Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Affinity purification of novel bispecific antibodies recognising carcinoembryonic antigen and doxorubicin

We have developed a method which combines Protein A affinity chromatography and HPLC analytical and semi-preparative hydroxyapatite affinity chromatography to purify bispecific antibodies (BsMabs) from hybrid-hybridomas secreting antibodies recognising carcinoembryonic antigen (CEA) and the chemotherapeutic drug doxorubicin (Dox). Elution of the HPLC hydroxyapatite columns with a 60–360 mM phosphate buffer gradient was found to give better separation than elution with a 60–180 mM phosphate buffer gradient. Careful monitoring of HPLC fractions by enzyme linked immunosorbent assays for anti-CEA, anti-Dox and dual anti-CEA/anti-Dox activity, and pooling of fractions on the basis of these results, enabled the purification of novel BsMabs for use in in vitro and preclinical in vivo experiments.     

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.     

The use of Fab-masking antigens to enhance the activity of immobilized antibodies

This study demonstrates that masking the Feb regions of a monoclonal antibody (Mab) with synthetic antigens prior to covalent immobilization efficiency. Water-soluble adducts of poly(2-methyloxazoline) polymers and a syntheticpeptide epitope for the Mab were constructed. These synthetic antigens are referred to as Fab-masking antigents (FMAs). The antibody used in this study is a Ca(2+)-dependent murine monoclonal lgG directed against the plasma protein, human protein C (hPC). The FMAs were pre-equilibrated with Mab in the presence of calcium prior to immobilization and were then removed by EDTA, which destabilized the FMA-Mab complexes. The antigen binding efficiency and accessibility of the Fab domain of the immobilized antibody was significantly increased for Mab immobilized in the presence of FMA relative to those Mab immobilized without FMA. The increase in binding efficiency was most pronounced for the largest FMA employed. No appreciable differences were detected in the avidity of hPC-Mab complexes formed by immunosorbents produced by either masked or unmaked antibody. These results provide evidence that orientgation may play an important role in the binding activity of immobilized antibodies.     

Study of antibody/antigen binding kinetics by total internal reflection ellipsometry

Total internal reflection ellipsometry (TIRE) has been applied for the investigation of (i) kinetics of biosensing layer formation, which was based on the immobilization of fragmented and intact antibodies, and (ii) kinetics of antigen interaction with the immobilized antibodies. It has been demonstrated that ellipsometric parameter Δ(t) showed much higher sensitivity at the initial phase of Au-protein and protein-protein interaction, while the parameter Ψ(t) was more sensitive when the steady-state conditions were established. A new method, which taking into consideration this feature and nonlinear change of Δ(t) and Ψ(t) parameters during various stages of biological layer formation process, was used for the calculation of antibody and antigen adsorption/interaction kinetics. The obtained results were analyzed using a model, which took into account partial reversibility during the formation of both antibody and antigen based monolayers. It was shown that the immobilization rate of antibody during the preparation of the sensing layer was similar for the formation of both intact and fragmented antibody based layers; however, the residence time was 25 times longer for intact antibody based layer formation in comparison to that of fragmented antibody based layer formation. On the contrary, residence time of antigen interaction with immobilized antibodies was about 8 times longer for the sensor based on fragmented antibodies. Moreover, it has been determined that the structural differences of immobilized antibodies (fragmented or intact) significantly influence antibody-antigen interaction rate, the major difference being in the residence time of antigen interaction with both types of immobilized antibodies.     

Isolation of anti-carcinoembryonic antigen (CEA), specific immunoglobulins and their use in immunocytochemical and enzyme-immunoassays (ELISA)

Anti-carcinoembryonic (CEA) polyclonal antibodies in sheep and rabbits were raised using purified CEA from acid extracts of human colon adenocarcinoma. CEA was purified by gel filtration on Sepharose 4B CL and chromatography on DEAE-Sephadex A50. The antiserum was adsorbed with human serum and perchloric acid extract from normal colon. Anti-CEA IgG was purified from monospecific antiserum by ion-exchange chromatography and its specificity was tested on cryostat sections from colon adenocarcinoma by the indirect immunoperoxidase technique. The specific reaction was compared with that obtained by using a similar technique and two CEA specific monoclonal antibodies. An anti-CEA IgG peroxidase conjugate was obtained allowing to establish a "sandwich" ELISA-CEA system with two antibodies. CEA determinations were made in a group of 15 normal controls (mean value 4.8 +/- 0.4 ng/ml) and in 30 colorectal tumor patients (mean value 26.6 +/- 2.15 ng/ml). The anti-CEA antibodies are proven useful in immunocytochemical and ELISA techniques and may be further used in radioimaging of tumors.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue

Summary A highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.     

The expression of Lewisx on carcinoembryonic antigen (CEA)-related glycoproteins of normal and inflamed oesophageal squamous mucosa

Carcinoembryonic antigen (CEA)-related antigens were detected histologically in normal and inflamed oesophageal squamous mucosa using polyclonal anti-CEA antisera and monoclonal antibodies recognizing CEA or NCAs (non-specific cross-reacting antigens). Expression was limited to the surface of more mature squames. Immunoblotting of detergent extracts of oesophageal mucosa separated on polyacrylamide gels using polyclonal anti-CEA antisera showed a number of CEA-related proteins, of 195, 145, and 80 kDa. CEA-specific monoclonal antibodies recognized only the 195-kDa glycoprotein. The lower molecular weight species were recognized by anti-NCA antibody DD9 and a CD66 antibody. The carboyhydrate antigen Lewis^x (Le^x, CD15), previously shown to be a marker of mature squames, was present predominantly on a subpopulation of the 195-kDa antigen and was demonstrable on the higher molecular weight component of a doublet recognized by the CEA antibodies. Expression of Le^x carbohydrate antigens in inflamed oesophageal squamous mucosa was shown to be significantly reduced relative to the expression seen in normal tissue. A suprabasal layer of CEA-positive, Le^x-negative cells became apparent in inflamed tissue showing altered glycosylation of the CEA under these conditions. It is postulated that CEA plays a role in maintaining the integrity of the squamous mucosa.     

