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C J Wikstrand

Publications and source records attributed to C J Wikstrand.

At least 37 records · Page 2Linked to original sources
PubMed1995

Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts.

Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene are characteristics of many types of tumors. One class of EGFR mutations, EGFRvIII, is characterized by an in-frame deletion resulting in a truncated external domain of the receptor. EGFRvIII was first identified in a subset of gliomas and has since been found in some non-small cell lung carcinomas and breast carcinomas. mAbs specific for this variant form of EGFR but unreactive with the wild-type EGFR have been reported from our laboratory. This study further characterizes three of these antibodies. We determined, via radiolabeling techniques and immunofluorescence microscopy, that, after cell binding in vitro, the anti-EGFRvIII-specific mAbs internalize at 37 degrees C. Furthermore, subsequent to internalization, the antibodies were processed intracellularly, presumably by lysosomal degradation. We also examined the use of an alternative radiolabeling procedure that uses nonmetabolizable radio-iodinated tyramine cellobiose. Our results show that the tyramine cellobiose labeling method allows for greater tumor cell retention of radiolabel in vitro (76% for tyramine cellobiose and 27% for Iodo-Gen after 24 h). Paired-label biodistribution studies in athymic mice indicate that anti-EGFRvIII mAb L8A4 localizes specifically to EGFRvIII-expressing tumor xenografts with a maximum of 34.3 +/- 7.6% injected dose/g when labeled using tyramine cellobiose compared with a maximum of 14.9 +/- 4.3% injected dose/g using Iodo-Gen; similar results were obtained with mAb H10. These results suggest that the anti-EGFRvIII mAbs may serve as potential carriers for radioconjugate- and immunotoxin-based therapies for tumors expressing EGFRvIII.

Animals↗
PubMed1995

Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.

Despite molecular biological advances in understanding human cancers, translation into therapy has been less forthcoming; targeting neoplastic cells still requires that tumor-specific markers, preferably those on the cell surface, be identified. The epidermal growth factor receptor (EGFR) exists in a deletion-mutant form, EGFRvIII, which has been identified by genetic and immunological means in a subset of gliomas and non-small cell lung carcinomas. Specific polyvalent antisera to the extracellular portion of the variant were readily induced, but immunization using a synthetic linear peptide representing the unique EGFRvIII primary sequence has been unsuccessful in mice or macaques. We report here five specific monoclonal antibodies (mAbs) developed through long-term immunization protocols using the EGFRvIII-specific synthetic peptide and the intact variant in different formats that maintained secondary and tertiary conformation. These mAbs identify the EGFRvIII on the cell surface with relatively high affinity (KA range, 0.13 to 2.5 x 10(9) M-1) by live cell Scatchard analysis. These mAbs are specific for EGFRvIII as determined by RIA, ELISA, Western blot, analytical flow cytometry, autophosphorylation, and immunohistochemistry. Isolating specific mAbs enabled us to analyze normal and neoplastic human tissue and establish that EGFRvIII is truly tumor specific for subsets of breast carcinomas and for previously reported non-small cell lung carcinomas and gliomas. Also, this receptor is not expressed by any normal human tissues thus far examined, including elements of the peripheral, central nervous, and lymphoid systems. With mAbs, we identified a higher incidence of EGFRvIII positivity in gliomas than previously described and identified an EGFRvIII-positive subset of breast tumors; also, we observed that the EGFRvIII epitope is not expressed in normal tissues, and we demonstrated the localizing and therapeutic potential of the mAbs for tumors expressing this epitope. Our observations strongly warrant development of this mAb-antigen system as therapy for breast, lung, and central nervous system tumors.

Amino Acid Sequence↗
PubMed1995

Tumor antigens in astrocytic gliomas.

