mediated activation is not affected by soluble CEA up to 25 micrograms/ml demonstrating the usefulness of this chimeric receptor for specific cellular activation by membrane-bound CEA even in the presence of high concentrations of CEA, as found in patients during progression of the disease.

A chimeric receptor that selectively targets membrane-bound carcinoembryonic antigen (mCEA) in the presence of soluble CEA

Backvround: Chimeric T cell recptors with specificity for defined tumor associated antigens are valuable tools to target T cells to tumor cells. The function of these receptors in the presence of soluble antigen, however, is crucial because the tumor associated antigen is often shed into the serum of cancer patients. Methods: For this reason we have generated a chimeric T cell receptor with specificity for the membrane bound form of the carcinoembryonic antigen (CEA). This receptor (humBW431/26-CH2/3-T) is composed of a humanized single chain antibody (humBW431/26), that was fused to the Fc domains (CH2/3) of the human lgG1 and the signaling unit of the Fc~R1 receptor t chain ('/). Results: After transfection this chimeric receptor converted MD45 T cells to specificity for CEA+ tumor cells. Only immobilized, especially membrane-bound CEA, could crosslink the chimeric receptor resulting in cellular activation with IL-2 release and cytolytic activity towards CEA+ tumor cells. Cellular activation by membrane bound antigen was also efficient in the presence of high amounts of soluble CEA (up to 25 lag/ml whereas an anti-idiotypic antibody could completely block T cell activation even at low concentrations (0,1 lag/ml). Conclusion: Our data demonstrate, that the chimeric T cell receptor humBW431/26-CH2/3-y has selective specificity for membrane-bound CEA and that targeting of tumor cells by chimeric T cell receptors can be performed even in the presence of high amounts of soluble antigen.

The carcinoembryonic antigen (CEA) modulates effector-target cell interaction by binding to activated lymphocytes.

We and others have shown that the carcinoembryonic antigen (CEA) modulates the susceptibility of human colorectal carcinoma cells to cytotoxic lymphocytes. We now demonstrate that the density of the CEA molecules on the tumor cell surface has a determining influence on its protective function. In contrast, CEA released by tumor cells has no protective effect for CEA-negative cells. To elucidate the responsible mechanism, we analyzed the binding properties of CEA to lymphokine activated killer (LAK) cells. In agreement with our observation that only membrane-bound CEA provides protection, we found that intercellular contact between LAK cells and CEA-expressing tumor cells is required for binding of CEA to LAK cells. Using FACScan analysis, we demonstrate the presence of CEA on lymphocytes co-cultured with CEA-transfected cells but not after co-culture with their parental cells and after incubation with soluble CEA. Interestingly, following overnight co-culture the amount of bound CEA on LAK cells was identical regardless of adherence on tumor cells or loss of contact with tumor cells. This indicates that CEA is released from the tumor cells after binding to LAK cells. Our results suggest that tumor cells can modulate effector cell adhesion by regulating the turnover rate of CEA on the tumor cell membrane.

Electrically promoted protein production by mammalian cells cultured on the electrode surface

Protein production of mammalian cells has been promoted by applying a small constant potential to the surface of an electrode on which cells are cultured. Human carcinoma line of MKN45 cells were cultured on the surface of a platinum-coated plastic plate electrode. Low d.c. voltage of constant potential was applied to the electrode during 4-day culture to modulate the production of carcinoembryonic antigen (CEA). The amounts of both secreted and membrane-bound CEA were dependent on the applied potential during culture. Secreted CEA was more than twice in amount in the potential range from 0.2 V to 0.6 V vs. Ag/Agcl as compared with that of normal culture. In the potential range, CEA was also increased in membrane-bound form. The potential-controlled cell culture may have an enhanced effect on protein production.

Carcinoembryonic antigen is anchored to membranes by covalent attachment to a glycosylphosphatidylinositol moiety: identification of the ethanolamine linkage site.

The COOH-terminal amino acid of carcinoembryonic antigen (CEA) is shown to covalently link with ethanolamine, evidence consistent with the anchorage of CEA to the plasma membrane through a phosphatidylinositol-glycan tail. Purified CEA was digested with trypsin, and the resulting peptides were isolated by reverse-phase HPLC. Tryptic hexapeptide T12, terminating atypically with alanine, corresponded in sequence (Ser-Ile-Thr-Val-Ser-Ala) with the last six residues (637-642) of the third repeating domain in the mature CEA protein. Mass determination of the hexapeptide by fast atom bombardment mass spectrometry suggested the presence of an additional ethanolamine moiety. This finding and the absence of the subsequent 26 hydrophobic residues predicted by cDNA sequence is evidence that hexapeptide T12 is the COOH-terminal peptide of mature CEA. A synthetic peptide identical to hexapeptide T12 was prepared, and ethanolamine was coupled to its COOH-terminal alanine; chromatographic properties of this synthetic ethanolamine-coupled peptide and peptide T12 were the same. B/E-linked-scan mass spectral analysis of the ethanolamine-coupled synthetic peptide and peptide T12 revealed a fragment ion series consistent with the presence of a COOH-terminal ethanolamine. Release of membrane-bound CEA from the CEA-expressing cell line LS 174T was shown by indirect immunofluorescence and flow cytometry after treatment with phosphatidylinositol-specific phospholipase C. We conclude that CEA is processed posttranslationally to remove the hydrophobic COOH-terminal residues (643-668) with subsequent addition of an ethanolamine-glycosylphosphatidylinositol moiety and that treatment of a colonic cell line with phosphatidylinositol-specific phospholipase C releases membrane-bound CEA.