Addison's disease presenting with muscle spasm.

Primary hypoadrenalism has various causes and protean manifestation. We report a young female patient who presented with severe muscle spasm as her primary complaint. On evaluation she was found to be a case of Addison's disease secondary to adrenal tuberculosis. Her muscle spasm disappeared rapidly with replacement dose of glucocorticoid.

Combination of CTL-associated antigen-4 blockade and depletion of CD25 regulatory T cells enhance tumour immunity of dendritic cell-based vaccine in a mouse model of colon cancer.

Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via CTL-associated antigen-4 (CTLA-4) blockade or removal of CD4(+) CD25(+) regulatory T (Treg) cells appears to be a promising strategy for cancer immunotherapy. In this study, we tested the hypothesis that the combination of CTLA-4 blockade and depletion of Treg cells would improve the potency of dendritic cell (DC)-based vaccine in a clinically relevant mouse model, which is transgenic for both carcinoembryonic antigen (CEA) and HLA-A2 for the treatment of colon carcinoma in a therapeutic setting. We found that administration of anti-CD25 antibody prior to vaccination or systemic administration of anti-CTLA-4 antibody with the vaccine improved tumour-free survival against CEA-expressing tumours compared with mice immunized with DC-based vaccine alone. However, the efficacy of the vaccine proved to be most effective when anti-CTLA-4 antibody was combined with Treg inhibition. This vaccination strategy dramatically improved the tumour-free survival and allowed the development of long-lasting immune responses. The combined vaccination strategy resulted in increased secretion of IFN-gamma and enhanced HLA-A2-restricted CEA-specific CTL responses. Furthermore, coadministration of anti-CD25 and anti-CTLA-4 antibodies along with the vaccine was effective against more advanced tumours. These results provide evidence that simultaneous blockade of T-cell regulatory pathways is a promising approach for the induction of therapeutic antitumour immunity against CEA(+) colon carcinoma.

A novel Gymnema sylvestre extract stimulates insulin secretion from human islets in vivo and in vitro.

Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

Galactosyltransferase activity in metastasizing and nonmetastasizing rat mammary carcinomas and its possible relationship with tumor cell surface antigen shedding.

In order to study the mechanism of tumor cell surface antigen shedding, galactosyltransferase levels were compared in 5 spontaneously metastasizing and 3 nonmetastasizing rat mammary tumors. The enzyme activity both with or without exogenous acceptors was higher in the metastasizing group. This difference did not seem to be due to the variation in levels of degrading enzymes such as pyrophosphatase or beta-galactosidase found in these tumors. Little difference in the biochemical properties of the enzyme was found between the two groups. Most of the enzyme activity (60-70%) was recivered in the microsomal frctosyltransferase was assayed in "purified" plasma membrane fractions, 70% of the activity was associated with the plasma membrane vesicles, in which the enzyme was enriched by factors of 10-40. The number of galactose acceptor sites on the plasma membranes increased in parallel to the metastasizing capacity, indicating the presence of larger numbers of incomplete glycopeptides on their cell surfaces. These findings seemed to indicate that the greater turnover of glycoprotein in the spontaneously metastasizing tumor cell surface was caused by the shedding of surface antigens into the systemic circulation of the host.

Adenosine-3',5'-cyclic monophosphate levels and adenosine-3',5'-cyclic monophosphate phosphodiesterase activity in metastasizing and nonmetastasizing rat mammary carcinomas.

The adenosine-3, 5-cyclic monophosphate phosphodiesterase (cPDE) activity in the homogenates of 6 spontaneously metastasizing, nonimmunogenic, glycocalyx-shedding rat mammary carcinomas (MT) was assayed and compared with four histologically and growth rate-matched nonmetastasizing, immunogenic MT. The levels of this enzyme were 2.5 times higher in the nonmetastasizing tumors. To rule out the possibility of the presence of inhibitor(s) or stimulator(s) of cPDE, homogenates from a nonmetastasizing and from a widely metastasizing tumor were mixed. cPDE from both nonmetastasizing and metastasizing MT showed two apparent Km and two corresponding Vmax. The activity of the enzyme at concentrations of 1 muM (low Km) and 100 muM (high Km) adenosine-3, 5-cyclic monophosphate (cAMP) decreased in parallel with increasing metastasizing capacity. About 50% of the low and the high Km cPDE was in the cytosol in both groups, whereas the rest was particulate. The proportion of low and high Km activity was similar in all the fractions except in the plasma membrane of the metastasizing tumors where the percent of low Km enzyme was three times higher than that of the high Km. The steady-state levels of cAMP were 1.3-2.0 times higher in the metastasizing tumors, inversely proportional to their cPDE activities.