Monoclonal antibodies against carcinoembryonic antigen (CEA) discriminate between CEA produced by gastrointestinal tumors and CEA produced by other tumors

Carcinoembryonic antigen (CEA) is a common tumor marker. It is not specific since many tumor types express it. We report here the use of a series of monoclonal anti-CEA antibodies with apparent restricted activity. These antibodies reacted histochemically with CEA produced by tumors originating in the gastrointestinal tract, but they did not react with CEA-secreting tumors of nongastrointestinal origin. We have used this phenomenon to accurately diagnose the tumor of origin in 20 patients who had a CEA-producing metastatic tumor, but in whom the primary origin was clinically not yet discovered. The use of such antibodies for diagnostic purposes may be an adjunct to other methods in evaluating tumors of unknown origin.     

Optimization of methods of immobilization of anti-immunoglobulins. Equilibrium parameters of the interaction of immobilized antibodies with an antigen

Methods for immobilization of anti-immunoglobulins on insoluble supports were optimized, and the interaction of immunoadsorbents obtained with [125I]-labeled rabbit IgG was investigated. It was shown that this interaction can be adequately described by a rather simple equilibrium model which reflects the interaction of a monovalent antigen with two independent types of binding sites. Within the framework of this model the association constants as well as the concentrations of high affinity binding sites which influence the capacity and efficiency of the separation system were determined. Optimization of the immobilization methods implicated a study on the role of certain functional groups of the antibody involved in the formation of covalent bonds, on the effect of the spacer arm length on the properties of immobilized antibody as well as on the role of the degree of immobilization. It was found that immunoadsorbents obtained after antibody immobilization via lysine or tyrosine residues on matrices with a specific spacer group are the optimal ones.     

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay

A novel simultaneous measurement method for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.     

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1–7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1–5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.Some of the studies described in this paper were in partial fulfillment of requirements for the completion of Dr. Irvine's dissertation at the George Washington University     

Cell adhesion activity of non-specific cross-reacting antigen (NCA) and carcinoembryonic antigen (CEA) expressed on CHO cell surface: homophilic and heterophilic adhesion

Cell adhesion activity of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) has been analysed by using CHO cells which had been transfected with cDNAs and are ectopically expressing each antigen on their surface. CEA expressing CHO tended to aggregate easily within 30 min after being suspended by trypsinization. Cell adhesion assay between 51Cr labelled cells and monolayered cells showed both homophilic and heterophilic interaction, the extent of which was CEA-CEA much greater than CEA-NCA greater than NCA-NCA. These reactions were completely inhibited by Fab' fragment of anti-CEA antibody. The results strongly suggested that CEA and NCA function as Ca++ independent cell adhesion molecules by homophilic and heterophilic interactions.     

Nature of the inhibitory serum factor in mice injected with isologous antibodies conjugated with cellulose

The formation of antigen-specific serum inhibitory factor was induced by injection of covalently bound to cellulose syngeneic antibodies to sheep red blood cells into (CBA X C57BL/6)F1 mice. This factor was absorbed by cellulose immunosorbents immobilized with antibodies against sheep red blood cells and with rabbit antibodies against mouse gamma-globulin and was not absorbed by immunosorbents immobilized with immunoglobulins of intact mice or immunoglobulin containing antibodies against rat red blood cells. These data, and evidence obtained by the authors previously, indicate that inhibitory factor of the serum is likely to be due to idiotypic antibodies.     

Combined radioimmunotherapy and chemotherapy of breast tumors with Y-90-labeled anti-Her2 and anti-CEA antibodies with taxol

Because breast cancer cells often express either Her2/neu or carcinoembryonic antigen (CEA) or both, these tumor markers are good targets for radioimmunotherapy using Y-90-labeled antibodies. We performed studies on nude mice bearing xenografts from MCF7, a cell line that has low Her2 and CEA expression, to more accurately reflect the more usual situation in breast cancer. Although uptake of In-111 anti-CEA into tumors was lower than that for In-111-labeled anti-Her2, radioimmunotherapy (RIT) with Y-90 anti-CEA was equivalent to that of Y-90 anti-Her2. When either Y-90 antibody was combined with a split-dose treatment with Taxol, the antitumor effect was greater than with either agent alone. When Y-90 anti-CEA was combined with a single dose of Taxol, the results were equivalent to the split-dose regimen. RIT plus cold Herceptin had no additional effects on tumor size reduction over RIT alone. When animals were first treated with Y-90 anti-Her2 and imaged 1-2 weeks later with In-111 anti-CEA or anti-Her2, tumor uptake was higher for anti-CEA and improved over tumor uptake with no prior RIT. These results suggest that a split dose of RIT with anti-Her2 antibody followed by anti-CEA antibody would be more effective than a single dose of either. This prediction was partially confirmed in a controlled study comparing single- vs split-dose anti-Her2 RIT followed by either anti-Her2 or anti-CEA RIT. These studies suggest that combined RIT and Taxol therapy are suitable in breast cancers expressing either low amounts of Her2 or CEA, thus expanding the number of eligible patients for combined therapies. They further suggest that split-dose RIT using different combinations of Y-90-labeled antibodies is effective in antitumor therapy.