anti-CEA Monoclonal Antibody Research Articles

CEA-immunoscintigraphy with 99m-technetium correlates with tumour cell differentiation in colorectal cancer

The clinical usefulness of immunoscintigraphy with the monoclonal anti-CEA (carcinoembryonic antigen) antibody BW431 26 , directly labelled with 99m-Technetium in targeting colorectal carcinomas was investigated in 43 patients. In addition, tumour cell grading and CEA-expression were examined immunohistochemically. Best imaging results were obtained in pelvic tumour lesions (sensitivity 80%). Tumour grading correlated with radioimmunoimaging, well differentiated tumours being detectable at a higher rate ( P = 0.09). Immunoscintigraphy preceded the findings of conventional diagnostic methods in 3 patients. In 4 cases immunoscintigraphy was decisive for patients management. Therefore, immunoscintigraphy with 99m-Technetium is valuable in directing patients management if conventional diagnostic methods remain undecisive.

Potential contribution of 131I-Labelled monoclonal anti-CEA antibodies in the treatment of liver metastases from colorectal carcinomas: Pretherapeutic study with dose recovery in resected tissues

20 patients with liver metastases from colorectal carcinoma undergoing laparotomy received 15-60 mg intravenously, either intact or fragments of, anti-carcinoembryonic antigen (anti-CEA) monoclonal antibodies labelled with 0.55-1.48 GBq (15-40 mCi) of 131I, 3-8 days prior to operation. The uptake measured per gram of metastases ranged from 0.33 to 6.6 x 10(-3%) of injected dose. Tumour to liver uptake ratios ranged from 2 to 33. The radiation dose, estimated in 6 patients (3 of each group), for an extrapolated dose of 3.7 GBq (100 mCi) of 131I ranged from 0.3 to 0.8 Gy in normal liver or spleen (an acceptable estimate for bone marrow radiation dose) and from 3.4 to 8.2 Gy to the hepatic metastases, indicating that probably other therapeutic modalities should be associated with radioimmunotherapy.

The diagnosis and evaluation of colorectal CA using radio—Labelled anti-CEA monoclonal antibody fragments

The diagnosis and evaluation of colorectal CA using radio—Labelled anti-CEA monoclonal antibody fragments

D14 anti-CEA monoclonal antibody in the estimation of neoplastic sequence in human colon

D14 anti-CEA monoclonal antibody in the estimation of neoplastic sequence in human colon

Evaluation of an Improved Dot-immunobinding Assay for Carcinoembryonic Antigen Determination in Nipple Discharge in Early Breast Cancer: Results of a Multicenter Study

We have previously reported carcinoembryonic antigen (CEA) measurement in nipple discharge to be a useful adjunct in the diagnosis of non-palpable breast cancer. We have now developed a "microdot-immunobinding assay" using a specially constructed device to screen efficiently large numbers of patients with nipple discharge for non-palpable breast cancer. The method is as follows: a sample of nipple discharge is placed on a solid phase monoclonal anti-CEA antibody and, if CEA is present in the discharge, it will be detected by a second monoclonal anti-CEA antibody conjugated with alkaline phosphatase. The use of bromochloroindolyl phosphate as a chromogen results in a stable color reaction that can be semiquantitatively analyzed with the naked eye. CEA levels determined by this microdot assay correlated well with those determined using the earlier Elmotec assay. To determine the accuracy of the method, a collaborative study involving 11 institutes in Japan was organized. The CEA levels in nipple discharges from 77 patients undergoing surgery, 44 of whom were diagnosed as having breast cancer, were assayed. The results were that 17 of the 23 patients with palpable breast cancer, and 16 of the 21 patients with non-palpable breast cancer exhibited CEA values > 400 ng/ml, a cut-off value determined in a previous study. The overall accuracy (78%) of this test for diagnosing non-palpable breast cancer was higher than that obtained from ductography or cytology. The system may thus be of use in the screening of early breast cancer.

Localization of CEA messenger RNA by in situ hybridization in normal colonic mucosa and colorectal adenocarcinomas.