Gliomas affect 15,000 to 17,000 Americans every year and carry a dismal prognosis. The potential of immunologically mediated diagnosis and therapy, although greatly enhanced since the advent of monoclonal antibodies, has not been fully realized due to significant problems, most especially the challenge of identifying antigenic molecules specific to glial tumors. Other problematic issues include antigen-associated factors such as heterogeneity, modulation, shedding, and cross-reactivity with normal cells, and factors associated with therapeutic agent delivery, typically variable tumor perfusion and unfavorable diffusional forces in tumor microenvironment. An understanding of these problems called for the delineation of operationally specific antigens (tumor-associated antigens not expressed by the normal central nervous system) combined with the use of compartmental therapeutic approaches to increase the specificity of therapy. Numerous antigens have been identified and are classified as extracellular/matrix-associated, membrane-associated, and intracellular antigens. Nevertheless, only a few have been demonstrated to be of significant therapeutic and diagnostic utility. These few include the extracellular matrix-associated antigens tenascin and GP 240, defined by the monoclonal antibodies 81C6 and Mel-14, both of which are now in Phase I clinical trials, and membrane-associated ganglioside molecules, primarily 3', 6'-isoLD1, defined by the antibody DMAb-22. Recent identification of the overexpression of a deletion variant of the epidermal growth factor receptor (EGFRvIII) in up to 50% of the more malignant glial tumors and the subsequent creation of monoclonal antibodies that are specific to this molecule and do not recognize the wild-type EGFR provide the most exciting development yet in the design of specific antiglioma immunoconjugates. In addition, the tumor-specific nature of EGFRvIII combined with improved knowledge of immune mechanisms, especially in the context of the central nervous system, will facilitate the design of highly selective cell-mediated therapeutic approaches with a view toward obtaining tumor-specific immunity.

Animals↗
PubMed1995

Phase I studies of treatment of malignant gliomas and neoplastic meningitis with 131I-radiolabeled monoclonal antibodies anti-tenascin 81C6 and anti-chondroitin proteoglycan sulfate Me1-14 F (ab')2--a preliminary report.

The advent of monoclonal antibody (MAb) technology has made Ehrlich's postulate of the 'magic bullet' an attainable goal. Although specific localization of polyvalent antibodies to human gliomas was demonstrated in the 1960s, the lack of specific, high affinity antibody populations and of defined target antigens of sufficient density precluded therapeutic applications. Not until the identification of operationally specific tumor-associated antigens (present in tumor tissue but not normal central nervous system tissue); production of homogeneous, high affinity MAbs to such antigens; and the use of compartmental administration (intrathecal or intracystic), has the promise of passive immunotherapy of primary and metastatic central nervous system neoplasms been recognized. We report here preliminary data from Phase I studies of the compartmental administration of the anti-tenascin MAb 81C6 and F(ab2)2 fragments of MAb Me1-14, which recognizes the proteoglycan chondroitin sulfate-associated protein of gliomas and melanomas, to patients with primary central nervous system tumors or tumors metastatic to the central nervous system. Phase I dose escalation studies of intracystically administered 131I-labeled anti-tenascin MAb 81C6 to either spontaneous cysts of recurrent gliomas or surgically created cystic resection cavities have resulted in striking responses. Of five patients with recurrent cystic gliomas treated, four had partial responses, clinically or radiographically. Similarly, in patients with surgically created resection cavities, a partial response at the treatment site and extended stable disease status has been obtained following intracystic administration of 131I-labeled 81C6. No evidence of hematologic or neurologic toxicity has been observed in either patient population, with the exception of transient exacerbation of a pre-existing seizure disorder in a single patient. Dosimetry calculations indicated high intracystic retention for four to six weeks with little or no systemic dissemination; estimated total doses intracystically ranged from 12,700-70,290 rad. Intrathecal administration of labeled MAbs to patients with neoplastic meningitis is more difficult to assess in terms of clinical responsiveness. Of patients so treated with either 131I-labeled 81C6 or 131I-labeled Me1-14 (F(ab)2, cerebrospinal fluid and radiographic responses have been achieved, and survival prolongation through maintenance of stable disease has been observed in several cases. Initial results from pHase I dose escalation trials are encouraging in terms of the proportion of cases of disease stabilization and partial and complete responses obtained. Importantly, neurotoxicity has been virtually nonexistent, and hematologic toxicity rare and rapidly responsive to treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1995

Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene.