Biochemical properties of cyclic nucleotide phosphodiesterase in metastasizing and nonmetastasizing rat mammary carcinomas.

The biochemical properties of cyclic nucleotide phosphodiesterases in a nonmetastasizing and a spontaneously metastasizing rat mammary carcinoma were compared. The phosphooiesterases in both tumors had a pH optimum of around 8.0 and preferentially hydrolysed cyclic purine nucleotides. The rate of hydrolysis of purine nucleotides in the nonmetastasizing tumor was two times higher than in the metastasizing tumor, but the rate of pyrimidine nucleotide hydrolysis was equal in both tumors. Theophylline, caffeine, and D,L-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro20-1724) inhibited the enzyme activity in both tumors; the percent inhibition was the same by each inhibitor. The cyclic nucleotie phosphodiesterase activity in either tumor was stimulated by Mg++, Mn++, and Co++ and suppressed by Ca++, Zn,++, and Ni++. EDTA inhibited the activity below the basal level (activity in the absence of added cation), an this inhibition could be recovered up to the basal level by an equimolar quantity of either Mn++ or Mg++. Further stimulation of the enzyme activity with increasing concentrations of divalent cations was observed only with Mn++. Similar effects were observe with ethylene glycol bis(beta-aminoethyl ether)-tn,n-tetraacetic acid. The stimulatory cations affected both the low and high Michaelis constant (tkm) enzymes in these tumors by increasing the maximum velocity. In the low Km enzyme, the Km was also slightly increased. Neither guanosine 3',5'-cyclic monophosphate nor adenosine 3',5'-cyclic monophosphate had any effect on the hydrolysis of the other at physiologic levels.

Fucosyltransferase activity in metastasizing and nonmetastasizing rat mammary carcinomas.

Fucosyltransferase levels in 6 established strains of spontaneously metastasizing rat mammary tumors (STMT-058, MT-449, DMBA-4, SMT-077, TMT-081, and SMT-2A) were compared with 4 nonmetastasizing strains (MT-W9B, MT-W9A, MT-100, and MT-66) as controls. Two acceptors were prepared from fetuin for the assay, one by acid hydrolysis of N-acetylneuraminic acid and the other by the stepwise removal of N-acetylneuraminic acid and penultimate galactose by Smith degradation. The enzyme that transfers fucose to the first acceptor was designated fucosyltransferase A, whereas the one that uses the second acceptor was designated fucosyltransferase B. Both types of fucosyltransferases were found in this rat mammary tumor system. Whereas the levels of fucosyltransferase A in the 2 tumor groups were comparable, those of fucosyltransferase B were sixfold to sevenfold higher in the metastasizing tumors. This difference in the level of fucosyltransferase B was not caused either by differential hydrolysis of GDP-fucose by pyrophosphatase in the 2 groups or by hydrolysis of the product by fucosidases. Presence of any other inhibitor(s) or activator(s) of fucosyltransferase was excluded by mixing experiments. Optimal conditions for the assay of this enzyme were determined in a representative strain from each group. Under all circumstances, the activity of fucosyltransferase B was higher in the metastasizing tumors. The enzyme was inhibited by nucleoside diphosphates and triphosphates, and guanosine nucleotides were the most efficient inhibitors. Subcellular distributions of the two fucosyltransferases were similar, 35-50% of the enzyme activity being present in the crude microsomes. When plasma membrane factions were prepared from the microsomes, the major part (50-70%) of the enzyme was associated with the light and heavy plasma membrane fractions. Increased activity of fucosyltransferase B in the group of metastasizing tumors may have reflected faster synthesis and shedding of fucose-containing glycoprotein antigens. Similar molecules possibly were also synthesized in the nonmetastasizing cells but at a much slower rate, because the antigen is not easily lost from the cell surface. Any alteration of the specificity of this focosyltransferase in the metastasizing tumors, in addition, may have caused antigen modulation.

Biochemical and immunologic characterization of galactosyltransferase purified from the ascites of ovarian cancer patients.

