Characterization of N-glycolylneuraminic acid-containing gangliosides as tumor-associated Hanganutziu-Deicher antigen in human colon cancer

Hanganutziu-Deicher (HD) antigen-active N-glycolylneuraminic acid (NeuGc)-containing gangliosides were isolated and characterized from human colon cancer tissues. The antigenic gangliosides were detected by thin-layer chromatography by our newly developed method (H. Higashi, Y. Fukui, S. Ueda, S. Kato, Y. Hirabayashi, M. Matsumoto, and M. Naiki. J. Biochem., 95: 1517-1520, 1984) of enzyme immunostaining using affinity-purified chicken antibody against hematoside containing NeuGc (II3NeuGc-LacCer) and horseradish peroxidase-conjugated rabbit anti-chicken lgG. One to six species of the antigenic gangliosides were isolated from seven of 16 cases of colon cancer, whereas no antigenic compound was detected in all apparently normal colorectal tissues from 17 individuals without colorectal cancer. Tissues from different patients showed different patterns of molecular species of the antigenic gangliosides. Densitometric determination indicated that HD antigenic sialic acid, NeuGc, accounted for about 1% or less of the total lipid-bound sialic acids. Four species of antigenic gangliosides were identified as hematoside and hematoside-containing O-acyl ester (II3NeuGc-LacCer and II3 4- or 7-O-acyl-NeuGc-LacCer), GM2-containing NeuGc (II3NeuGc-GgOse3Cer), and sialylparagloboside (IV3NeuGc-nLcOse4Cer) by their behaviors on 2-dimensional thin-layer chromatography, and the effects of mild alkaline treatment, sialidase treatment, periodate oxidation, and endo-beta-galactosidase treatment.     

Establishment of a human monoclonal antibody to Hanganutziu-Deicher antigen as a tumor-associated carbohydrate antigen

We have established a human-human hybridoma producing a monoclonal antibody against a tumor-associated carbohydrate-specific antigen, Hanganutziu-Deicher (HD) antigen. Human spleen lymphocytes from a patient with esophageal varices complicated with liver cirrhosis were cultured in serum-free medium and co-stimulated with both anti-human mu-chain antibodies and supernatants of concanavalin A-stimulated human spleen cell culture (ConA sup). The activated lymphocytes were subsequently primed in vitro with particulate HD3 antigen and fused with a parent hybrid myeloma cell line, KR-12. A hybridoma, 1F43E31G7 produced anti-HD human monoclonal antibody (IgM lambda). This monoclonal antibody reacted strongly with N-glycolylneuraminyl alpha 2-3 lactosylceramide (HD3) and slightly with N-glycolylneuraminyl alpha 2-3 lactoneotetraosylceramide (HD5), but did not react with N-glycolylneuraminyl alpha 2-3 lactoneohexaosylceramide (HD7), N-acetylneuraminyl alpha 2-3 lactosylceramide (GM3) and other derivatives of HD3 prepared by chemical modification of the sialic acid residue of HD3, which indicates that the monoclonal antibody is directed precisely toward the terminal sialic acid and whole structure of HD3.     

Detection of tumor-associated antigen in human melanoma cell line supernatants.

Spent tissue culture medium (CDM-S) removed from a single cell line of human malignant melanoma grown in serum-free CDM, contained tumor-associated antigenic activity. Antibodies to CDM-S measured by complement fixation were detected in 44% (31/70) melanoma, 55% (15/27) sarcoma, 63% (24/38) carcinoma and 15% (11/72) normal sera. Delayed cutaneous hypersensitivity reactions (DCHR) were demonstrated in 4/5 melanoma patients at a 500 mug dose, 3/5 at a 100 mug dose and in 1/7 carcinoma patients at the 500 mug dose. One ml of CDM-S was shown to contain antigen equivalent to that obtained from the membranes of 2.9 X 10(7) tissue-cultured melanoma cells. After purification, 84% (16/19) sera from melanoma patients, 66% (12/18) from sarcoma and carcinoma patients and 8% (2/26) from normal controls were positive to the antigen by complement fixation.     