The type III deletion-mutant gene for the epidermal growth factor receptor (EGFRvIII) is frequently expressed in glioblastomas and in breast and non-small cell lung carcinomas. To understand its contribution to the malignant phenotype in humans, we transfected NR6 cells with the mammalian vector pH beta APr-1-neo containing cDNA for either EGFRvIII or wild-type EGFR. Western blot analyses showed that NR6 transfected with wild-type EGFR (NR6W) contained a normal-sized protein (170 kilodaltons); cells transfected with EGFRvIII (NR6M) contained a truncated protein (145 kilodaltons). NR6W cells demonstrated a saturation binding curve with 125I-labeled EGF (affinity, 1.8 x 10(8); r2 = 0.96). NR6M cells, however, showed a low but consistent level of 125I-labeled EGF binding (affinity, 4 x 10(7); r2 = 0.99) compared with NR6, which lacked binding. The population doubling time was shorter for NR6M (0.64 days) than for NR6W (1.1 days) and NR6V (2.27 days). Soft agar focus formation assay by NR6M was 4- to 5-fold higher than that by NR6W. In nude mice, NR6M (1 x 10(7) cells), without exogenous ligand, formed tumors within 12 days; no tumors were observed over 90 days in mice receiving identical doses of NR6W, NR6V, or NR6 cells. EGF stimulated autophosphorylation of receptor in NR6W (4- to 9-fold) but caused only slight (1.8- to 1.9-fold) to no enhancement in NR6M. Further, there was no difference in constitutive tyrosine kinase activity between NR6M and NR6W. Our results clearly indicate that EGFRvIII functions as an oncoprotein, but its intrinsic tyrosine kinase activity may not be responsible for its biological function.

3T3 Cells↗
PubMed1995

Immunotoxins that target an oncogenic mutant epidermal growth factor receptor expressed in human tumors.

Human cancers arise from a series of mutations, many of which direct the expression of mutant proteins with altered functions. These aberrant proteins are attractive targets for new therapeutic agents. One such protein is a mutant epidermal growth factor receptor (EGFRvIII) that has an in-frame deletion near the NH2 terminus of its extracellular domain. This protein was first identified in human gliomas, but has also been shown to be present in lung and breast carcinomas. The deletion results in a receptor with constitutive tyrosine kinase activity that enhances the tumorigenicity of glioblastomas in vivo. The deletion also creates a tumor-specific cell-surface sequence at the deletion junction. Three specific anti-EGFRvIII mAbs have been isolated following immunization with a mixture of a deletion junction synthetic peptide and EGFRvIII as present on cell membranes. We have constructed immunotoxins by conjugating a modified version of Pseudomonas exotoxin A to these mAbs. Immunotoxins were tested on cells that had been transfected with cDNA for the EGFRvIII receptor and expressed receptor protein at 5 x 10(5) receptors/cell. All three immunotoxins were cytotoxic to these cells, with 50% inhibition of protein synthesis occurring in a 15-50 pM range. The immunotoxins specifically targeted EGFRvIII, as their cytotoxicity could be blocked by their respective free antibody. They showed little or no cytotoxicity to cells expressing high levels of normal epidermal growth factor receptors, demonstrating that they are able to discriminate between cells expressing the mutant receptor and those expressing the wild-type receptor. Immunotoxins targeted to mutant epidermal growth factor receptors are promising candidates for further development as tumor cell-specific therapeutic agents.

3T3 Cells↗
PubMed1994

Generation and characterization of a mouse/human chimeric antibody directed against extracellular matrix protein tenascin.

