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This study was designed to test the effect of supplementation of several antioxidants, including alpha-tocopherol, on the clinical reduction of premalignant oral lesions. Samples of oral mucosa and serum were taken from baseline to 9 months of supplementation from patients with premalignant oral lesions and analyzed for alpha-tocopherol by HPLC. Statistical increases in both serum and tissue alpha-tocopherol were found after supplementation. There was no statistical relationship between alpha-tocopherol and beta-carotene levels.

The baculovirus expression system has been used for the production of a variety of proteins, including antibodies. Two single-gene constructs encoding single-chain immunoglobulins have recently been developed. The antibody employed was monoclonal antibody (MAb) CC49 which reacts with the pancarcinoma antigen, tumor associated glycoprotein, TAG-72. One, single-chain construct designated SCA delta CLCH1 (SCIg), consists of the CC49 sFv covalently joined to the human Fc (gamma 1) through the hinge region. The other, SCA delta CLCH1-IL-2 (SCIg-IL-2), has a human IL-2 molecule attached to the carboxyl end of the SCIg. These constructs have been used to test the feasibility of producing biologically active antibodies using the baculovirus expression system. Both constructs have been successfully expressed in insect cells and purified. The baculovirus recombinant single-chain antibodies have been designated, bV-SCA delta CLCH1 (bV-SCIg) and bV-SCA delta CLCH1-IL-2 (bV-SCIg-IL-2) they have been shown to be secreted in the culture supernatant as dimeric molecules of approximately 115 kDa and 140 kDa, respectively. The specificity and antibody dependent cellular cytolytic activity of the baculovirus recombinant single-chain antibodies were shown to be similar to that of the myeloma derived molecules. Glycosylation analysis showed that baculovirus derived proteins were N-glycosylated, but carried few if any high mannose residues. The biological activity of the IL-2 moiety was retained in bV-SCIg-IL-2, as evidenced by its stimulatory effect on the proliferation of the IL-2 dependent cell line HT-2. The observation that a significantly shorter time is required to develop baculovirus recombinant molecules as compared to myeloma derived molecules and that insect cells express single chain MAbs at acceptable levels may have implications for the production of these molecules for clinical use.

Human prostate-specific antigen (PSA), a 33- to 34-kDa serine proteinase with extensive homology to glandular kallikrein, is a single-chain glycoprotein that contains 7% carbohydrate. The presence of PSA in the serum of patients with prostatic cancer is widely employed as a marker of disease status. PSA has also been thought of as a possible target for use in active specific immunotherapy protocols. To date, the source of PSA employed has been seminal fluid from different individuals; this has raised concerns about differences among PSA batches for standardization of assays. This report is the first description of the production and the purification of a recombinant source of PSA using a baculovirus expression system. A baculovirus recombinant of the cDNA encoding the full length PSA was expressed in insect cells yielding two major immunoreactive products of 31 and 29 kDa. The latter size conforms to the molecular weight of a core preprotein deduced from the sequence of the cDNA insert. The larger protein represents the N-linked glycosylated form of the preprotein. Western blot analysis showed that both the glycosylated and aglycosylated forms of PSA reacted with a polyclonal and two different monoclonal antibodies specific for PSA. bV-PSA, like commercially available PSA, showed also low-molecular-weight immunoreactive products when culture supernatants were concentrated or taken through steps of purification. bV-PSA was purified to a final product consisting of a major 29-kDa protein and a minor 31-kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoembryonic antigen (CEA), a glycosylated protein of Mr 180, is one of the most widely studied oncofetal antigens. A majority of gastrointestinal cancers as well as breast and non-small-cell lung carcinomas express CEA. CEA thus represents a potential target for immunotherapy of several carcinoma types. A recombinant vaccinia-CEA virus (rV-CEA) was previously shown to induce anti-tumor activity in an experimental murine model after three rV-CEA inoculations. However, because the majority of cancer patients have received a previous smallpox vaccination, a long-lasting immune memory and/or induced anamnestic responses against vaccinia proteins may prevent repetitive boosting with the recombinant vaccinia virus expressing CEA. Therefore, other types of vaccines may be required to boost the anti-CEA immune response; one such schema would be the use of purified CEA as a boost in hosts given one administration of rV-CEA. Commercially available sources of CEA are usually derived from liver metastases extracts and are sometimes contaminated with nonspecific cross-reactive antigen. We have previously generated a recombinant source of full-length human CEA using a baculovirus expression system (bV-CEA). bV-CEA was shown to be glycosylated differently than native CEA, but it contains at least 10 epitopes found on native CEA (nCEA). Moreover, bV-CEA was able to induce a humoral response against CEA present on human colorectal cancer cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoembryonic antigen (CEA), a glycosylated protein of M(r) 180 kDa, is one of the most widely used human tumor markers. A majority of gastrointestinal cancers as well as breast and nonsmall cell lung carcinomas express CEA. We have previously described a recombinant baculovirus BVCEA-140 expressing the full-length human CEA and a variant, BVCEA-16, that encodes only the NH2-terminal domain, as well as a recombinant (BVNCA) expressing the closely related molecule nonspecific cross-reactive antigen (NCA). We have now compared a panel of 24 anti-CEA and anti-NCA monoclonal antibodies (MAbs) for their ability to bind to these recombinant CEA and NCA proteins, as well as with a new 60 kDa subgenomic form designated BVCEA-60. The epitope mapping studies indicate that all the CEA specific MAbs can recognize BVCEA-140. We also compared the sugar composition of BVCEA-140 to native CEA, using a lectin-linked immunoradiometric assay. The results demonstrated that both the native and recombinant baculovirus CEA contain simple high-mannose carbohydrates as well as biantennary and biantennary hybrid complexes. However, native CEA also contains triantennary and tetraantennary complex sugars, while the recombinant CEA molecule does not. Immunogenicity of the recombinant CEA molecules was demonstrated in mice. ELISA and Western blot analyses were used to determine the cross-reactivity of the anti-CEA sera. Mice immunized with BVCEA-140 elicit antibodies that are reactive to native CEA. When the BVCEA-16 was used as an immunogen, the antisera failed to detect native CEA or BVCEA-140. These studies demonstrate that minor sugar differences exist between native and baculovirus-derived CEA. However, epitope mapping with a panel of 24 anti-CEA MAbs (recognizing at least 10 CEA epitopes) stowed virtual immunologic identity between these two molecules. Moreover, BVCEA-140 appears to be a more potent humoral immunogen in mice than native CEA. These purified recombinant proteins can thus serve as standards in CEA serum assays for the possible detection and characterization of cell-mediated immune responses to CEA and as a potential source of immunogen (primary or for boosting) for active specific immunotherapy protocols of human carcinomas.