Occurrence of tumor-associated ganglioside antigens with Hanganutziu-Deicher antigenic activity on human melanomas

The specificity of antibody to NeuGc alpha 2-3Gal beta 1-4Glc-cer (GM3(NeuGc] was carefully reexamined by the method of enzyme-immunostaining on a thin layer plate. The affinity-purified antibody was found to react with NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuGc] and NeuGc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuGc-NeuAc], but not with NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuGc)) or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc-cer (GD3(NeuAc-NeuAc]. From this result together with the previous results, it (GD3(NeuAc-NeuAc], From this result together with the previous results, it could be concluded that the antibody recognizes the outer portion of molecular species of sialic acids in the gangliosides. By using this antibody, the expression of Hanganutziu-Deicher (HD) gangliosides could be demonstrated in human malignant melanoma. The molecular species were different among individuals examined. Among HD-antigenic gangliosides, GM3(NeuGc) was commonly found in melanoma tissues. One of the patients examined expressed GD3(NeuGc-NeuGc) and GD 3(NeuGc-NeuAc), which may be characteristic gangliosides in human melanomas, since these gangliosides could not be detected in human colon cancer or human fetal tissues.     

Detection of a tumor-associated nucleoprotein antigen in mink lung cells transformed by two different viral oncogenes

Salt-precipitated chromatin was prepared from cultured MvlLu line mink lung cells and from these cells transformed by either of the oncogenes v-mos (MIMS-102 line) or v-fes (F3C7 line). Xenoantisera were raised to chromatin from each of the three cell types and cross-tested in microcomplement fixation assays to determine immunospecificity. Chromatin from cells transformed by either v-mos or v-fes revealed antigenic profiles statistically indistinguishable (P less than 0.2 to 0.5) from one another with their respective cross-tested antisera, but did not react significantly with antisera to chromatin from the untransformed parental cell line. Likewise, little cross-reaction was observed with chromatin from the untransformed cells and antisera raised to chromatin from either of the oncogenically transformed lines (P less than 0.001), although each chromatin demonstrated high reactivity with its homologous antiserum preparation. These immunological data are consistent with the observed normal or transformed characteristics for each cell type, including morphology, anchorage-independent growth, and growth in the absence of serum.     

Detection of a polyoma virus-induced tumor-associated membrane antigen in mouse cells by the macrophage migration inhibition test

Soluble membrane fractions derived from polyoma tumor cells trigger lymphocytes, derived from polyoma-immunized animals, but not from nonimmunized controls, to release the lymphokine, macrophage migration-inhibitory factor. The reaction can be blocked by sera from polyoma-bearing animals. Absorption of these sera with polyoma cells, but not with nonpolyoma cell lines, abrogates this activity. These findings suggest that there is a polyoma virus-induced membrane component that can induce polyoma-specific macrophage migration inhibition.     

Specific expression of unusual GM2 ganglioside with Hanganutziu-Deicher antigen activity on human colon cancers

This paper reports the presence of GM2 ganglioside containing N-glycolylneuraminic acid (NeuGc) in human colon cancer tissues. GM2(NeuGc) was detected by two-dimensional thin layer chromatography (2d-TLC)/enzyme-immunostaining using affinity-purified chicken antibody against GM3(NeuGc) and horseradish peroxidase-conjugated rabbit anti-chicken IgG antibody. Like usual GM2 ganglioside containing N-acetylneuraminic acid (NeuAc) isolated from Tay-Sachs brain, GM2(NeuGc) in colon cancer could be converted into GM3(NeuGc) by human kidney beta-N-acetylhexosaminidase A in the presence of a GM2-specific activator protein isolated from guinea pig kidney. Three of 7 specimens of Hanganutziu-Deicher (HD) antigen-positive human colon cancer tissues so far examined expressed this unique ganglioside. In order to detect and determine specifically GM2(NeuGc) on human colon cancers, specific antibody against GM2 (NeuGc) has been prepared by immunizing chickens. By a sensitive TLC/immunostaining method using the antibody, the amounts of the antigen were determined to be 0.3-3% of total lipid-bound sialic acid. NeuGc-containing gangliosides were also detected in meconium and fetal intestinal tissues. Three species of antigenic gangliosides in pooled meconium were tentatively identified as GM3(NeuGc), sialylparagloboside and sialylhexaosylceramide on the basis of their migration positions on 2d-TLC and the results of endo-beta-galactosidase treatment. GM3(NeuGc) was the sole HD-active ganglioside in fetal intestinal tissue from one of 3 individuals tested; the other two showed no HD-active ganglioside at all. GM2(NeuGc), however, could not be detected in either meconium or fetal tissues so far examined, suggesting that this unique ganglioside is a tumor-specific antigen, at least for human intestinal tissues.     