The murine anti-tenascin monoclonal antibody 81C6, following iodination, has been shown to be an efficient localizing and therapeutic agent in both subcutaneous and intracranial human glioma xenograft models in athymic mice and rats. Similarly, effective monoclonal antibody 81C6 localization has been demonstrated in glioma patients, and Phase I trials with the intact murine IgG2b kappa molecule are currently in progress. In order to maximize the potential for repeated administration by minimizing murine Fc-mediated immunogenicity and reducing Fc-mediated immune effects, we created murine 81C6 variable region/human IgG2 chimeric monoclonal antibodies by the molecular cloning of the variable region genes of mouse 81C6 and their genetic linkage to human constant region exons. The resulting chimeric constructs were introduced into SP2/0 cells, and stable transfectomas were selected by G418 and mycophenolic acid resistance. The resistant clones were screened for anti-tenascin activity on tenascin-coated plates by enzyme-linked immunosorbent assay. The N-terminal amino acid sequence of both heavy and light chains of the purified chimeric 81C6 antibody matched exactly with that of the native mouse 81C6 as well as with that deduced from the nucleotide sequence. The production level of chimeric 81C6 (13.9 mg/ml) from ascites in the highest expressing transfectoma was much higher than that of native mouse 81C6 (2.5 mg/ml). The chimeric antibody showed the same specificity and equivalent affinity for human intact tenascin or tenascin-expressing cells as the native mouse 81C6 antibody. Direct comparison of radioiodinated chimeric and radioiodinated mouse 81C6 biodistribution in subcutaneous and intracranial xenograft-bearing mice showed higher tumor-to-normal tissue ratios for chimeric 81C6 as compared with native mouse 81C6. The improved localizing and clearance characteristics of chimeric 81C6 in xenograft model systems suggests that chimeric 81C6 would be an improved reagent for intracompartmental therapy of tenascin-expressing tumors in the human central nervous system.

Animals↗
PubMed1994

Detection of glioma-associated gangliosides GM2, GD2, GD3, 3'-isoLM1 3',6'-isoLD1 in central nervous system tumors in vitro and in vivo using epitope-defined monoclonal antibodies.

In this study, MAbs to the 'conventional' gangliosides expressed by human gliomas were generated and used to detect ganglioside species previously unisolated or defined in normal adult CNS tissue. Despite the marked phenotypic and genotypic heterogeneity shown by glioma cell lines (Bigner et al., 1981), the ganglioside phenotype of these cell lines is remarkably consistent qualitatively, if not quantitatively, in the ganglioside species expressed (Table V). The majority of cell lines and tumor samples express GM2, GD2 and GD3; this does not provide a diagnostic advantage (Vick et al., 1992). Nevertheless, as the relative amounts of these gangliosides in tumor as compared with normal adult CNS tissue is considerable, such reagents might be considered in compartmental immunotherapeutic approaches. Since GD2 and GD3 have been determined to mediate tumoricidal activity with human effector cells via specific antiganglioside epitope MAbs (Thurin et al., 1987; Kushner and Cheung, 1991; Barker et al., 1991; Reisfeld, 1993), cell-mediated approaches, as well as targeted immunoglobulin therapies, are also possible. The prospect of a more targeted approach with little or no effect on normal CNS tissue is now possible via the 'oncofetal' epitopes characteristic of 3'-isoLM1 and 3',6'-isoLD1. Several factors recommend the use of these moieties for compartmental immunotherapy; the inability to detect them within the adult CNS; the relatively high frequency of expression of 3'-isoLM1 and 3',6'-isoLD1, especially in human tumor samples (50-100%, depending upon the series and assay); and the existence of specific MAbs reactive with these epitopes. Current technology is being applied to these MAbs to transfer the specific recognition capacity of existing murine MAbs into various human framework structures of any desired immunoglobulin class, and thereby, biologic function. The variety of effector functions, the stability in affinity, labeling capacity, and the exquisite sensitivity of these MAbs for these glioma-distinctive epitopes is an exciting and promising approach for immunotherapy of human CNS tumors.

Antibodies, Monoclonal↗
PubMed1994

Mouse/human chimeric Me1-14 antibody: genomic cloning of the variable region genes, linkage to human constant region genes, expression, and characterization.

