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CA 125 is found in body fluids in a variety of molecular weight forms. The largest species are found in normal abdominal fluid and cervical mucus. The present study therefore incorporated CA 125 derived from these sources as well as ascites fluid to investigate if the source of CA 125 influenced epitope characterization. Ascites-derived CA 125 varied in size from about 190 to about 2,700 kD. Cervical mucus-derived CA 125 treated with ultrasound changed its apparent size from more than 20,000 to 700 kD. Epitope mapping of antibodies was not grossly influenced by the size or source of CA 125 used as target. However, low-molecular-weight CA 125, i.e. ascites fractions CA 17/E, CA 17/F and CA 10/7, did show differences in certain assay combinations and cross-inhibition patterns which probably can be explained by steric effects due to the smaller size compared with the most abundant forms of CA 125 present in serum and other body fluids. The specificity of six new monoclonal antibodies to CA 125 was tested by cross-inhibition and immunometric assay combinations and compared to reference antibodies. One antibody, X306, belonged to the OC125-like antibodies. Four antibodies, X52, X75, X325 and VK8, were M11-like. The sixth antibody, 7C12, reacted with an epitope which was difficult to define. This antibody was inhibited by M11-like antibodies and OV197. However, used as an inhibitor, 7C12 inhibited only itself. We grouped it as an OV197-like antibody, but clearly different from OV197. The topography of epitopes was studied by analyzing all antibody pairs in immunoradiometric assays. These results confirmed the grouping of antibodies described above and are in accordance with previous findings that the highest signal is obtained using an OC125-like antibody or OV197 on the solid phase and an M11-like antibody as tracer. The composition of the sample in terms of high- and low-molecular-weight species of CA 125 was measured, with different responses depending on the antibody pair used. This might be one reason for discrepancies between assay results for CA 125 using different assays., (Copyright 2002 S. Karger AG, Basel)

CA125 is an ovarian cancer antigen that is the basis for a widely used serum assay for the monitoring of patients with ovarian cancer; however, detailed information on its biochemical and molecular nature is lacking. We now report the isolation of a long, but partial, cDNA that corresponds to the CA125 antigen. A rabbit polyclonal antibody produced to purified CA125 antigen was used to screen a lambdaZAP cDNA library from OVCAR-3 cells in Escherichia coli. The longest insert from the 54 positive isolated clones had a 5797-base pair sequence containing a stop codon and a poly(A) sequence but no clear 5' initiation sequence. The deduced amino acid sequence has many of the attributes of a mucin molecule and was designated CA125/MUC16 (gene MUC16). These features include a high serine, threonine, and proline content in an N-terminal region of nine partially conserved tandem repeats (156 amino acids each) and a C-terminal region non-tandem repeat sequence containing a possible transmembrane region and a potential tyrosine phosphorylation site. Northern blotting showed that the level of MUC16 mRNA correlated with the expression of CA125 in a panel of cell lines. The molecular cloning of the CA125 antigen will lead to a better understanding of its role in ovarian cancer.

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewis(y) (Le(y)), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Le(y) as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Le(y) conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Le(y)-serine epitopes and the Pam(3)Cys immunostimulating moiety was found to be superior to a similar construct containing only one Le(y)-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Le(y)-serine Pam(3)Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

To provide further information on the biochemical nature of the cellular and secreted forms of the mucin-like CA 125 ovarian cancer antigen. Pulse-chase experiments were performed in the NIH:OVCAR-3 ovarian cancer cell line with [(35)S]Met/Cys radiolabeling. After pulsing the cells with radioisotope for 30 min and analyzing cell lysates by immunoprecipitation with anti-CA 125 antibodies, a doublet species (form B, approximately 400 kD) and a ladder of slower-moving components were detected by SDS-PAGE and autoradiography. After a 4-hour chase period, a much larger species (form A) became evident. With further culture, the B form disappeared and the A form accumulated, suggesting a 'precursor-product' relationship between the two forms. The putative precursor species did not appear in the culture supernatant, but secretion of the mature species (form A) began after about 1 h of synthesis. Lectin-binding experiments demonstrated that the B form is a glycoprotein and not an early apomucin precursor. In contrast to other reports, no smaller species of the mature form of CA 125 were detected in this study. Trypsin digestion severely degraded the antigen but discrete smaller fragments were not formed. CA 125 antigen is synthesized through a glycosylated 400-kD precursor species. The mature form of the antigen appears in the cell after about 1 h of synthesis and in the culture medium after 1-4 h., (Copyright 2001 S. Karger AG, Basel)

On the basis of the observation that tumor cells exhibit aberrant glycosylation, carbohydrate vaccines are being developed for the active immunotherapy of cancer. Potential targets include members of the ABH Lewis blood group family (e.g., Lewis(y)), members of the mucin core family (e.g., Tn, sialyl Tn and T-F) and gangliosides (e.g., GM2). Most studies have used glycoconjugate vaccines in which a synthetic oligosaccharide is coupled to a protein carrier, for example, KLH, but completely synthetic constructs are also being examined. Preclinical studies in mice have shown the effectiveness of some of these vaccines. Clinical studies are under way in a number of human cancers but the results are still being evaluated.

This article traces reseach on the chemistry and immunochemistry of blood group A, B, H, and Lewis antigens from early work on the identification of soluble sources of these antigens, through the elucidation of the structures of the carbohydrate epitopes responsible for these specificities, to recent work on exploring their possible use as cancer vaccines. The various approaches used in the isolation of oligosaccharides from mucins for use in structural studies are discussed, as are recent efforts in the chemical systhesis of blood group-active oligosaccharides.