Carcinoembryonic antigen (CEA) mRNA expression was studied in 14 cases of normal colorectal mucosa and colonic adenocarcinomas using in situ hybridization with a 32P-labelled cDNA probe to the unique 3'-untranslated region of CEA. This approach has the advantage that the target mRNA remains in the cell of origin, whereas there is considerable ambiguity in immunocytochemistry data for CEA because the protein is secreted. Furthermore, the specific cDNA probe overcomes potential problems of immunological cross-reactivity with other members of the CEA family. The results demonstrated that abundant, heterogeneously distributed CEA mRNA was present in colorectal adenocarcinomas, with the highest levels in cells lining glandular structures. Parallel immunohistochemistry with anti-CEA monoclonal antibody A5B7 showed that the regions of tumours with the highest levels of CEA mRNA also had the highest CEA protein levels, suggesting that the heterogeneous distribution reflects CEA expression rather than differential secretion of the protein. In the normal colonic mucosa, CEA mRNA expression was observed in surface epithelial cells and goblet cells of the upper crypts, with very low hybridization in the mid crypt and at the base. This crypt-surface distribution was identical to that observed for CEA protein. In situ hybridization therefore confirms that high levels of CEA mRNA are expressed in differentiated surface epithelial cells of the normal colon.

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.

Detection of carcinoembryonic antigen in colonic effluent by specific anti-CEA monoclonal antibodies

To characterize the CEA in colonic effluent, anti-CEA monoclonal antibody COL-4 was used in a qualitative radioimmunoassay in both fractionated and unfractionated colonic effluent. Both effuent samples and tissue extracts, were subjected to Western blotting and tissue sections to immunohistochemistry. Quantitative levels of colonic effluent CEA were determined by a kit (Abbott-EIA). Higher mean values of COL-4 binding activity were seen only in patients with a past history of polyps (P < 0.01). Quantitated CEA correlated with the presence of colorectal cancer (CRC) as compared to normal subjects, (1133 ± 875 vs. 459 ng/ml ± 602, P < 0.05) but not when standardized for protein content. COL-4 reacted with an 180 000 M r CEA in the effluent and activity was associated with membrane fraction of the effluent, but bore no relation to the immunohistological staining. We conclude that CEA is detectable in colonic effluent and is membrane associated, but the overlapping values in effluent samples do not make this a useful test in the diagnosis of CRC.

Application of Genetically-Engineered Anti-CEA Antibodies for Potential Immunotherapy of Colorectal Cancer

Hitherto anti-CEA monoclonal antibodies (MAbs), normally of mouse origin, have been used primarily for clinical diagnosis of colorectal cancer, either as a tumor marker in serum to monitor tumor recurrence, or latterly as a means to localize in vivo CEA-bearing tumors, and metastases in patients. In vivo diagnosis using mouse anti-CEA MAbs has so far had limited clinical utility because the antibodies elicit a strong anti-mouse immunoglobulin immune response on repeated administration in man. This problem has been addressed by the development of various strategies for "humanization" of mouse anti-CEA MAbs by genetic manipulation of immunoglobulin genes. Such humanized, engineered antibodies markedly attenuate the antigenic response directed against the MAb, such that safe, repeated administration to patients has become feasible. Such humanized anti-CEA antibodies can thus be radioactively-labelled and applied for in vivo monitoring and detection of recurrent malignant disease, or used for therapeutic strategies which similarly take advantage of the ability of the antibodies to target cytotoxic agents selectively to tumor cells. The application of these novel procedures for manipulating MAb structure presents entirely new opportunities for diagnosis and treatment of human colorectal cancer.

Anti-CEA and other antibodies in the study of gastrointestinal tumors.