Murine monoclonal antibody Me1-14, which recognizes an epitope on chondroitin proteoglycan sulfate expressed in malignant glioma and melanoma, has been used for radioimmunolocalization and therapy both in animal models and in patients. Here, we report the generation, characterization, and in vivo biodistribution of mouse/human chimeric Me1-14. Rearranged immunoglobulin genes from the Me1-14 hybridoma were identified by Southern blot analysis. Putative rearranged light- and heavy-chain genes were cloned from Lambda-ZapII Me1-14 genomic libraries and sequenced for nucleotide analysis. One of the putative heavy-chain Eco RI fragments (3.5 kb) had all the features of an intact variable region, including a functional leader sequence, in-frame V-D and D-J junctions, and cysteines 22 and 92. The deduced amino acid sequence from the heavy-chain variable region gene showed considerable homology with the invariant protein sequence of the mouse heavy-chain subgroup IIIB. Like the heavy-chain gene, one of the putative rearranged kappa-chain Hind III fragments (4 kb) had all of the characteristics of the functional variable region, and the deduced amino acid sequence showed homology to the invariant sequence of kappa-chain group V. The variable region genes for heavy- and light-chains were linked to human constant region exons in the expression vectors at the unique sites and cotransfected into mouse SP2/0 cells. The production level of chimeric Me1-14 from ascites in the highest expressing transfectoma was 1.8 mg/ml. The chimeric Me1-14 antibody exhibited the same specificity and similar affinity as that of parent Me1-14. Direct comparison of radioiodinated chimeric and murine Me1-14 in paired-label biodistribution analysis in subcutaneous xenograft-bearing mice showed higher tumor-to-normal organ ratios for chimeric Me1-14 IgG2, suggesting that this chimeric Me1-14 may be potentially useful in vivo for diagnostic and therapeutic purposes in patients.

Amino Acid Sequence↗
PubMed1993

Lactotetraose series ganglioside 3',6'-isoLD1 in tumors of central nervous and other systems in vitro and in vivo.

Two monoclonal antibodies, DMAb-21 and DMAb-22, directed against the lactotetraose series ganglioside-associated epitope IV3NeuAc,III6-NeuAcLcOse4Cer (3',6'-isoLD1), were found to define the minimum binding epitope NeuAc(or NeuGc)alpha 2-3Gal beta 1-3(NeuAc or NeuGc)alpha 2-6GlcNAc. The distribution of 3',6'-isoLD1 in cultured cell lines and derived xenografts of primary tumors of the human central nervous system and of embryonal or neuroectodermal tumor derivation was determined. Only 4 of 26 cell lines, 3 teratomas and 1 pancreatic adenocarcinoma, expressed detectable 3',6'-isoLD1 when cultured in vitro; none of 14 tested glioma lines, including 2 that expressed the monosialo-precursor IV3NeuAcLcOse4Cer in vitro, expressed detectable levels. Expression of 3',6'-isoLD1 was more frequent when neoplastic cells were grown in xenograft form in athymic mice; 4 of 10 glioma and 2 of 2 teratoma xenograft ganglioside extracts were positive for 3',6'-isoLD1. The absence of 3',6'-isoLD1 in cultured tumor cells of the central nervous system and its proportional increased presence in tumor cells of the same origin grown in vivo further supports previous studies suggesting that ganglioside expression may be modified by environmental forces. The expression of lacto series gangliosides both in vitro and in vivo by teratoma and pancreatic adenocarcinoma cells, as opposed to only in vivo expression by glioma cells, suggests that tissue-specific forces may also exist. Immunohistochemical localization of 3',6'-isoLD1 in frozen sections of primary central nervous system neoplasms including those of glial and nonglial origin was performed; 20 of 30 (67%) of glial tumors were positive. Among nonglial tumors, 21 of 34 (62%) of epithelial cancers were reactive with anti-3',6'-isoLD1 monoclonal antibodies; notably negative were carcinomas of the ovary and lung carcinomas of all subtypes. Lymphomas and infiltrative lymphocytes were uniformly negative. The restriction of 3',6'-isoLD1 expression within the human central nervous system to periods of fetal-neonatal astroglial proliferation, to intense reactive astrocytosis, and to primary neoplasms, and the production of specific monoclonal antibodies to this epitope provide a specific complex for immunolocalization and, eventually, immunotherapy.

Animals↗
PubMed1993

Investigation of a synthetic peptide as immunogen for a variant epidermal growth factor receptor associated with gliomas.