As the initial step in developing carbohydrate-based vaccines for the treatment of ovarian cancer patients in an adjuvant setting, 25 patients were immunized with a Lewis(y) pentasaccharide (Le(y))-keyhole limpet hemocyanin (KLH)-conjugate vaccine together with the immunological adjuvant QS-21. Four different doses of the vaccine, containing 3, 10, 30, and 60 microg of carbohydrate were administered s.c. at 0, 1, 2, 3, 7, and 19 weeks to groups of 6 patients. Sera taken from the patients at regular intervals were assayed by ELISA for reactivity with naturally occurring forms of Le(y) (Le(y)-ceramide and Le(y) mucin) and by flow cytometry and a complement-dependent cytoxicity assay for reactivity with Le(y)-expressing tumor cells. The majority of the patients (16/24) produced anti-Le(y) antibodies as assessed by ELISA, and a proportion of these had strong anti-tumor cell reactivity as assessed by flow cytometry and complement-dependent cytotoxicity. One serum, analyzed in detail, was shown to react with glycolipids but not with glycoproteins or mucins expressed by ovarian cancer cell line OVCAR-3. The vaccine was well tolerated and no gastrointestinal, hematologic, renal, or hepatic toxicity related to the vaccine was observed. On the basis of this study, Le(y)-KLH should be a suitable component for a polyvalent vaccine under consideration for the therapy of epithelial cancers., (Copyright 2000 Wiley-Liss, Inc.)

Ovarian cyst fluid has been a valuable source of the mucins (traditionally termed "blood group substances") that were used for the elucidation of the structures of the ABO Lewis blood group determinants, but the identity of the mucin peptide core(s) carrying these carbohydrate specificities is not known. An ovarian cyst fluid mucin was purified, deglycosylated with HF and digested with trypsin or chymotrypsin to yield a number of peptides. Amino acid sequencing of these peptides yielded five different sequences which showed complete or partial homology to the MUC-6 apomucin deduced from DNA sequencing. As no other sequences were identified, it is concluded that MUC-6 is the major mucin core structure of ovarian cyst fluid mucin.

The band 1q21 is among the most common sites affected by chromosomal translocations in lymphoid, myeloid, epithelial, and sarcomatous lesions. In non-Hodgkin's lymphoma (NHL), translocations and duplications affecting this chromosomal site are frequently, but not exclusively, seen in association with primary abnormalities such as the t(14;18)(q32;q21) and t(8;14)(q24;q32) translocations, suggesting a role for 1q21 rearrangements in tumor progression. We report here the characterization and cloning of breakpoints in a case of extranodal ascitic B-cell lymphoma with a t(1;14)(q21;q32) translocation. The breakpoints on the der(1) and der(14) chromosomes were mapped by fluorescence in situ hybridization and Southern blot analysis and cloned using an IGHG (Cgamma) probe. The translocation linked the IGHG4 switch (Sgamma4) sequences of the productively rearranged allele to chromosome 1 sequences downstream of MUC1, leaving the MUC1 transcriptional unit intact. MUC1 was markedly overexpressed in the tumor at the mRNA and protein levels relative to lymphoma cell lines lacking a 1q21 rearrangement. Presumably, MUC1 transcription is aberrantly regulated by the IGHA (Calpha) 3' enhancer element retained on the same chromosome. Screening of a panel of B-cell lymphomas by Southern blot analysis identified a subset with a 3' MUC1 breakpoint and another with low-level amplification of MUC1. MUC-1 mucin has previously been shown to be frequently overexpressed in human epithelial cancers and to be associated with tumor progression and poor clinical outcome. Thus, MUC1 activation by chromosomal translocation, rearrangement, and amplification, identified here for the first time in NHL, is consistent with its suggested role in tumorigenesis. (Blood. 2000;95:2666-2671)

We have previously reported on a carbohydrate-based vaccine program for immunotherapy in cancer patients. One such vaccine, based on the globo H antigen conjugated to the protein keyhole limpet hemocyanin (KLH), has been in clinical evaluation. Although this and other carbohydrate vaccines have been shown to induce antibody responses, there are currently no quantitative data on the antibody levels achieved in immunized patients by these or other anti-cancer vaccines. We report herein an efficient route to complex synthetic oligosaccharides attached to an affinity matrix for identifying and isolating antibodies elicited against such a carbohydrate-based vaccine in humans. Pre- and postvaccination profiles from serum samples of patients immunized with globo H-KLH were compared. All anti-globo H antibody activity was efficiently separated from other serum constituents. The isolated antibodies were readily quantified, and their specificities were analyzed. Since no comparable data were available on antibodies resulting from the vaccination of other cancer patients, we compared the observed levels with those quoted in studies with bacterial polysaccharide vaccines that had been quantified. Remarkably, cancer patients immunized with globo H-KLH produce anti-globo H antibody levels often exceeding those formed by immunization with bacterial polysaccharides. In addition, substantial quantities of both IgG and IgM antibodies were elicited, clearly indicating a class switch to IgG. Taken together, these analyses serve to clarify several aspects of the immune response to the vaccine and give several new insights to the carbohydrate-based vaccination strategy. Furthermore, antibodies so isolated could well have applications in clinical therapy.

The biologic functions of gangliosides G(M3) and G(D3) in the attachment of human melanoma cells to extracellular matrix proteins (type I and IV collagens, fibronectin, and laminin) were investigated by using the G(D3)-deficient mutant clone (SK-MEL-28-N1) and the parent cell line SK-MEL28. SK-MEL-28-N1 (N1) (high G(M3) expression: G(M3), 97.3%; G(D3), 0%) was selected by treating SK-MEL-28 (high G(D3) but low G(M3): G(M3), 6.5%, G(D3), 93.5%) with an anti-G(D3) monoclonal antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells. The N1 clone showed significantly higher ability to adhere to type I and IV collagens and laminin than the parent clone SK-MEL-28. In the N1 clone, the expression of alpha2beta1 and alpha3beta1 integrin receptors was increased, whereas in SK-MEL-28, their expression was very low or undetectable. The treatment with monoclonal antibodies directed specifically to G(D3) expressed on SK-MEL-28 inhibited the cell attachment to type IV collagen (33% inhibition of control), fibronectin (59%), and laminin (71%). These findings suggest that gangliosides G(M3) (by influencing integrin receptor levels) and G(D3) (by interacting directly with matrix proteins) might play some functional roles in attachment to extracellular matrix proteins and thereby enhance the metastatic potency of melanoma cells.