Characterization of sialosylated Lewisx as a new tumor-associated antigen

A monoclonal antibody CSLEX1 which reacts with sialosyl Lex but not with sialosyl Lea has been produced. The CSLEX1 antigen has a tissue distribution similar to that of Lex, appearing characteristically in the proximal tubules of the kidney and on granulocytes. It is tumor associated in that 14 of 34 (41%) of tumor lines tested reacted with the CSLEX1 antibody, and 50 of 74 (68%) of tumor tissues tested reacted with the antibody. Loss of immunoperoxidase staining of tissues after neuraminidase treatment showed that the antibody is reacting to sialyl derivatives. The antibody reacted in solid-phase radioimmunoassay to sialosyllactofucopentaosyl(III)ceramide and sialosyldifucosylganglioside (6B). These results indicate that the CSLEX1 epitope has the following structure: (formula: see text) This structure had not previously been known to be tumor associated.     

Characterization of gangliosides in human uveal melanoma cells

We have evaluated the ganglioside composition of 20 primary uveal melanomas, of 2 cell lines derived from 2 uveal melanomas, of a liver metastasis from an uveal melanoma, and of 8 normal choroids. The results show that normal choroid tissue has a ganglioside content similar to the primary tumors of uveal melanoma except for GD1a, GD1b, and GT1b, which are present only on normal choroid tissues. On the other hand, the uveal melanomas have similarities with cutaneous melanomas, since GM3 (74%) and GD3 (25%) are found in both tissues and are present in about the same amounts. However, GM1 was found in 50%, GM2 in 20% and GD2 in none of the uveal melanomas. According to data published by others, cutaneous melanoma biopsies have no GM1, whereas GM2 is present in 100% and GD2 in 71% of tumor tissues. Transplantation of the 2 cell lines subcutaneously into nude mice resulted in the growth of tumors which had a ganglioside profile larger than that of the primary tumors. GM3 was significantly diminished and GD3 significantly increased in the primary uveal melanoma from patients who had received radiotherapy before enucleation compared with those who did not have radiotherapy. These results show that uveal melanomas contain gangliosides that could be used as targets for monoclonal antibody therapy.     

Detection of Gangliosides as N-Glycolylneuraminic Acid-specific Tumor-associated Hanganutziu-Deicher Antigen in Human Retinoblastoma Cells

Gangliosides were shown to bear the tumor-associated N -glycolylneuraminic acid (NeuGc)-specific Hanganutziu-Deicher (HD) antigen expressed in human retinoblastoma cells. HD antigenie gangliosides were detected by thin-layer chromatography/enzyme-immunostaining using affinity-purified chicken antibody against GM3 containing NeuGc and horseradish peroxidase-conjugated anti-chicken IgG. One to four species of the antigenic gangliosides were detected from all of 4 cell lines, Y79, WERI-Rb1, TOTL1, and YK, as well as freshly cultured retinoblastoma cells and isolated tumor tissue. All cases contained GMS(NeuGc) as an HD antigen. No HD antigenic ganglioside was detected in normal retinal tissues by the same procedure.     