Galactosyltransferase appears to be a promising marker for ovarian carcinoma. For an understanding of its role in this cancer, the enzyme was purified from the ascites of ovarian cancer patients, and its biochemical and immunologic properties were studied. For adequate recovery and stability, Triton X-100 (0.01%) was necessary in all the buffers used for the purification of this enzyme. Immunoglobulins were not detectable in this preparation, which showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis under nondenaturing conditions resolved the enzyme into two to three active components. An antiserum in rabbits, however, produced a single precipitin line suggesting a single determinant. By chromatography in concanavalin A-Sepharose 4B, the enzyme can be resolved into two components (F-1 and F-2). Purified galactosyltransferase and components F-1 and F-2 all catalyzed the transfer of galactose from UDP-galactose to alkali-stable beta-N-glycosidic acceptors, as well as to alkali-labile beta-O-glycosidic mucin-type acceptors. In addition, they catalyzed the N-acetyllactosamine synthetase reaction and, in the presence of alpha-lactalbumin, the lactose synthetase reaction. Galactosyltransferase and components F-1 and F-2 differed in their sensitivity to alpha-lactalbumin-induced inhibition of N-acetyllactosamine synthesis. Galactosyltransferase in the malignant ascites exists as different isoforms, which do not differ significantly in major biochemical and immunologic properties.

Characterization of products synthesized by galactosyltransferase purified from the ascites of ovarian cancer patients.

Galactosyltransferase purified from the ascites of ovarian cancer patients can use to an equal extent N-glycosidic glycoproteins, such as asialoagalactofetuin, and O-glycosidic mucin, such as asialo bovine submaxillary mucin (BSM), as acceptors. Thermal treatment and substrate competition experiments demonstrated that the same enzyme catalyzed the transfer of galactose to both types of acceptors. Alkaline borohydride treatment showed that, while the product with asialoagalactofetuin was totally resistant, about 90% of the product with asialo BSM was hydrolyzed by this treatment. Gel filtration of the released oligosaccharides on a calibrated Biogel P-2 column showed three peaks. One major oligosaccharide (O-2) of size 5.7 glucose U and two minor peaks (O-1 and O-3) of sizes 8.7 and 3.7 glucose U, respectively, were obtained. The oligosaccharides were doubly labeled, first by incubation with uridine-diphosphate [14C]galactose, followed by alkali treatment in the presence of [3H]borohydride. The doubly labeled oligosaccharides were separately purified by gel filtration and ion-exchange chromatography and digested with various exoglycosidases. The digested products were characterized by gel filtration and paper chromatography in three different systems. From these results, the structures of these oligosaccharides were computed as follows: O-1 = beta-galactosyl-beta-N-acetylglucosamine-galactosaminitol (sialic acid); O-2 = beta-galactose-beta-N-acetylglucosamine-galactosaminitol; O-3 = beta-galactose-galactosaminitol. These results suggest that the galactosyltransferase from the ascites of ovarian cancer patients catalyzes the transfer of galactose to N-acetylglucosamine, irrespective of whether it is a part of an N-glycan or an O-glycan.

Effect of hyperthermia on activity of three glycosyltransferases in Chinese hamster ovary cells.

We measured activities of three glycosyltransferases at various times during heat-induced thermotolerance development. Glycosyltransferases are normally located in the Golgi apparatus and catalyze cellular glycosylation reactions. UDP-Gal:N-acetylglucosamine beta 1,4-galactosyltransferase (beta 1,4-GalT) is known to participate in the formation of N-linked glycoproteins; when compared to cell survival, beta 1,4-GalT activity was significantly more heat resistant (50% loss of activity: 80 min, 45 degrees C) and showed little elevation at a time when thermotolerance was fully expressed. However, beta 1,4-GalT activity increased twofold by 24-h postheating when thermotolerance had begun to decay. Activity of beta 1,4-GalT was compared with glycosyltransferase activities that are considered to be specific for O-linked glycoproteins: UDP-Gal:N-acetylgalactosamine-beta 1,3-galactosyltransferase (beta 1,3-GalT), and UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (Gal-NAcT). Heat-inactivation experiments with heating times up to 60 min at 45 degrees C failed to reduce either activity below that of unheated control cells. Instead both beta 1,3-GalT and GalNAcT activity increased approximately twofold immediately after 10 min at 45 degrees C. Activity of beta 1,3-GalT rapidly decreased with time after heating and returned to control levels by 6-h postheating. In contrast, GalNAcT activity continued to increase with time after 10 min at 45 degrees C, and was 4.5-fold above unheated controls by 6-h postheating. GalNAcT activity returned to control levels 24- to 48-h postheating. A comparison with the cellular survival response showed that GalNAcT activity preceded thermotolerance expression by 2-4 h and also decayed more rapidly than heat resistance in thermotolerant cells. These data, together with other published results, suggest that expression of thermotolerance may be associated with enhanced glycosylation of intracellular proteins.