The complex carbohydrate molecule globo H hexasaccharide has been synthesized, conjugated to keyhole limpet hemocyanin, and administered with the immunologic adjuvant QS-21 as a vaccine for patients with prostate cancer who have relapsed after primary therapies such as radiation or surgery. Globo H is one of several candidate antigens present on prostate cancer cells that can serve as targets for immune recognition and treatment strategies. The vaccine, given as five subcutaneous vaccinations over 26 weeks, has been shown to be safe and capable of inducing specific high-titer IgM antibodies against globo H. Its immunogenicity was confirmed in prostate cancer patients with a broad range of stages and tumor burdens. Observations of several patients who had evidence of disease relapse restricted to a rising biochemical marker, prostate-specific antigen (PSA), indicated that a treatment effect could occur within 3 months after completion of the vaccine therapy. This effect was manifested as a decline of the slope of the log of PSA concentration vs. time plot after treatment compared with values before treatment. Five patients continue to have stable PSA slope profiles in the absence of any radiographic evidence of disease for more than 2 years. The concept of using PSA slope profiles in assessing early treatment effects in biological therapies such as vaccines awaits further validation in phase II and III trials. The use of a variety of lesser known candidate glycoprotein and carbohydrate antigens in prostate cancer serves as a focus for the development of a multivalent vaccine of the treatment of relapsed prostate cancer in patients with minimal tumor burden.

Expression of blood group-related carbohydrate antigens was examined in frozen sections from a series of ovarian carcinomas of different histological types using an indirect immunoperoxidase technique. Antigenic specificities belonging to the O(H) and Lewis blood group families (H-1, H-2, Le(a), sLe(a), Le(x), sLe(x), Le(b) and Le(y)) or the mucin-core family (Tn, sTn and TF) were studied. A distinct difference in antigen expression between mucinous and other ovarian carcinomas (serous and endometrioid) was observed. Specifically, mucinous tumors tended to express sTn, Le(a) and sLe(a) strongly and homogeneously, whereas serous and endometrioid tumors rarely expressed these specificities and, in contrast, expressed Le(y) and H type 2 antigen strongly. When expressed in serous tumors, sTn was usually distributed in a heterogeneous pattern, whereas sTn expression in mucinous tumors was much more homogeneous. The distribution of Le(y) in serous tumors was noticeably homogeneous. H-1, Le(x), sLe(x), Le(b), TF and Tn specificities were rarely expressed in any type of ovarian carcinoma. Our results provide further support for the different histogenesis of mucinous and non-mucinous tumors and indicate alternative differentiation pathways for the 3 pathological subtypes of ovarian tumor. They also provide the basis for the choice of carbohydrate antigens for active and passive immunotherapy of ovarian carcinomas.

Sialyl-Tn (STn) is an O-serine- or O-threonine-linked disaccharide [NeuAcalpha(2-->6)GalNAcalpha-O-Ser/Thr) expressed on mucins of most types of adenocarcinoma as single STn or clustered STn [STn(c)] epitopes. Though STn is expressed on some normal tissues it is relatively tumor-specific, especially in the clustered conformation. Clinical trials with STn-keyhole limpet hemocyanin (KLH) conjugate vaccines, prepared using reductive amination with a two-carbon linker group, have resulted in high titers against STn but lower titers against natural forms of STn (ovine submaxillary mucin, or tumor cells). To obtain antibodies of more appropriate specificity, we attempted to prepare STn(c)-KLH conjugates to establish their immunogenicity in mice in preparation for clinical trials; however, conjugation efficiency was poor when the same two-carbon linker was used, presumably because of steric hindrance. STn-KLH and STn(c)-KLH conjugates were prepared using the regular two-carbon or the recently developed more efficient longer heterobifunctional 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linkers, and the resulting immunogenicities in mice were compared. The highest titers against STn were seen with the STn-KLH conjugate with the two-carbon linker, and the highest titers against STn(c) were seen with STn(c)-KLH with the MMCCH linker. Conjugation with MMCCH resulted in the highest conjugation efficiency (yield) and the highest titers against ovine submaxillary mucin and STn-positive tumor cells, and is the method of choice for the preparation of STn(c) vaccine for clinical trials.

Most human neuroblastoma tumors are characterized by the high expression of GD2 (or GD2 and/or GM2) gangliosides, whereas melanomas characteristically express GD3 ganglioside. The molecular basis for these patterns was investigated by examining the relationship between ganglioside levels, glycosyltransferase (GM2/GD2 synthase and GD3 synthase) activity, and corresponding mRNA levels in a panel of human neuroblastoma and melanoma cell lines. In general, the ganglioside patterns could be explained by the levels of the transferases and their mRNA, indicating control at the level of transcription. A key role was noted for GD3 synthase. Notably, it was found that neuroblastoma cell lines with high GD2 ganglioside levels had low levels of GD3, its synthase, and mRNA for the enzyme even though this step provides the substrate for GD2 synthesis. The key role for GD3 synthase was also examined by stably transfecting GD3 synthase cDNA into a neuroblastoma cell line (SH-SY5Y) not expressing GD3 and GD2. The resulting cell line had high levels of GD2 ganglioside and altered morphology and growth characteristics.

Globo H (Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal alpha1 --> 4Galbeta1 --> 4Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc alpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 3Gal. An isomeric structure with an internal alphaGalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.

STn (NeuAcalpha2 --> 6GalNAc alpha-O-Ser/Thr) is a carbohydrate epitope overexpressed in various human carcinomas. Clinical trials are underway using synthetic STn or STn trimeric glycopeptides [STn, cluster; STn(c)] conjugated with keyhole limpet hemocyanin (KLH) as active specific immunotherapy for these cancers. These vaccines have been prepared by conjugating a crotyl ethyl amide derivative of STn or STn(c) to KLH by direct reductive amination after ozonolysis. In the case of STn(c) the conjugation efficiency and the resulting epitope ratios were low. This may be due to steric hinderance of the short spacer arm. To overcome these difficulties, without resynthesis, the STn(c) glycopeptide was modified by attachment of an MMCCH (4-(4-N-maieimidomethyl) cyclohexane-1-carboxyl hydrazide) spacer arm to the aldehyde derivative, and then conjugated with thiolated KLH. This method gave a higher epitope ratio and yield than the direct method. The STn(c)-MMCCH-KLH conjugate induced high titer antibodies in mice against STn(c). This method may be generally applicable for large synthetic oligosaccharides.