Localization of gastrointestinal tumors by means of labeled monoclonal antibodies is a new, sensitive and suitable technique currently used in several centers. Encouraging results have been documented with several monoclonal antibodies by different authors. This article reviews our experience with radioimmunoscintigraphy in 59 patients with colorectal cancer in follow-up, using 131I and 111In labeled B72.3, and in 16 patients with primary gastrointestinal tumors using 99mTc anti-CEA monoclonal antibody (type F023C5). The sensitivity of both B72.3 and anti-CEA was greater than 70% either for primary tumors and abdominal recurrences or distant metastases except hepatic ones. A significant gradient in antibody uptake was measured on surgical biopsies between tumors and normal tissues allowing a good in vivo contrast for gamma detection. We have defined the impact of some factors affecting in vivo tumor targeting. In fact, pharmacodynamics of MAbs, percentage of injected dose bound to tissues were measured, and in particular antigenic content in tumor nodules was quantified. Furthermore, the results of RIS were compared to those obtained by CT and other imaging modalities.

Gamma-interferon enhancement of carcinoembryonic antigen expression in human colon carcinoma xenografts.

Athymic nu/nu mice bearing a subcutaneous human colon cancer xenograft (WiDr, low CEA expression) were treated with gamma-interferon (gamma IFN) at varying doses, frequencies, and periods of duration. CEA content (micrograms/g) and uptake of radiolabeled anti-CEA monoclonal antibody (MAB) (percent injected dose per gram, % ID/g) were measured at 48 h following administration of the MAB, The optimal enhancement of tumor CEA content and tumor localization of [111In] anti-CEA monoclonal antibody (MAB) was seen at gamma IFN doses of 100,000 U i.p. every 8 h for 4 days (4.7 micrograms/g; 29% ID/g) compared to control animals (0.9 micrograms/g; 10% ID/g). The effects of gamma IFN on CEA content and MAB localization were less pronounced when administered (a) at lower doses: 5,000 to 50,000 U i.p. every 8 h, (b) at varying frequencies: 300,000 U/day delivered in divided doses every 4 or 24 h, or (c) for varying periods: 2 or 6 days of therapy. In each case, the biologic effects on tumor CEA content and uptake of [111In]MAB correlated closely with the serum gamma IFN level. Therefore, we conclude that enhancement of in vivo CEA expression by gamma IFN may have clinical relevance for tumor imaging and therapy using radiolabeled monoclonal antibodies.

Capture of recombinant ricin A chain by a bispecific anti-RTA:anti-CEA monoclonal antibody pre-targeted to a human gastric carcinoma xenograft in nude mice.

A bispecific antibody against carcinoembryonic antigen (CEA) and ricin A chain has been shown to localise in a CEA-producing human tumour xenograft. When labelled recombinant ricin A chain was subsequently injected there was uptake into tumours, indicating that this bispecific antibody can capture or carry toxin into tumour deposits.

Radioimmunoscintigraphy of colorectal carcinoma with a 99Tcm-labelled anti-CEA monoclonal antibody (BW 431/26).

Radioimmunoscintigraphy (RIS) with BW 431/26 monoclonal antibody (MoAb) labelled with 99Tcm (962 MBq) has been performed in 64 patients with colorectal carcinoma, one of them with two independent tumours. The group consisted of 46 primary lesions, 15 pelvic recurrences and four suspected recurrences which were shown to be liver metastases. Imaging of liver was obtained in all patients, but surgical liver examination was performed in only 56 of them. Planar scans were obtained at 4 and 24 h postinjection. Tomographic images were also performed in five patients. The final diagnosis was confirmed in all patients by their clinical course and by findings at surgery and pathology. A comparative study between the RIS results and the final diagnosis gave a global sensitivity in primary tumours and pelvic recurrences of 59.7% with an accuracy of 59.0%. When rectal tumours were excluded, the results were 81.1 and 84.9%, respectively. In liver metastases the sensitivity was 50%, with an accuracy of 85.7% and a specificity of 100%. No correlation has been found between CEA serum levels and lesion detection. In conclusion, RIS is a useful technique for the study and localization of colorectal tumours, being also indicated in patients with normal carcinoembryonic antigen (CEA) serum levels and clinical suspicion of illness.

Imaging of medullary carcinoma of the thyroid using 111In-labelled anti-CEA monoclonal antibody fragments.