Identification of a human cancer-associated antigen defined with monoclonal antibody.

Splenic lymphocytes of BALB/c mice immunized with a glycoprotein-enriched fraction of human ovarian adenocarcinoma were fused with the mouse myeloma cell line P3/NS1/1-Ag4 in the presence of polyethylene glycol (Mr 4,000). The hybrid cultures were screened in an indirect solid-phase radioimmunoassay for the production of relevant antibodies. Hybrids that produced antibodies which bound to the glycoprotein-enriched fractions of ovarian tumors but not to the similar fractions prepared from pooled normal ovary or sera were cloned twice by the limiting dilution method. Two such clones designated 4F4 and 7A10 were expanded in culture and also were grown in mice as ascitic tumors. The immunoglobulin isotype of the clones was of immunoglobulin G1 subclass with kappa light chains. Immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect the target antigen in 125I-labeled glycoprotein-enriched fractions of ovarian tumors. A single-chain Mr 48,000 peptide was identified by both clones 4F4 and 7A10. This antigen, which showed binding to concanavalin A-Sepharose, was designated gp48. Monoclonal antibodies against gp48 reacted significantly in radioimmunoassay to approximately 90% of human ovarian tumors and 60% of other tumors, both benign and malignant, but not to normal adult tissues or sera. Quantitative absorption analyses indicated that although the antigen was present in small amounts in some normal adult tissues such as cervix and intestine, it was present in much higher concentrations in most ovarian tumors, in some other tumors, and in fetal intestine and liver. Immunoperoxidase staining of formalin-fixed paraffin-embedded sections of solid ovarian adenocarcinomas revealed strong epithelial reactivity. Monoclonal antibodies to gp48 may be of value for the follow-up and immunotherapy of a variety of human tumors.

Glycosyltransferase and glycosidase activities in ovarian cancer patients.

In order to elucidate the mechanism of appearance of abnormal glycoproteins in cancer, activities of glycoprotein glycosyltransferases and glycosidases were determined in the homogenates prepared from normal ovaries and ovarian epithelial adenocarcinoma. Significantly high activities (more than normal mean + 2 S.D.) of these enzymes were found as follows: galactosyltransferase and sialytransferase in 100%; fucosyltransferase 1 (exogenous acceptor, fetuin minus sialic acid and galactose) in 86%; fucosyltransferase 2 (fetuin minus sialic acid, acceptor) in 45%; N-acetylglucosaminyltransferase 1 (ovalbumin acceptor) in 53%; N-acetylglucosaminyltransferase 2 (ribonuclease A as acceptor) in 10% of the samples analyzed. Among the glycosidases, substantially elevated activities above normal controls were found as follows: N-acetyl-beta-D-glucosaminidase in 85%; N-acetyl-beta-D-galactosaminidase in 63%; N-acetyl-beta-D-galactosidase in 50%, and those of alpha-L-fucosidase in 35% of the tumors. In serum of these cancer patients, only levels of galactosyltransferase were consistently elevated compared to controls. Increases in serum levels for other transferases were as follows: fucosyl-1, 10%; fucosyl-2, 60%; sialyl-, 20%; N-acetylglucosaminyl-1, 90%; N-acetylglycosaminyl-2, in 80% of the serum samples from ovarian carcinoma patients. Galactosyltransferase thus appears to be an excellent marker for ovarian carcinoma.

Murine monoclonal antibodies against galactosyltransferase from the ascites of ovarian cancer patients.