Many human carcinomas overexpress the Lewis(y) (Le[y]) blood-group epitope [Fucalpha1-->2Galbeta1-->4 (Fucalpha1-->3)GlcNAcbeta1-->3Gal-]. With a view to developing Le(y) based vaccines we have examined the immunogenicity of Le(y)-protein conjugates in mice. Le(y) pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Le(y) oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Le(y) oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Le(y) epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Le(y) (MCF-7). These experiments suggest that Le(y)-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients.

The panel of ISOBM TD-4 Workshop antibodies was tested against a range of carbohydrate antigens consisting of (a) ovarian cyst mucins expressing A, B, H, Le(a), Le(x), Le(b), Le(y) and precursor (nonfucosylated) specificities, (b) ovine submaxillary mucin (OSM, expressing sialyl Tn) and desialylated OSM (expressing Tn) and (c) synthetic glycoconjugates (Le(a)-PAA and Le(x)-PAA). The assay used was an ELISA method using rabbit anti-mouse Ig as the second reagent. Thirty-five of the 51 antibodies were unreactive with this group of antigens and presumably react with MUC1 peptide or a carbohydrate specificity not represented. Two MAbs (127 and 128) detected Le(x) antigen and one MAb (157) reacted with A blood group. MAb 151 (and possibly 152) detected sialyl Tn epitopes. Three antibodies (139, 149 and 168) reacted with nonfucosylated structures (possibly LNT or LNneoT). A number of MAbs (137, 145, 162, 163 and 164) reacted widely with the panel of antigens; whether these are nonspecific reactions due to 'sticky' antibodies or caused by the presence of MUC1 peptide in the antigen preparations is uncertain.

Monoclonal antibody (mAb) MX35 reacts with approximately 90% of ovarian epithelial cancers and has been studied in localization and biodistribution trials in ovarian cancer patients. This study shows that mAb MX35 recognizes a cell surface antigen of about 95,000 D on OVCAR-3 ovarian cancer cells. The antigen could be immunoprecipitated from lysates of cells metabolically labeled with [3H]glucosamine and it bound to concanavalin A and wheat germ agglutinin lectins, showing that it is a glycoprotein. MX35 antigen can also be detected in detergent lysates of OVCAR-3 cells by Western blotting. Using this technique the MX35 epitope(s) was shown to be heat stable but susceptible to reduction by 2-mercaptoethanol. Protease digestion of the antigen resulted in smaller fragments (42-52 kDa) that still reacted with antibody. We conclude that MX35 antigen is a 95 kDa glycoprotein, stabilized by disulfide bonds, with a large protease-resistant region that carries the MX35 epitopes., (Copyright 1997 Academic Press.)

Blood group-related antigens have been attractive targets for immunotherapy of cancer since their initial identification as cancer-related antigens. However, available information on the relative expression of most of these antigens on human malignant and normal tissues has been insufficient for selecting optimal antigens and tumors for immune attack. In this study, the distribution of the blood group-related antigens TF, Tn, sTn, Le(a), sialyl Le(a), Le(b), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on 13 types of cancer and 16 normal tissues was compared. Our results show that sTn is strongly expressed on cancers of breast, colon, stomach, ovary, prostate and uterus; Tn on prostate cancer; TF on cancers of breast, colon, ovary, prostate and uterus; Le(y) on the cancers of colon, lung, pancreas and ovary; Le(a) and Le(x) on gastric cancer; and sialyl Le(a) and sialyl Le(x) on colon cancer. The complete absence of these antigens on cancers of neuroectodermal or mesodermal origin including melanoma, sarcoma, neuroblastoma and B cell lymphoma is as striking as their widespread presence on tumors of epithelial origin. Normal tissues were also tested. Tn and Le(b) were only detected on gastric and ovarian epithelia; sTn on Leydig cells of testis in addition to gastric and ovarian epithelia; Le(x) and sialyl Le(x) on polymorphonuclear leukocytes; and TF, Le(a), sialyl Le(a), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on epithelia from a variety of tissues.

Understanding the distribution of tumor-associated antigens on cancers and normal tissues is essential for selection of targets for cancer immunotherapy. Seven carbohydrate antigens, potential targets for immunotherapy, were studied using a panel of well-characterized MAbs by immunohistochemistry on cryostat-cut tissue sections of 13 types of cancers and 18 normal tissues. GD2 and GD3 were present on most cancers of neuroectodermal origin and GD2 was also present on B cell lymphomas. 9-O-acetyl-GD3 was detected only on melanoma while fucosyl GM1 was detected only on small cell lung cancers (SCLC). Surprisingly, GM2 was strongly expressed on all tested tumors, including cancers of neuroectodermal origin and cancers of epithelial origin. Polysialic acid was primarily expressed on SCLC and neuroblastomas. Globo H was present on most cancers of epithelial origin. These antigens were also identified in normal tissues. Fucosyl GM1 was not expressed significantly on any of the normal tissues analyzed. GD3, GD2, GM2 and polysialic acid were detected in normal brain to varying degrees. GM2 and Globo H were expressed on the luminal surface of epithelia of a variety of organs. The unexpected expression of GM2 on a broad range of cancers and normal epithelial tissues was confirmed by loss after methanol fixation and by immune thin layer chromatography.

Our objective was to quantify the targeting of the monoclonal antibody (mAb) MX35 F(ab')2 to micrometastatic epithelial ovarian cancer. This mAb detects a Mr 95,000 glycoprotein with homogeneous distribution on 80% of ovarian tumor specimens. Six patients with minimal residual disease from an imaging trial were injected with 2 or 10 mg of 131I- and 125I-labeled mAb MX35 F(ab')2. Biopsied samples were removed at second-look laparotomy 1-5 days post-i.v. or -i.p. infusion of antibody. Serial cryostat sections were stained by indirect immunoperoxidase method for antigen distribution and exposed to storage phosphor screens for quantitative autoradiography. Coregistration of tumor histology, antigen expression, and radionuclide distribution demonstrated specific localization in micrometastatic tumor foci (50 micrometer to 1 mm) found within tissue stroma. The radiolabeled antibody uptake determined by well scintillation counts ranged between 5.2 and 223.5 x 10(-4) percentage of injected dose/g of tumor tissue for 131I. Specific localization of mAb in tumor was determined by tumor:normal tissue (fat) ratios ranging from 0.9:1 to 35.9:1 for 131I. The high resolution and linear response of the storage phosphor screen imager was used to estimate the radionuclide activity localized in each micrometastatic site. Quantitation of phosphor screen response revealed microCi/g values of 0.026-0.341 for normal tissue and 0.184-6.092 for tumor biopsies, evaluated 4 or 5 days post-antibody injection. The tumor:normal tissue (adjacent to tumor) ratios were between 1 and 4 times greater using the phosphor screen method than well counter measurements, but even larger variations of ratios up to 20:1 were observed between tumor cell foci and stromal cells within the same tissue section. This study has demonstrated that mAb MX35 F(ab')2 localizes to the micrometastatic ovarian carcinoma deposits within the peritoneal cavity. The dosimetry results suggest a therapeutic potential for this antibody in patients with minimal residual disease (<5 mm).