The distribution of 111In-labelled anti-carcinoembryonic antigen (CEA) monoclonal antibody fragments [F(Ab')2] was studied in five patients either with known inoperable medullary carcinoma of the thyroid (MCT) or evidence of recurrence/metastases based on elevated calcitonin (hCT) levels. All five cases had elevated serum CEA levels and positive immunohistochemical stains for both hCT and CEA prior to scintigraphy. In two patients with identified inoperable disease both planar and SPECT scans were positive. In the remaining three patients, where the recurrence/metastatic sites were unknown, SPECT images were positive in two. Of these, only one had positive planar images. These results indicate that 111In-labelled anti-CEA F(ab')2 scintigraphy, especially in conjunction with SPECT, is useful for the diagnostic evaluation of patients with MCT. The limiting factor of this technique is the high level of non-specific uptake, particularly in the liver, but improvements in the specificity of newer anti-CEA antibodies and the ability to label these with 99Tcm is addressing this problem.

Reaction profiles of seven enzyme immunoassay kits for carcinoembryonic antigen (CEA) analyzed with purified preparations of CEA and related normal antigens

Antigenic reactivities of 4 purified CEA preparations and 4 different CEA-related normal antigens (NCA from lungs, NCA-2 from meconium, and NFA-1 and NFA-2 from adult feces) were comparatively analyzed with seven commercially available EIA kits [Abbott CEA-EIA Monoclonal, D-ZYME CEA, Imzyne CEA, CEA MITSUI II, CEA Roche EIA, Immunoball-CEA (N), and Glaozyme CEA]. All kits employ a sandwich-type solid-phase method using polystyrene beads and monoclonal anti-CEA antibodies as either capture antibody and/or tracer antibody. In general, all CEAs reacted well with these assay kits. Reactivity differences in weight among the CEA preparations were, however, observed in all but one assay kit (D-ZYME CEA) in which all CEAs showed fairly homogeneous reactivity. The degree of reaction intensity among the preparations used varied depending on the assay kits used. NCA, which is well known to be partially cross-reactive with CEA, revealed a negligible reactivity in all kits. NFA-1 which is also partially cross-reactive with CEA but antigenically unrelated to NCA, showed very strong reactivity in 2 kits [CEA Roche EIA and Immunoball-CEA (N)] and weak reactivity in another kit (CEA MITSUI II). NCA-2 in meconium and NFA-2 in normal adult feces, which both have immunochemical and physicochemical properties very similar to those of CEA, reacted to a greater or lesser extent with all kits. However, one assay kit (D-ZYME CEA), whose reactivity to different CEAs was fairly homogeneous, could discriminate CEAs from NCA-2 and NFA-2. Although the reactivity of NFA-2 was less than that of any CEA in all kits, that of NCA-2 was higher than that of some CEAs in 3 kits (Abbott CEA-EIA Monoclonal, CEA MITSUI II, and CEA Roche EIA). These differences in reactivity and specificity of currently available EIA kits for CEA should be borne in mind when selecting an assay kit.

Epitope mapping of the carcinoembryonic antigen with various related recombinant proteins expressed in chinese hamster ovary cells and 25 distinct monoclonal antibodies