Glycoprotein:galactosyltransferase is a promising enzyme marker for ovarian carcinoma. Five stable murine hybridoma monoclones that produce homogeneous antibodies against galactosyltransferase from the ascites of ovarian cancer patients have been established. Three of the monoclonal antibodies produced were immunoglobulin G1 isotype, while two were immunoglobulin M. All the antibodies showed linear Scatchard binding plots and had very high affinity for galactosyltransferase with equilibrium dissociation constants (Kd) ranging between 0.16 X 10(-9) M and 0.97 X 10(-9) M. Two of the monoclonal antibodies recognized adjacent epitopes on the enzyme molecule, two antibodies recognized two other unique epitopes, while the epitope recognized by the fifth was uncertain. Following polyacrylamide gel electrophoresis of the purified enzyme, in the presence of sodium dodecyl sulfate, the separated proteins were transferred to nitrocellulose filters (transblotting) and galactosyltransferase was detected on the filters by immunoperoxidase staining after treatment with monoclonal antibodies. A band at Mr 47,000 was detected by all of the monoclonal antibodies. One of them can detect, in addition, two bands at Mr 52,000 and Mr 54,000. Purified galactosyltransferase catalyzed the transfer of galactose to four types of acceptors: (a) alkali-stable N-glycosidic glycoproteins; (b) alkali-labile mucin-type acceptors; (c) N-acetylglucosamine; and (d) glucose in the presence of alpha-lactalbumin. All these transfer activities of the enzyme were present in the immunoprecipitates of the monoclonal antibodies. Transblotting of the enzyme from nondenaturing slab gels produced diffused stain patterns. Assay of the enzyme using the four types of acceptors in gel slices showed overlapping activity profiles, which coincided with the stained area on the filters, suggesting that the reactions are catalyzed by the same protein.

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity in the ovarian cancer patient.

Uridine 5'-diphosphate-galactose:glycoprotein galactosyltransferase activity was demonstrated in homogenates of normal ovary and ovarian epithelial adenocarcinomas. The specific activity of the enzyme in ovarian tumors was 3 to 5 times higher than in normal ovaries when the enzyme was assayed under identical conditions. The glycoprotein fetuin, from which terminal sialic acid and penultimate galactose were removed (fetuin minus N-acetylneuraminis acid and galactose), acted as an excellent exogenous acceptor. Galactosyltransferase from normal ovary and ovarian tumor cells had similar properties. Both required Mn2+ and Triton X-100 and had broad pH optima between 5.5 and 7. Galactosyltransferase activity was also measured in serum samples from ovarian cancer patients and normal healthy individuals in the presence of fetuin minus N-acetylneuraminic acid and galactose as exogenous acceptor. The enzyme levels were significantly elevated in the sera of ovarian cancer patients as compared to normal controls. The differences in the levels of this enzyme in the tissues and sera of normal individuals and ovarian cancer patients were not due to differential levels of the degrading enzymes such as uridine 5'-diphosphate-galactose pyrophosphatase or beta-D-galactosidase. Serial determinations were carried out on the sera of 5 ovarian cancer patients over a long period of time. The serum level of galactosyltransferase activity appeared to correlate with tumor volume as well as with the clinical status of the patient, which suggests possible leakage of the tumor enzyme into the host sera. Serial determination of this enzyme level in ovarian cancer patients seems promising in measuring tumor progression or success of therapeutic approaches.

Immunoperoxidase localization of a high-molecular-weight mucin recognized by monoclonal antibody 1D3.

The distribution of an antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya, M., Chatterjee, S.K., Barlow, J. J., and Fuji, H. Cancer Res., 42: 1650-1654, 1982) was investigated in various types of human malignant and normal adult tissues by indirect immunoperoxidase assay in fixed paraffin-embedded sections. One hundred percent of ovarian mucinous cystadenocarcinomas expressed high levels of the antigen with intense staining of 80 to 100% of the tumoral area, thus confirming our previous finding with radioimmunoassay and absorption analyses. About 51% of colonic carcinomas, 33% of gastric carcinomas, and 22% of pancreatic carcinomas were also positive for this high-molecular-weight mucoprotein antigen. All other ovarian and nonovarian carcinomas tested including carcinoma of lung, breast, endometrium, cervix, and prostate were not stained by 1D3. In addition, sarcomas, melanomas, and lymphomas also did not express any detectable level of the antigen. When surveyed against various normal adult tissues, 1D3 had reactivity limited to the colon. Normal colon, however, exhibited reduced staining intensities compared to tumors or to the apparently normal colon adjacent to tumors. The antigen thus appears to be a colorectal tissue-specific antigen showing increased levels in ovarian mucinous cystadenocarcinomas and in some gastrointestinal tumors. 1D3 antigen is a potential tumor marker for mucinous ovarian and colonic tumors.

Monoclonal antibodies recognizing tumor-associated antigen of human ovarian mucinous cystadenocarcinomas.