Mucins, including MUC-1, are generally considered to be products of epithelial tissues and of their tumors. To examine the possible expression of MUC-1 in other cell types, a panel of human epithelial and non-epithelial tumor cell lines was studied by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, immunocytology and radioimmunoprecipitation. Using the highly sensitive RT-PCR method, products corresponding to the non-repetitive 5' and 3' MUC-1 sequences were detected in all the cell lines examined. Amplified products lacking the tandem repeat region of MUC-1, including a new short form (designated MUC-1/Z) different from the previously reported MUC-1/Y protein, were also detected in most cell lines tested. Northern blot analysis, using a probe to the variable number tandem repeat (VNTR) region, confirmed the presence of MUC-1 mRNA in the astrocytoma, melanoma and neuroblastoma cell lines studied. MUC-1 protein was detected by immunocytology in these cell lines using monoclonal antibody (MAb) 139H2. Immunoprecipitation analysis with [3H]-glucosamine-labeled cell lysates and MAb 139H2 or an antibody to the cytoplasmic domain, CT-1, detected MUC-1 protein in 2 epithelial cell lines, an astrocytoma cell line (SK-MG-4) but not in the melanoma and neuroblastoma cell lines studied. Northern blot analysis using a probe to the 3' end of MUC-1 mRNA, confirmed the presence of MUC-1 mucin and also identified short products corresponding to the size of the short variant forms. Protein products corresponding to the MUC-1/Y and MUC-1/Z variant forms were not observed using either [3H]-glucosamine-labeled OVCAR-3 cells or [3H]-amino acid-labeled MCF-7 cells and either CT-1 antibody or MAb 232A1, detecting an epitope to the C-terminal region. Thus, depending on the sensitivity of the assay used, varying amounts of MUC-1 mRNA and protein could be detected in non-epithelial tumor cell lines. Although the amounts of MUC-1 in these cell lines are much lower than in carcinomas, it is possible that MUC-1 mucin serves a similar function in non-epithelial as in epithelial cells. The possible role of MUC-1/Y and MUC-1/Z variant forms in these cell lines is not understood.

The alpha2,3 sialyltransferase, alpha2,3 SAT (O), catalyzes the transfer of sialic acid to Galbeta1,3 N-acetyl-D-galactosamine (GalNAc) (core-1) in mucin type O-glycosylation, and thus terminates chain extension. A Core-2 branch can also be formed from core-1 by the core-2 beta1,6 N-acetyl-d-glucosamine transferase (beta1,6 GlcNAc T) that leads to chain extension. Increased levels of the alpha2,3 SAT (O) and decreased levels of the core-2 beta1,6 GlcNAc T are seen in breast cancer cells and correlate with differences in the structure of the O-glycans synthesized (Brockhausen et al., 1995; Lloyd et al., 1996). Since in mucin type O-glycosylation sugars are added individually and sequentially in the Golgi apparatus, the position of the transferases, as well as their activity, can determine the final structure of the O-glycans synthesized. A cDNA coding for the human alpha2,3 SAT (O) tagged with an immunoreactive epitope from the myc gene has been used to map the position of the glycosyltransferase in nontumorigenic (MTSV1-7) and malignant (T47D) breast epithelial cell lines. Transfectants were analyzed for expression of the enzyme at the level of message and protein, as well as for enzymic activity. In T47D cells, which do not express core-2 beta1,6 GlcNAc T, the increased activity of the sialyltransferase correlated with increased sialylation of core-1 O-glycans on the epithelial mucin MUC1. Furthermore, in MTSV1-7 cells, which do express core-2 beta1,6 GlcNAc T, an increase in sialylated core-1 structures is accompanied by a reduction in the ratio of GlcNAc: GalNAc in the O-glycans attached to MUC1, implying a decrease in branching. Using quantitative immunoelectron microscopy, the sialyltransferase was mapped to the medial- and trans-Golgi cisternae, with some being present in the TGN. The data represent the first fine mapping of a sialyltransferase specifically active in O-glycosylation and demonstrate that the structure of O-glycans synthesized by a cell can be manipulated by transfecting with recombinant glycosyltransferases.

A new murine monoclonal antibody (MAb VK-8), detecting the CA 125 ovarian cancer antigen, was used to purify this antigen from OVCAR-3 ovarian cancer cells by affinity chromatography. The biochemical properties of the purified antigen are characteristic of a mucin-type glycoprotein: (1) the molecule is highly glycosylated (77% w/w), mainly with galactose, N-acetylglucosamine, and N-acetylgalactosamine, (2) the protein moiety is rich in serine, threonine and proline, (3) many of the serine and threonine residues are glycosylated, (4) the glycan chains are almost entirely O-linked, with core 2 [Galbeta1 --> 3(GlcNAcbeta1 --> 6)GalNAc] structures predominating and (5) these chains carry fucosylated Type 2 (Le(y) and Le(x) and H type 2) blood group structures. The antigen exhibited a very high m.w. (> 10(3) kDa) in aqueous buffer as well as in urea, but was degraded by proteolytic enzymes to smaller fragments that no longer reacted with the antibody. Although this result, and other immunochemical data, indicate that OC125, the original MAb to CA125, and VK-8 antibodies detect epitopes on the protein portion of the molecule, the involvement of carbohydrate cannot be ruled out. Further insight into the structure and function of the CA125 antigen will come from cloning the gene coding for the peptide backbone, and from more detailed carbohydrate structural analysis.