Sialylated Lewis x as a tumor-associated antigen in stomach cancer

Sialylated Lewis (S-Lex) has been studied histologically and serologically in stomach cancer by the CSLEX1 monoclonal antibody. S-Lex was expressed in 73.9% of 46 stomach cancer tissues, 29.4% of metaplastic parts adjacent to cancer, and none of six gastric ulcer tissues including metaplasia. Serologically positive percentages were as follows: 26.0% of 100 stomach cancers, 0.9% of 322 benign diseases, and 0.7% of 280 healthy donors in the sera, as well as 72.4% of 29 ascites of stomach cancers and 5.3% of 17 effusions of benign diseases. These findings demonstrate that S-Lex possesses a potential usefulness as a tumor marker in stomach cancer.     

Quantitative determination of N-glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as a tumor-associated sialic acid by gas chromatography-mass spectrometry

N-Glycolylneuraminic acid (NeuGc) is distributed in most animals except humans and chickens. However, human and chicken cancerous tissues often synthesize this heterophilic sialic acid as a tumor-associated Hanganutziu-Deicher antigen [M. Naiki and H. Higashi, Adv. Exp. Med. Biol., 152: 445-456, 1982; H. Higashi et al., Cancer Res., 45: 3796-3802, 1985]. In this paper, NeuGc in human cancerous tissues and chicken Marek's disease lymphoma cell lines was determined quantitatively with gas chromatography-mass spectrometry analysis using mass fragmentography. The detectable limit of NeuGc was 40 pg (0.12 pmol) in each injection using 5 ng of trideuteriomethyl ester trideuteriomethyl glycoside of the sialic acid as an internal standard sample when a pair of ions at m/e 386 and 389 was chosen for ion monitoring. NeuGc was detected in ganglioside-rich fractions of various human cancerous tissues from 5 of 8 patients examined but was not detected in glycosphingolipids of normal human tissues. The contents of NeuGc in these cancerous tissues ranged from 0.02 to 0.5% of the total sialic acid content. NeuGc was also detected in freeze-dried samples of 5 different cell lines from chicken Marek's disease lymphomas but was not detected in a cell line from chicken lymphoid leukosis lymphoma and normal chicken skeletal muscle tissue. The contents of NeuGc in the positive cell lines ranged from 0.03 to 0.11% of the total sialic acid content. These results indicate that NeuGc can be synthesized in both humans and chickens in some cancers.     

Isolation of a soluble tumor-associated antigen from human melanoma.

Human melanoma-associated antigen was solubilized from fresh surgical specimens by 3 M KC1 extraction. The antigenicity of this extract was demonstrated by delayed cutaneous hypersensitivity responses and inhibition of complement fixation. Twenty-one of 33 melanoma patients had delayed cutaneous hypersensitivity responses to melanoma antigen, while only four of 28 reacted to autologous muscle. Although KC1 extracts did not fix complement directly, they reacted with antibody, thus inhibiting complement fixation by the autologous melanoma antigen extracted from tissue culture supernatants. The soluble antigenic moiety was then purified by fractionation on a G-150 Sephedex column and polyacrylamide gels, and the antigenic activity was monitored by delayed cutaneous hypersensitivity responses in melanoma patients. A 20-fold purification was achieved. Solubilization of tumor antigens with 3 M KC1 provides tumor-associated antigen of high activity which is amenable to further biochemical purification.     

Purification and characterization of a human lung tumor-associated antigen.

A human lung tumor-associated antigen was purified from a saline extract of a lung adenocarcinoma. The antigen was demonstrated in extracts of lung adenocarcinoma. The antigen was demonstrated in extracts of lung tumors with the use of an absorbed antiserum by double-diffusion immunoprecipitation. The antiserum did not react with extracts of normal lung or other normal tissues, and the antigen was immunologically distinct from other tumor-associated antigens. Purification was achieved by antibody affinity chromatography and preparative polyacrylamide gel electrophoresis. Isolation procedures were monitored by immunoreactivity with absorbed monospecific antiserum. The antigen was labeled with 125I and judged homogeneous by 1) polyacrylamide gel elecrophoresis in detergent and nondetergent gels, 2) molecular sieve chromatography, 3) ion exchange chromatography, and 4) sucrose gradient sedimentation analysis. A molecular weight of 77,000 was calculated from the s20.w value of 4.24S and from the D20.w value of 5.0X10(-7) cm2/sec. Sodium dodecyl sulfate gel electrophoresis indicated a subunit molecular weight of 42,000. The Stokes radius of the antigen was 40 A and the frictional ratio was 1.42, indicating a nonspherical molecule. The purified radioiodinated antigen could be quantitatively precipitated with specific antiserum.     