Spleen cells from BALB/c mice immunized with human ovarian cystadenocarcinoma extract were fused with the mouse myeloma cell line P3/NSI/1-Ag 4 in the presence of polyethylene glycol (Mr 4000). Of the 46 hybrids obtained, four secreted antibodies preferentially reactive to the immunizing ovarian tumor extract. Two of these hybrids, which showed no reaction with normal controls, were selected for cloning by the limiting dilution method. The numerous clones obtained from each hybrid were screened against a panel of five ovarian tumor extracts, pooled normal ovary extracts, and pooled normal human sera. One clone from each hybrid that showed specificity for ovarian mucinous cystadenocarcinomas was recloned to assure monoclonality and to establish a permanent hybridoma cell line. The antibodies secreted by these cell lines were of IgG1 subclass with kappa light chains. These antibody-producing hybridomas were selected for further analysis of the antibody specificity by a solid-phase radioimmunoassay and quantitative absorption tests. The monoclonal antibodies recognized an antigenic determinant present only in mucinous cystadenocarcinomas of the ovary. These did not react with any other gynecological or nongynecological tumor thus far tested. The antigen was not demonstrable in any normal adult tissues tested. Among fetal tissues examined, only fetal intestine extract showed a positive reaction. The antigenic determinant recognized by these monoclonal antibodies was unrelated to carcinoembryonic antigen, normal glycoprotein, normal human serum components, or human ABO blood group materials. These antibodies, which have relatively high affinity and can be produced in large amounts, will be useful for the isolation and immunochemical characterization of this antigen. The purified antigen and the specific antibodies could be then combined in a sensitive radioimmunoassay for the early detection of the antigen in the sera and body fluids of patients with ovarian mucinous cystadenocarcinomas.

Induction of antitumor immunity by an anti-idiotype antibody mimicking carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen expressed on most gastrointestinal adenocarcinomas and is a putative target for cancer immunotherapy. We developed a murine monoclonal anti-idiotype (anti-Id) antibody, 3H1, which mimics a specific epitope of CEA, for cancer immunotherapy. In this study, the efficacy of 3H1 as a tumor vaccine was evaluated in a murine tumor model. In this model, the murine colorectal cancer cell line MC-38 was transduced with the human CEA gene and injected into syngeneic C57BL/6 (H-2b) mice. Immunization of naive mice with 3H1 conjugated with keyhole limpet hemocyanin Freund's adjuvant induced humoral and cellular anti-3H1 as well as anti-CEA immunity. Mice immunized with 3H1 were protected against a challenge with lethal doses of MC-38-cea, whereas no protection was observed when 3H1 vaccinated mice were challenged with CEA negative MC-38 cells or when mice were vaccinated with an unrelated anti-Id antibody and challenged with MC-38-cea cells (P < 0.003). These data demonstrate that the 3H1 vaccine can induce protective CEA-specific antitumor immunity.

Molecular mimicry of carcinoembryonic antigen by peptides derived from the structure of an anti-idiotype antibody.

Our goal was to use carcinoembryonic antigen (CEA) as a target for immunotherapy in CEA-positive cancer patients who are all immune tolerant to the native antigen. We isolated and characterized an anti-idiotype monoclonal antibody 3H1, which mimics a distinct and specific epitope of the Mr 180,000 CEA and can be used as a surrogate for CEA. In Phase Ib clinical trials in a group of 23 advanced colorectal cancer patients, 3H1 induced both humoral and cellular anti-3H1 responses, as well as anti-CEA immunity. To study the cellular immunity invoked by 3H1 at the molecular level, we have cloned and sequenced the cDNAs encoding the variable heavy and light chains of 3H1 and deduced the amino acid sequences of the encoded proteins. To identify any cross-reactive peptides of 3H1 and CEA, we compared the amino acid sequences of 3H1 with those of CEA and found several regions of homology in 3H1 heavy and light chain variable domains, as well as in the framework regions. To search for potential cross-reactive T-cell epitopes, a number of peptides were synthesized based on 3H1/CEA homology and were used as stimulants in cell proliferation assays, using peripheral blood mononuclear cells from a group of 3H1-immunized CEA-positive cancer patients in the adjuvant setting. Two partially homologous peptides, designated LCD-2 (from 3H1) and CEA-B (from CEA), were identified in 10 of 21 adjuvant patients by strong proliferation responses (stimulation index, 3-50-fold), which were extensively studied in five of these individuals over an extended period of time (12-24 months). We saw no correlation with the MHC class I haplotype of the patients. Analysis of the subtype of the responding T cells demonstrated that primarily CD4+ T cells were stimulated by both 3H1 and 3H1-derived peptides. Interleukin 2, interleukin 4, and IFN-gamma were assayed in the culture medium of peripheral blood mononuclear cells stimulated with 3H1, CEA, and LCD-2 to determine the T-cell helper subset induced by these stimulants. The in vitro responses were mainly associated with secretion of IFN-gamma, which suggested that the induced T cells were most likely CD4+ Th1 type. Future studies will include the design of second-generation LCD-2 and CEA peptides to further enhance antigenicity, to characterize the responding T-cell populations more fully, and to test refined peptides for immunogenicity.