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAcalpha2-3Galbeta1-3GalNAc-ol, Galbeta1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

T, Tn, and sialyated Tn (sTn) are pancarcinoma antigens, and increased expression of these carbohydrate epitopes has been correlated with a poor prognosis in several epithelial malignancies. Ten murine monoclonal antibodies have been generated to these antigens, and compared by ELISA and immunohistochemistry to established mAbs reactive with these antigens. Nine mAbs (3 IgM and 6 IgG) reactive with synthetic T-human serum albumin (T-HSA) were produced after immunizing BALB/c mice with a synthetic T-keyhole limpet hemocyanin glycoconjugate (T-KLH). An additional IgM mAb (145.22) was produced in mice immunized with erythrocytes isolated from a patient with Tn syndrome. Three IgM and six IgG1 mAbs reactive with T-HSA did not react with natural T antigen present on desialyated glycophorin. All three IgM and several IgG1 mAbs, however, did react with LS-174T, a mucinous colon carcinoma cell line, 647V, a human bladder carcinoma cell line, and TA3Ha, a murine mammary carcinoma cell line as well as fresh frozen colon carcinomas. MAb 145.22 reacted with both natural and synthetic sources of sTn and Tn, as well as with LS-174T cells and mucin deposits in 10/11 colon carcinomas on fresh-frozen sections. MAb B72.3 reacted strongly with ovine submaxillary mucin (OSM) and sTn-HSA, while mAb CC49, a second-generation mAb to TAG-72 carcinoma mucin, reacted strongly with OSM, less strongly with desialyated OSM, and only weakly with sTn-HSA, suggesting that the epitope specificity for mAb CC49 is distinct from that of B72.3.

We have selected GD3-deficient human melanoma cell lines, in order to investigate the function of GD3 ganglioside. This was done by treating SK-MEL-28 cells with anti-GD3 antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells, resulting in the derivation of two cell lines deficient in the cell surface expression of GD3. Neither cell line (designated SK-MEL-28-N1 and SK-MEL-28-N2) had detectable cell surface expression of GD3 as analyzed with monoclonal antibody R24, and no GD3 was detectable in either cell line by glycolipid isolation, thin-layer chromatography, or resorcinol-HC1 spray, but thin-layer chromatography immunostaining with monoclonal antibody R24 showed the presence of low amounts of GD3 in both N1 and N2 (1/40 of the amount in the parent cell line in N1 and 1/500 in N2). In SK-MEL-28-N1, the residual GD3 was shown by immunofluorescence assays on permeabilized cells to be present in discrete intracellular organelles, suggesting that these cells have a defect in the transport of GD3 as well as in its synthesis. Both SK-MEL-28-N1 and -N2 had an increase in detectable GM3 expression. The mutant cell lines had altered cell morphology in comparison to the parent cell line and both had slower growth rates in vitro and lower tumorgenicity in nu/nu mice. These results indicate that GD3 ganglioside plays an important role in proliferation and growth of melanoma cells.

Human mucin MUC1 is abundantly expressed in some cancers of epithelia] origin and is largely restricted to the apical surface of secretory cells in normal tissues. It is, therefore, a potential target for cancer immunotherapy. In preparation for clinical trials, vaccines containing synthetic MUC1 peptides of different lengths and sequences mixed with various adjuvants or covalently attached, using different linker methods, to protein carrier keyhole limpet hemocyanin (KLH) were studied in mice. MUC1 peptides (containing 30 amino acids), plus adjuvants QS-21 or Bacillus Calmette-Guerin, were incapable of inducing antibody. However, MUC1 peptides conjugated to KLH (MUCl-KLH), plus QS-21, induced high titer antibody against the immunizing peptides and against MUC1-expressing tumor cells. Although T-cell responses, including delayed-type hypersensitivity, lymphocyte proliferation, and CTL, were not observed in mice immunized with these vaccines, significant protection from MUC1-expressing tumor cell challenge in mice immunized with MUC1-KLH was observed. Based on these studies, a vaccine containing MUC1-KLH conjugate prepared with m-maleimidobenzoyl-N-hydroxysuccinimide ester linker, plus QS-21, has been constructed for testing in clinical trials.

The expression of A, B, and H blood group antigens in epithelial ovarian cancer was evaluated in 137 patients with advanced disease by staining frozen sections with specific monoclonal antibodies using an indirect immunoperoxidase method. Expression of blood group antigens was observed in a proportion of ovarian carcinomas and in some areas of ovarian surface epithelium. Forty-eight percent of the tumors tested from 130 blood group A, B, or 0 individuals showed no expression of the appropriate blood group antigen, 32% had heterogeneous antigen expression, and 20% had strong expression. In the 7 blood group AB patients studied, no expression, heterogeneous expression of both antigens, or absence of one, but not the other antigen, was observed. No tumor showed A, B, or H antigen expression that was not compatible with the patient's blood group type. Histologic Grade 3 tumors showed absence of blood group antigen expression more often than did Grade 2 tumors. The presence or absence of A, B, or H antigen expression did not correlate with survival in this group of patients. This is in contrast to studies in other epithelial tumor types in which the normal epithelium synthesizes blood group antigens and loss of ABH antigen expression is observed in the corresponding tumors.

The expression of Ley blood group antigen in epithelial ovarian cancer tissues and cell lines has been studied using a Ley-specific monoclonal antibody (MAb 3S193). In ovarian cancer specimens, Ley was expressed in 75% of the 140 tumor specimens examined, with strong or moderate expression being observed in 56% of the samples. Seven of the 11 ovarian cancer cell lines studied were Ley-positive. Using immunochemical approaches, Ley epitopes were found to be expressed on 4 types of carrier molecules: CA125 ovarian cancer antigen, MUC-1 mucins, lower m.w. glycoproteins and glycolipids. In cell lines, Ley was more commonly expressed on MUC-1 mucin than on CA125, whereas in tumor specimens Ley was commonly found on both CA125 and MUC-1. The biochemical nature of the smaller Ley glycoproteins was not determined, but it was shown that they were not CEA and LAMP-1, known Ley carriers in some other tumor types. Glycolipids carrying Ley epitopes were detected in both ovarian cancer cell lines and tumor specimens. The presence of Ley epitopes on a number of different molecular carriers, including 2 major ovarian cancer antigens (CA125 and MUC-1), explains the high incidence of Ley in ovarian cancer. The high expression of Ley in ovarian cancer and the availability of specific murine and humanized MAbs make Ley an attractive candidate target for clinical studies.