Antigenic epitopes of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) were analysed in relation to their domain structures [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M for CEA and domains N, I (A1-B1), and M for NCA]. We reconstructed cDNAs for CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M (CEA-whole), NCA-N, NCA-N-I and NCA-N-I-M (NCA-whole), which were expressed in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. Their mol. wts judged from Western blotting were 17,000–26,000 for CEA-N, 70,000 for CEA-N-I, 150,000 for CEA-N-I-II, 165,000 for s-CEA-whole which was spontaneously released from cells into culture medium, 180,000 for p-CEA-whole which was solubilized with phosphatidylinositol specific phospholipase C (PI-PLC) from cells, 18,000–25,000 for NCA-N, 63,000 for NCA-N-I, and 96,000 for p-NCA-whole which was solubilized with PI-PLC from cells. The divergence of the observed mol. wts from those calculated from cDNA sequences seems to indicate that these recombinant proteins are highly N-glycosylated. By enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 25 distinct anti-CEA monoclonal antibodies (MAbs), each representative of 25 different subgroups within five groups (Groups 1–5) previously classified by us in terms of the reactivity with CEA and CEA-related antigens. Twenty-one MAbs previously shown to react with different protein epitopes of the CEA molecule allow to define six groups (A-F) of epitopes according to their expression by different domains of the CEA and NCA molecules. Among four epitopes common to CEA and NCA, two were found to be present on domain N (Group A) and two on domain I (Group B). Among 15 epitopes absent from NCA but expressed by CEA and normal fecal antigens (NFAs), four were on domain N (Group C), five on domain I (Group D) and six on domain II (Group E). Two epitopes were previously described as “CEA distinctive”, because they were recognized by MAbs reacting with CEA but not with the NFAs. These two epitopes (Group F) were found to be expressed by p-CEA-whole but not by s-CEA-whole. The latter results suggest that the Group F epitopes are located on a part of the domain III close to the anchoring device of the CEA molecule. Two of four MAbs reactive with sugar epitopes reacted with all the recombinant CEA proteins, suggesting the presence of some carbohydrate epitopes similar to those of CEA used for immunization. The epitope mapping reported here helps form the basis for understanding the relation between the clinical structure and antigenic activities of the CEA molecule.

Phase I/II Trial of 111In-Labeled Anti-CEA Monoclonal Antibody (111In-ZCE-025) in Diagnosis of Colorectal Cancer.

A phase I/II study of imaging with radiolabeled anti-CEA monoclonal antibody, 111In-ZCE-025, was carried out in patients with colorectal cancers as a joint research project at 5 hospitals in Japan. There were 24 patients, 22 of which were judged to be evaluable. Nineteen had primary lesions with or without metastatic lesions, two had local recurrences, and one had only metastatic lesions. On the patient basis, 21 of the 22 (95.5%) were judged to be positive. Of the 40 known lesions, planar imaging detected 24 (60.0%), whereas combined imaging (planar and single photon emission computed tomography, SPECT) detected 29 (72.5%) . Surgery disclosed another 22 new lesions, two (9.1 %) of which had been detected by the planar and four (18.2%) by the combined imagings. As for the newly visualized sites, on the other hand, planar imaging depicted 10, two of which were true positives (20.0%), whereas combined imaging depicted 20 and four of which were true positives (20.0%) . These low positive predictive values for newly visualized sites were due to many false positive lymph nodes.In conclusion, this method will be useful for preoperative screening and diagnosis of local recurrence of colorectal cancers. But the formation of HAMA should be taken into consideration when this method is repeatedly used. At present, for metastatic foci in lymph nodes, this method possesses only a complementary role.

Immunoscintigraphy using 111In-labeled F(ab')2 fragments of anticarcinoembryonic antigen monoclonal antibody for detecting recurrences of medullary thyroid carcinoma.

The only current possibility for curing medullary thyroid carcinoma (MTC), especially recurrences, is total surgical removal. Early positive diagnosis of recurrences is now possible by monitoring tumor markers such as thyrocalcitonin and carcinoembryonic antigen (CEA). However, preoperative topographic diagnosis of such recurrences remains an unresolved problem. Immunoscintigraphy (IS) using an anti-CEA monoclonal antibody is a new approach that complements morphological imaging, i.e. ultrasonography, computerized tomography, and magnetic resonance imaging. In this study, IS by means of an 111In-labeled anti-CEA monoclonal antibody F(ab')2 was performed nine times in eight patients. True positives were obtained five times (one case of cervical involvement confirmed by surgery, three cases of mediastinal involvement confirmed by computerized tomography, magnetic resonance imaging, and surgery, and one case of bone metastasis, one of them was revealed neither by x-ray nor by conventional bone scan). The remaining four tests gave a false positive, a true negative, a probably false negative, and one unconclusive result. We conclude that IS is helpful in diagnosing sites of MTC recurrence and should accompany other examinations in the evaluation of lesions.