Carcinoembryonic antigen (CEA), one of the most extensively studied human tumor-associated antigens, represents a potential target for passive as well as active immunotherapy. We describe here the first baculovirus recombinants expressing the human CEA gene. Eight baculovirus clones were isolated which expressed products of varying molecular weights; one clone, termed BVCEA-140, was shown to contain multiple CEA epitopes by reactivity to a panel of anti-CEA monoclonal antibodies. When purified protein isolated from this clone was deglycosylated, immunoreactive species ranging from M(r) 50,000 to M(r) 110,000 were found. Results of Southern blot analysis carried out on BVCEA-140 DNA were consistent with the hypothesis that these products result from the stable expression of variants which have recombined within the repeated domains of CEA. Other baculovirus recombinants expressing products comprising different portions of the CEA gene were also derived. One, termed BVCEA-35, was shown to be a recombination between the first 87 bases of domains I and III of the CEA gene. A variant, termed BVCEA-16, contained only the NH2-terminal domain of the CEA gene. Moreover, a recombinant expressing the closely related molecule nonspecific cross-reactive antigen was also derived. As shown here, commercially available preparations of CEA, which are derived from tumor biopsies or cell line supernatants, may contain nonspecific cross-reacting antigens and other contaminants. Thus, the recombinant CEA molecules described should have numerous uses including validation of the use of monoclonal antibodies as standards in CEA serum assays, the characterization of immune responses to CEA, the use as immunogen, and the study of structure function relationships.

We analyzed the immunohistochemical expression of three epitopes of the tumor-associated glycoprotein 72 (TAG-72) in whole cross-sections of primary colorectal carcinomas and in regional lymph node metastases using monoclonal antibodies (MAbs) B72.3, CC-49, and CC-83, which recognize distinct carbohydrate antigenic determinants. B72.3, CC-49, and CC-83 reacted with 13 of 27 (48%), 25 of 27 (92%), and 21 of 27 (77%) carcinomas, respectively. The immunoreactivity with lymph node metastases followed a similar pattern; MAb CC-49 was again the most reactive of the three antibodies, since it labeled 13 of 15 metastatic lesions. Positive reactions of the MAbs with the primary tumors were not always predictive of the immunorecognition of their metastases. Distinct areas within whole cross-sections of TAG-72-positive primary carcinomas demonstrated marked differences in the expression of the three epitopes. CC-49 tended to react with the highest number of areas and with the highest percentages of carcinoma cells within each area. In no instances did B72.3 demonstrate reactivity superior to that of either CC-49 or CC-83. Tumors negative for the CC-49 epitope in any area also did not express the other two TAG-72 epitopes. However, the comparison of the immunostaining obtained with each MAb in TAG-72-positive primary lesions revealed areas where CC-83 was clearly more reactive than CC-49. Moreover, one lymph node metastasis, negative for CC-49, was recognized by CC-83. Thus, the combined use of MAbs CC-49 and CC-83 resulted in additive immunostaining of primary and metastatic colorectal carcinoma cells. The study provides evidence of intratumoral heterogeneity in the glycosylation pattern of the TAG-72 antigen in colorectal cancer and emphasizes the advantages of cocktails of anti-tumor-associated antigen MAbs in the immunodetection of colorectal tumor cells.

The authors investigated by immunohistochemical study the drainage of three tumor-associated antigens in unaffected regional lymph nodes of colon cancer patients. The study was conducted using monoclonal antibodies (MoAb) directed against different epitopes of the tumor-associated glycoprotein, TAG-72 (CC-49, CC-83, B72.3), of the carcinoembryonic antigen (CEA) (COL-4, COL-12), and of the colon-associated antigen, CAA (anti-CAA). The authors detected immunohistochemical reactions of MoAb CC-49 and anti-CAA with antigen-presenting cells (APC), such as peritumoral and sinus macrophages and lymphatic endothelial cells and with specific areas of germinal centers in lymph nodes draining 11 of 24 colorectal carcinomas studied. The corresponding primary tumors expressed the TAG-72 and CAA antigens. No immunostaining was detectable in lymph nodes using the anti-CEA MoAb, even when the primary tumors strongly expressed the specific epitopes. In germinal centers of regional lymph nodes, the immunostaining was often distributed at the periphery with a characteristic crescentic or circular pattern, which strongly suggested the exposure of the specific epitopes defined by MoAb CC-49 and anti-CAA on follicular dendritic cells. This would indicate that these epitopes are selectively recognized and presented to germinal center B-cells. This phenomenon may have clinical and diagnostic implications.