GD3 Synthase (alpha 2,8sialyltransferase) (EC 2.4.99.8) cDNA has been cloned by eukaryotic cell expression cloning. Using this cDNA as a probe, the expression level of the gene in human cancer cell lines was analysed by Northern blotting and RT-PCR, then correlated with the ganglioside expression and enzyme activity. Melanoma cell lines showed extremely strong bands in Northern blot and RT-PCR/Southern analysis. The enzyme activity was also very high in melanomas as expected. Neuroblastoma and astrocytoma lines showed relatively low levels of the gene expression, whereas they expressed high levels of GD2. Although the mRNA level of the GD3 synthase gene and enzyme activity in individual cell lines correlated positively, some cell lines showed much higher activity than expected from the mRNA level. Among leukaemia lines, adult T cell leukaemia-associated (HTLV-I+) lines showed fairly high levels of the mRNA. On the other hand, T-ALL lines showed very low levels. In addition, GD3 and GD2 expression and mRNA level of the gene during T lymphocyte activation were analysed. Only GD3 expression was induced by any of the stimulatory reagents used, and corresponding up-regulation of the GD3 synthase gene was shown in RT-PCR/Southern analysis.

Previous studies (Y. Nagata, S. Yamashiro, J. Yodoi, K. O. Lloyd, H. Shiku, and K. Furukawa (1992) J. Biol. Chem. 267, 12082-12089) had isolated a putative cDNA coding for beta 1,4 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and demonstrated the presence of GM2 and/or GD2 gangliosides in melanoma cell lines stably transfected with this gene. We have now measured the levels of glycosyltransferase activities and mRNA levels in five transfected cell lines in comparison with their parent cell lines (mouse melanoma B16 and human melanoma MeWo). Membrane preparations from the transfected cell lines demonstrated de novo synthesis of GM2 or GD2 in in vitro assays using GM3 or GD3, respectively, as ganglioside acceptors. The enzyme levels, however, varied considerably among the different transfectants, as did the mRNA levels for the beta 1,4 Gal-NAc-transferase. The effect of the transfected gene on levels of preexisting glycosyltransferases involved in ganglioside biosynthesis was also measured and the ganglioside composition of the cell lines was determined. The level of beta 1,4 GalNAc-transferase expressed in the different cell lines was found to dramatically influence the overall ganglioside composition of the cell. In the transfected cell line with the highest levels of the transferase, for example, biosynthesis was almost completely redirected away from the b pathway to the a pathway with the resulting expression of only GM2. These data from this family of related cell lines clearly demonstrate the primary roles that relative glycosyltransferase levels play in determining the ganglioside composition of cells.

Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), dot-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins.

Sialyl Tn (sTn) is a mucin-associated carbohydrate antigen expressed in most types of human adenocarcinoma. Defining the configuration of tumor cell surface sTn recognized by antibodies is important for understanding the basis for the cancer cell specificity of sTn-reactive mAbs, for the development of more effective mAbs, and for designing cancer vaccines against sTn. In this study, we compared the immunogenicity of synthetic single sTn disaccharide epitopes and clusters [sTn(C)] of 3 sTn epitopes covalently linked via serine to keyhole limpet hemocyanin [KLH; sTn-KLH and sTn(C)-KLH, respectively]. The cell surface sTn configurations were analyzed with the use of sera from mice immunized with these neoglycoproteins and a panel of sTn-reactive mAb. Sera from mice immunized with sTn-KLH reacted in direct and inhibition assays with sTn-human serum albumin (HSA) but only weakly with sTn(C)-HSA, whereas sera from mice immunized with sTn(C)-KLH reacted with sTn(C)-HSA but not with sTn-HSA. Both anti-sTn and anti-sTn(C) sera reacted with ovine submaxillary mucin (a natural source of sTn) and with sTn-positive human tumor cell line LS-C but not with sTn-negative LS-B cells. With regard to the sTn-reactive mAbs, B72.3 reacted exclusively with clustered sTn, whereas mAb B195.3R11 reacted preferentially with unclustered sTn. Results with mAbs TKH2, B239.1, and CC49 were less clear, although all reacted more strongly with clustered sTn than with unclustered sTn. These results suggest that sTn is recognized at the tumor cell surface in at least two quite distinct configurations, as clustered and nonclustered arrays.

The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.

Melanomas and other cancers of neuroectodermal origin express multiple cell-surface gangliosides in patterns that vary significantly even within the same tumor type. Monoclonal antibodies (mAb) against four of these gangliosides (GM2, GD2, 9-O-acetyl-GD3 and GD3) were tested alone and in combination on 14 tumor cell lines (7 melanomas, 3 neuroblastomas, 3 sarcomas and 1 astrocytoma) using flow cytometry and complement-dependent cytotoxicity (CDC) assays. Increased tumor cell recognition and CDC resulting from the combination of three or four mAb were found in 14/14 tested cell lines, and this was most striking when each mAb was used at suboptimal concentration. At these concentrations, the average mean fluorescence intensity of the 14 cell lines with individual mAb was between 3.0 and 6.8 and increased to 10.8 and 18.8 with the three- and four-mAb mixtures. The average percentage CDC-specific release with individual mAb was 2.0%-8.3%, and 12.3% and 16.6% with the three- and four-mAb combinations. The number of cell lines showing significant mean fluorescence intensity and CDC increased from 2-8/14 with single mAb to 13-14/14 with the mixtures of three or four mAb. Our experimental results support the rationale for active immunization with a polyvalent ganglioside vaccine or passive therapy with a combination of mAb to different gangliosides in patients with tumors of neuroectodermal origin. In addition, our studies have demonstrated that 9-O-acetyl-GD3 is a surprisingly effective target for immune attack, although it is a minor constituent of these cells.