Usefulness of immunoscintigraphy in the follow-up of surgically treated gastrointestinal carcinomas using 111in-labelled anti-CEA monoclonal antibodies

Twenty-five patients treated surgically for gastrointestinal carcinomas (16 rectum—sigmoid colon, 6 colon, 3 stomach) were investigated by immunoscintigraphy (IS) using 111In-labelled anti-CEA antibody (mouse monoclonal F023C5) F(ab′) 2 fragments in order to visualize questionable abdominopelvic recurrences (excluding the liver). Fifteen (60%) patients showed a rise in serum CEA levels above 5 ng/ml. Planar scans and emission computed tomographic (ECT) imaging were carried out without reference to the results of computed tomographic (CT) scans and gastrointestinal endoscopic examinations done 1–2 weeks before IS. Final diagnoses were based on biopsies and autopsies (13 cases) or on follow-up findings over at least 2 years (12 cases). Sixteen patients had a final diagnosis of recurrent malignant disease in the extrahepatic abdomen or pelvis. Of these, six were correctly diagnosed by both IS and conventional diagnostic procedures, six by IS only and two by conventional methods only. Two tumour recurrences remained undetected by both diagnostic approaches. However, five tumour recurrences were detected by IS more than 4 months earlier than by any other diagnostic procedures performed during clinical follow-up. Of the nine disease-free patients (disregarding the liver) three were correctly identified by both IS and the other diagnostic methods, four by IS only and two by conventional diagnostic procedures only. Overall sensitivity (75%) and specificity (89%) of 111In-IS were higher than figures obtained using CT scanning and endoscopy (50% and 78% respectively). True positive IS was observed in 6/15 (40%) CEA seropositive patients and in 6/10 (60%) CEA seronegative patients. Our comparative study (performed blind) supports the main role of immunoscintigraphy with fragments of anti-CEA monoclonal antibodies in the early detection of extrahepatic recurrences of gastrointestinal carcinomas in both CEA seropositive and seronegative patients.

Reactivities of an anti-CEA peptide monoclonal antibody.

Synthetic peptides representing different areas of the CEA molecule were used as immunogens for the development of anti-CEA antibodies. Both polyclonal and monoclonal antibodies were generated using peptides composed of CEA amino acid positions 99-128 and 585-613, respectively. One MAb, designated CP4, generated using the CEA peptide 99-128, was chosen for a more detailed analysis of reactivity. MAb CP4 reacts in solid phase RIAs with CEA peptide 99-128 immunogen and purified native CEA. CP4 did not react with purified non-specific cross reacting antigen (NCA), even though there were two single amino acid differences between NCA and CEA in the 29 amino acid peptide. The affinity constants of CP4 for the CEA peptide 99-128 and native CEA are 4.07 x 10(9) M-1 and 5.75 x 10(8) M-1, respectively. When CP4 was reacted with purified CEA in Western blotting experiments, the Mr 180,000 glycoprotein characteristic of CEA was detected, but CP4 reacted to various size entities in tumor cell extracts. The results of liquid competition RIAs showed that the epitope that MAb CP4 recognized on native CEA is not available for binding when CEA is in solution. Physical (adsorption to a solid matrix) or chemical (deglycosylation or formalin-fixation) alteration of CEA is required for binding of CP4 to CEA. MAb CP4 reacted approximately 1,000-fold greater to deglycosylated CEA than native CEA. Immunohistochemical studies using formalin-fixed paraffin-embedded tissue sections demonstrated that, among carcinomas, CP4 reacts selectively with colorectal carcinomas, while normal colon is negative. Although stomach carcinoma is negative, dysplastic lesions and areas of intestinal metaplasia are reactive. Two of 7 normal stomach tissues showed focal cytoplasmic reactivity of the surface epithelium. CP4, therefore, appears to react with an epitope with highly restricted expression in colorectal carcinoma. These studies demonstrate the complexities in dealing with an anti-peptide MAb with reactivity to an epitope which is accessible only under certain conditions.