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.     

A tumor-associated antigen expressed in melanoma cells with lower malignant potential

The antigen K-1-2, detectable by a MAb is found in nevi and melanomas. It is associated with melanoma cells of low invasive and metastatic potential as shown by immunoperoxidase studies with cell lines, biopsies and autopsies: K-1-2 occurs in melanoma cell line SK-Mel 25, but not in cell line A-375. A-375 has a higher malignant potential than SK-Mel 25 because, in contrast to SK-Mel 25, it produces plasminogen activator and grows in nude mice. K-1-2 was frequently strongly expressed (greater than or equal to 50% cells positive) in flat (less than 1.5 mm) and less frequently in medium and thick primary tumors. In thick primary melanomas K-1-2 positive cells were confined to the junctional zone or to marginal, flat areas of the tumor. Only rarely does K-1-2 occur in metastases. Strong expression of the K-1-2 antigen was found less often in primary melanomas, which develop early metastases, than in tumors that had not metastasized during an observation period of 18 months. In 5 patients with disseminated metastatic disease, metastases strongly expressing K-1-2 and those negative for this marker or containing only a minor percentage of K-1-2 positive cells were observed simultaneously or at different times. These findings suggest that a change from high malignancy to low malignancy--as observed in animal systems--may also occur in human melanoma.     

Identification of CLUAP1 as a human osteosarcoma tumor-associated antigen recognized by the humoral immune system

Since the prognosis of human osteosarcoma in advanced stage remains poor, the development of new and effective therapies including immunotherapy is required. To identify tumor-associated antigens of osteosarcoma applicable to the immunotherapy of this malignancy, we employed the serological analysis of recombinant cDNA expression library (SEREX) technique that defines tumor antigens recognized by the humoral immune system. Screening a cDNA library derived from an osteosarcoma cell line MG63 with sera from osteosarcoma patients identified 43 positive clones, representing 14 distinct antigens. Among them, CLUAP1 (clusterin-associated protein 1) was highly expressed in osteosarcoma tissue samples and cell lines. Overexpression of CLUAP1 was observed in other malignancies including ovarian, colon, and lung cancers. Our results suggest that CLUAP1 may be useful as a prognostic/diagnostic marker and/or for a target of immunotherapy of osteosarcoma.     

Characterization of a human endocrine tissue and tumor-associated Ewing's sarcoma antigen

The histogenesis of Ewing's sarcoma (ES), the second most frequent primary bone tumor in humans, remains controversial. A new cell line (SIM-1) was derived from a peripheral neuroectodermal tumor (PNET) and used for the production of a monoclonal antibody (HBA-71), which recognizes a novel cell surface antigen of ES- and PNET-derived cells and paraffin-embedded tumor sections. The HBA-71 antigen expression is restricted to PNET/ES and the antigen was not detected on cell lines or tissue sections of any other tumor tested, with the exception of ependymoma. Three proteins with molecular weights of 300,000, 185,000, and 90,000 were isolated from SIM-1 membrane extracts by HBA-71 affinity chromatography. Trypsin treatment of intact SIM-1 cells destroys the HBA-71 epitope and cleaves off two proteins with molecular weights of 210,000 and 95,000. HBA-71 antigen expression is not influenced by treatment of ES cell lines with differentiation inducers. Within normal tissues reactivity was observed with the adenohypophysis, ependymal cells, endocrine pancreas, Sertoli, and ovary granulosa cells. The reagent links ES with PNET and provides a highly valuable probe for (a) the immunohistological differential diagnosis of ES/PNET using fresh tissue or paraffin sections from other small round cell tumors, (b) the histogenetic studies of ES/PNET, and (c) the in vivo diagnostic and therapeutic procedures in patients with ES and PNET.