Le(y)-reactive monoclonal antibodies (mAbs) were generated in mice by immunization with synthetic Le(y) neoglycoproteins or with Le(y)-expressing cells. Serological analysis indicated that mAbs raised against synthetic Le(y) (i) reacted strongly with synthetic Le(y) but poorly with natural Le(y), (ii) cross-reacted with Le(x) or H-type 2 structures, and (iii) were IgG1, IgG2a, or IgG2b. mAbs raised against Le(y)-expressing cells (i) reacted with both synthetic Le(y) and natural Le(y), (ii) were of two types: cross-reactive with Le(x) or H-type 2 structures or specific for Le(y), and (iii) were IgM or IgG3. One of the mAbs raised against natural Le(y), mAb 3S193 (IgG3), showed high specificity for Le(y) in ELISA tests with synthetic Le(y) and Le(y) containing glycoproteins and glycolipids; it also reacted strongly in rosetting assays and cytotoxic tests with Le(y)-expressing cells. mAb 3S193 did not lyse O, A, AB, and B human erythrocytes in the presence of human complement. In flow cytometry, there was weak reactivity with granulocytes, a reactivity also observed with two previously described highly specific Le(y) mouse mAbs--BR55-2 (IgG3) and B3 (IgG1). A humanized version of mAb 3S193 has been constructed, and the specificity pattern and reactivity for Le(y) remain very similar to mouse mAb 3S193.

The internalization and recycling of glycosphingolipids (GLs), added exogenously to cells, has been shown in a number of systems. In addition, a portion of the internalized GLs becomes elongated by further glycosylation, presumably by passage through the Golgi apparatus, and is recycled to the plasma membrane. We have previously shown (K. Furukawa, I. Thampoe, H. Yamaguchi, and K. O. Lloyd J. Immunol. 142, 848, 1989) that NeuGc-LacCer (GM3), added exogenously to cultured human melanoma cells, is converted to NeuAc-NeuGc-LacCer (GD3) and appears at the cell surface where it can be recognized by a monoclonal antibody (32-27M) specifically recognizing this structure. The mechanism of this process has been investigated by analyzing the effect of monensin and Brefeldin A (BFA), two drugs known to affect vesicular transport and Golgi function, on the recycling of NeuGc-LacCer. Using two different serological assays, BFA was shown to specifically inhibit the recycling process. BFA probably achieves this effect by inhibiting the transfer of endocytosed GL from endosomes and the trans Golgi network to the Golgi stacks by a retrograde route and thus prevents its entry into the biosynthetic compartments. Monensin had a similar, but less clear-cut, effect on GM3 recycling. These results have important implications for understanding the modulation of cell surface glycolipids.

For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.

As an adjunct to existing thin layer and column chromatographic methods for the identification of glycolipids a method that utilizes the high pH anion chromatographic (HPAEC) analysis of the oligosaccharides released from the glycolipids by endoglycoceramidase has been developed. Using a Dionex Carbo Pak PA1 column and elution with a linear gradient of sodium acetate in 0.2 M NaOH, the elution times of eight neutral and fourteen acidic oligosaccharides derived from glycolipids were determined. Under these conditions the neutral oligosaccharides were well separated from each other but some of the acidic oligosaccharides had overlapping elution times. The ganglioside-derived oligosaccharides could be further identified by treating them with sialidase or by mild acid hydrolysis and reanalysing the products by HPAEC. The method was applied to the analysis of mixed bovine brain gangliosides. The procedure provides an additional approach for the initial identification of glycolipids by analysing the component oligosaccharides rather than the intact glycolipids.

Background: Although expression of the HER-2/neu oncogene may be of some prognostic importance in advanced ovarian cancer, its role in early-stage disease has not been established. The current study examined the prevalence and significance of HER-2/neu expression in early epithelial ovarian cancer., Methods: The authors analyzed the expression of HER-2/neu on frozen tumor specimens from 40 patients with early epithelial ovarian cancer using the indirect immunoperoxidase technique with monoclonal antibodies that detect epitopes on the extracellular domain of the HER-2/neu protein. All patients underwent comprehensive surgical staging. HER-2/neu expression was graded as negative, weak, moderate (1+ to 2+), or strong (3+). Complete clinical data and long-term follow up were available for all patients., Results: The distribution of patients by stage was as follows: Stage IA, 6; IB, 0; IC, 14; IIA, 4; IIB, 6; IIC, 10. The mean patient age was 53 years. Fourteen patients had serous tumors; nine, endometrioid; eight, clear cell; eight, mucinous; and one, undifferentiated. Intratumoral heterogeneity of HER-2/neu expression was observed with most specimens. In eight specimens (20%), some areas of the tumor showed strong (3+) expression, beyond the level that can be seen in normal ovarian epithelium. Twenty-eight specimens (70%) showed moderate (1+ to 2+) staining, whereas four specimens (10%) showed negative or weak staining. At a mean follow-up time among surviving patients of 32 months, 15 patients (37%) have had cancer recurrence. No statistically significant relationship was found between HER-2/neu expression and survival, disease-free survival, stage, or grade. A significant increase was found in 3+ expression of HER-2/neu in clear cell tumors., Conclusion: Consistent HER-2/neu overexpression occurs infrequently in early ovarian cancer, making it unlikely that such overexpression is a general early event in ovarian carcinogenesis. HER-2/neu expression does not appear to be a strong prognostic marker in early epithelial ovarian cancer.

Cluster 13 was defined by 2 independently derived murine monoclonal antibodies (MAbs), RS7 (IgG1) and MR54 (IgG2a), which were raised against human squamous-cell carcinoma of the lung and a human ovarian-carcinoma cell line, respectively. Immunologic and biochemical evidence demonstrated that RS7 and MR54, as well as 2 additional MAbs, MR6 (IgG2a) and MR23 (IgG1), generated in the same fusion as MR54, recognize the same antigen, a 46- to 48-kDa glycoprotein. Evaluation of the expression of antigen on the surface of tumor cell lines, Western blotting analyses, competitive binding studies, and double-determinant ELISA assays, support this conclusion. Two distinct epitopes are defined by these MAbs. In order to further characterize this antigen, amino-acid-sequence analyses were performed on peptides derived from antigen purified by affinity chromatography with MAb RS7. The sequence data obtained from 2 peptides, which were independently generated by CNBr cleavage and trypsin digestion respectively indicated identity to GA733-1. The GA733-1 genomic DNA sequence predicted a type-1 membrane protein of 35 kDa, with 4 potential N-linked glycosylation sites. The GA733-1 protein product has not been identified previously, and MAbs to this tumor-associated antigen were not previously known.