Clinical and immune responses in resected colon cancer patients treated with anti-idiotype monoclonal antibody vaccine that mimics the carcinoembryonic antigen.

PURPOSE: We generated an anti-idiotype antibody, designated CeaVac, that is an internal image of the carcinoembryonic antigen (CEA). We previously demonstrated that the majority of patients with advanced colorectal cancer generate specific anti-CEA responses. The purpose of the current study was to treat patients with surgically resected colon cancer with CeaVac to determine the immune response and clinical outcome to treatment with vaccine. We also compared the immune responses between patients treated with fluorouracil (5-FU) chemotherapy regimens plus vaccine versus vaccine alone. PATIENTS AND METHODS: Thirty-two patients with resected Dukes' B, C, and D, and incompletely resected Dukes' D disease were treated with 2 mg of CeaVac every other week for four injections and then monthly until tumor recurrence or progression. Fourteen patients were treated concurrently with 5-FU chemotherapy regimens. RESULTS: All 32 patients entered onto this trial generated high-titer immunoglobulin G and T-cell proliferative immune responses against CEA. The 5-FU regimens did not have a qualitative or quantitative effect on the immune response. Three of 15 patients with Dukes' B and C disease progressed at 19, 24, and 35 months. Seven of eight patients with completely resected Dukes' D disease remained on study from 12 to 33 months; one patient with resected Dukes' D disease relapsed at 9 months. One patient with incompletely resected Dukes' D disease remained on study at 14 months without evidence of progression; eight experienced disease progression at 6 to 31 months. CONCLUSION: CeaVac consistently generated a potent anti-CEA humoral and cellular immune response in all 32 patients entered onto this trial. A number of very high-risk patients continue on study. 5-FU regimens, which are the standard of care for patients with Dukes' C disease, did not affect the immune response. These data warrant a phase III trial for patients with resected colon cancer.

Antigen mimicry by an anti-idiotypic antibody single chain variable fragment.

For the therapy of cancer patients whose disease is positive for Carcinoembryonic Antigen (CEA), we developed an active specific immunotherapy based on the idiotypic network. The anti-idiotype monoclonal antibody (mAb), 3H1 was generated by immunization of mice with the anti-CEA mAb, 8019. 3H1 mimics CEA both functionally and structurally and acts as a surrogate for CEA. To define the minimum structural requirements for antigen mimicry by 3H1, we constructed plasmid vectors for expression of single chain Fv (scFv) variants of 3H1 in Escherichia coli. Variable heavy (VH) and variable light (VL) chain domains of 3H1 were linked by a 15 amino acid linker (Ln), (Gly4Ser)3 in two constructs, VH-Ln-VL and VL-LnVH. Ln was omitted in two constructs, VH-VL and VL-VH. Each of the scFv constructs has a tag of six His [(His)6 tag] for purification by metal chelate affinity chromatography and detection by enzyme-linked immunoabsorbent assay (ELISA). Comparisons of the binding of 8019 to purified scFv proteins by ELISA and immunoblot experiments showed that only VH-Ln-VL had significant activity. VH-Ln-VL also showed maximum inhibition of binding of 8019 to CEA. Immunization of mice with naked VH-Ln-VL and VH-Ln-VL conjugated to keyhole limpet hemocyanin induced anti-CEA antibodies in mouse sera. Sera from immunized mice inhibited the binding of 8019 to 3H1 as well as CEA. Induction of anti-CEA antibodies in the immunized mice was confirmed by flow cytometric analysis using CEA positive MC-38cea cells. These results demonstrate that for antigen mimicry of 3H1 scFv, the presence of Ln is necessary and the domain order should be VH followed by VL.