We have previously demonstrated antibody production to a glioma-associated variant form of the human epidermal growth factor receptor in rabbits that had received a synthetic peptide mimicking the unique primary structure of the variant protein as immunogen. We report here the response of mice, rabbits, goats, and macaques immunized by various protocols to this peptide. Titers to both peptide- and cell-elaborated variant receptor were measured, and the capacity to recognize the variant receptor in human tumor samples was determined. Within the range of species and strains investigated, we demonstrated a variable species-associated response to the peptide (rabbits > mice > goats > rats > macaques). Rabbits and a single goat produced specific, high titer antibody activity to the variant receptor protein following immunization with peptide alone. Murine titers to the parent protein were not appreciable following peptide immunization alone; additional immunization with variant receptor as expressed on cell membranes was used to boost this response.

Amino Acid Sequence↗
PubMed1992

Expression of O6-methylguanine-DNA methyltransferase in six human medulloblastoma cell lines.

Six well characterized human medulloblastoma cell lines (D283 Med, Daoy, D341 Med, D384 Med, D425 Med, and D458 Med) were examined for the expression of O6-methylguanine-DNA methyltransferase (MGMT) by activity and Western and Northern blot analysis. High levels of MGMT activity were present in D283 Med, Daoy, D341 Med, and D384 Med (1.36, 0.80, 1.68, and 1.62 pmol/mg of protein, respectively), but negligible MGMT activity was detected in D425 Med and D458 Med (0.06 and 0.05 pmol/mg of protein, respectively), which were derived separately at different times from the same patient. The presence of MGMT protein and its transcript was demonstrated in D283 Med, Daoy, D341 Med, and D384 Med, but both the protein and the mRNA were undetectable in D425 Med and D458 Med. Nevertheless, all six cell lines contained an apparently unaltered MGMT gene, as determined by Southern blot analysis. The absence of MGMT activity in D425 Med and D458 Med is likely due to the absence of the protein, resulting from a lack of transcription of the MGMT gene. The varying levels of expression of MGMT in medulloblastoma cells found in this study should provide a molecular basis for drug design and selection in chemotherapy of this tumor.

Blotting, Northern↗
PubMed1992

Monoclonal antibodies to malignant human gliomas.

Operationally specific monoclonal antibodies (MAbs) reactive with tumor but not normal adult tissues offer great potential for diagnosis and therapy of CNS neoplasms. Two targets for specific MAb localization were chosen for this study: (1) glioma-associated gangliosides GM2 [II3NeuAc-GgOse3Cer], GD2 [II3(NeuAc)2-GgOse3Cer], GD3[II3(NeuAc)2-LacCer], 3'-isoLM1 [IV3NeuAc-LcOse4Cer], and 3',6'-isoLD1 [IV3NeuAc,III6NeuAc-LcOse4Cer] and (2) epidermal growth factor receptor (EGFR) variant molecules. Epitopic specificity of isolated ganglioside hybridomas was determined with FAB-MS defined ganglioside standards. All MAb are IgM. Assay of 14 cytologic specimens and 31 frozen sections of primary CNS neoplasms revealed staining with anti-GD3 (14/14, 31/31), anti-GM2 (9/14, 26/31), and anti-GD2 (6/14, 24/30), respectively. 3'-isoLM1 and 3',6' isoLD1, which exhibit a restricted oncofetal expression pattern and are not detectable in adult human brain, are present in 15/31 primary CNS neoplasms and in 1/8 human glioma xenografts, as detected by MAbs SL-50 and DMAb-14, respectively. EGFR proteins, the second target, have unique amino acid spans resulting from gene deletion in the amplified EGFR gene present in subsets of malignant human gliomas. Antibodies against EGFR deletion-mutant Type III show highly restricted activity with a subset of glioma biopsies (6/35) expressing the mutant EGFR. These reagents should be useful for in vitro and in vivo diagnosis and, potentially, for treatment of malignant brain tumors.

Adult↗
PubMed1992

Tenascin expression in human glioma cell lines and normal tissues.

Tenascin expression was evaluated in 21 human glioma cell lines and in normal adult tissue extracts by Western and Northern blotting. The cell lines differed in their relative expression of tenascin in the cell-associated and supernatant compartments. Glioma cell line tenascin production was not uniformly stimulated by changes in fetal bovine serum concentration in the growth media. In most glioma cell lines and normal tissue extracts, reducing Western blots and Northern blots revealed two tenascin species, respectively: a major 340 kDa polypeptide and a 9 kb RNA transcript accompanied by a less intense 250 kDa polypeptide and 7 kDa RNA species. In U-87 MG and in normal adult kidney extracts, however, the 250 kDa band and 7 kb transcript were more prominent. Quantitation of tenascin in the glioma lines revealed variable levels that were significantly higher than those in the tissue extracts.

Animals↗
PubMed1991

Anti-GM2 monoclonal antibodies induce necrosis in GM2-rich cultures of a human glioma cell line.

The effects of four anti-GM2 monoclonal antibodies (DMAb-1, DMAb-2, DMAb-3, and DMAb-5) were studied on spheroid cultures from a human glioma cell line (D-54 MG) that is known to express high levels of GM2. The spheroids developed central necrosis 48 h after antibody exposures at concentrations greater than 6 micrograms/ml. No necrosis was found with antibodies that had been absorbed with GM2 prior to exposure or with unrelated cytotoxic antibodies. Immunohistochemistry showed that the necrosis started shortly after the antibodies were evenly distributed throughout the spheroids. Light and transmission electron microscopy revealed that a small portion of the cells, mainly in the periphery of the spheroids, was unaffected by antibody exposure. New monolayer cultures established from antibody-treated cells expressed a 50% lower GM2 content as shown by flow cytometry and determination of ganglioside content throughout at least 12 passages. Thus, the GM2-rich D-54 MG cell line has subpopulations of cells with lower GM2 content. Spheroids obtained from this subpopulation developed only minor necrosis after antibody treatment. These results show that GM2 antibodies cause severe necrosis of GM2-containing glioma cells in vitro, but the effect depends on the concentration of antigen, and a threshold number of GM2 molecules is required.

Antibodies, Monoclonal↗
PubMed1991

Glycolipids and glycosyltransferases in permanent cell lines established from human medulloblastomas.

Medulloblastoma biopsies are heterogenous and might contain normal brain tissue, which limits the usefulness of such tumor material for biochemical analyses. We have, therefore, examined the gangliosides and their metabolism using the medulloblastoma cell lines. Daoy and D341 Med, cultured both in vitro and as xenografts in nude mice. The ganglioside patterns in the Daoy showed a switch from a high GM2, 70% (mol% of total ganglioside sialic acid) and low lactoseries gangliosides (2%) content in monolayer cultures, to a high proportion of lactoseries gangliosides (50%) and virtually no GM2 (1%) in xenografts, but an increased proportion of other a-series gangliosides. The D341 Med showed a similar change regarding the lacto-series gangliosides from 1% in suspension culture to 10% in xenografts. The activity of five glycosyltransferases, GM3, GD3, GM2, GM1 and LA2 synthases, did not parallel the ganglioside patterns and could not account for the noted variations therein. In the Daoy cell line the LA2 synthase as well as the GM2 synthase activity was relatively high in both culture systems, despite the marked difference in the expression of GM2 and the lactoseries gangliosides. These results suggest that environmental factors play a crucial role for the in vivo activity of the glycosyltransferases.

Animals↗
PubMed1991

Medulloblastoma: tumor biological and clinical perspectives.

Medulloblastoma is the most common central nervous system malignancy of childhood, with approximately 350 new cases seen in the United States each year. Although the low incidence of this tumor, compared with adult neoplasms such as breast or colon carcinoma, has limited laboratory and clinical research, recent studies have greatly enhanced our understanding of the biology, phenotype, genotype, and therapy of medulloblastoma. Further efforts to integrate laboratory and clinical studies of this tumor may allow design of novel therapeutic approaches resulting in enhanced disease-free survival.

Animals↗