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Biomedical subjects

I Hellström

Publications and source records attributed to I Hellström.

At least 55 records · Page 3Linked to original sources
PubMed1994

Crystallization and preliminary X-ray analysis of the monoclonal anti-tumor antibody BR96 and its complex with the Lewis Y determinant.

The monoclonal anti-tumor antibody BR96 binds a tetrasaccharide, Lewis y (Le(y)), in vitro and recognizes a Le(y)-bearing or Le(y)-related tumor-associated antigen in vivo. The Fab of the murine monoclonal antibody, mBR96 (IgG3, kappa), and the Fab' of its human chimera, cBR96 (IgG1, kappa), and their complexes with Le(y) have been screened for crystallization conditions. Crystals suitable for X-ray diffraction have been obtained for uncomplexed cBR96 Fab', cBR96 Fab' in complex with Le(y) and mBR96 Fab in complex with Le(y). The symmetry of the cBR96 Fab' crystals is consistent with space group P2(1)2(1)2, a = 61.1 A; b = 174.3 A; c = 45.6 A; the symmetry of the cBR96 Fab'-Le(y) complex crystals with space group P4(3)2(1)2 (or its enantiomorph), a = b = 82.2 A; c = 167.1 A and the symmetry of the mBR96 Fab-Le(y) complex crystals with space group P2(1)2(1)2(1), a = 69.4 A; b = 84.9 A; c = 86.8 A.

Animals↗
PubMed1994

Anti-tumor antibody BR96 blocks cell migration and binds to a lysosomal membrane glycoprotein on cell surface microspikes and ruffled membranes.

BR 96 is an internalizing antibody that binds to Lewis Y (Le(y)), a carbohydrate determinant expressed at high levels on many human carcinomas (Hellström, I., H. J. Garrigues, U. Garrigues, and K. E. Hellström. 1990. Cancer Res. 50:2183-2190). Breast carcinoma cell lines grown to confluence bind less BR96 than subconfluent cultures (Garrigues, J., U. Garrigues, I. Hellström, and K. E. Hellström. 1993. Am. J. Path. 142:607-622). However, when the confluent cells are induced to migrate by scratch wounding, they again bind BR96 suggesting that antigens bearing the Le(y) determinant may promote cell migration. In the present study, BR96 was found to be highly enriched on microspikes and ruffled membranes, cell surface structures involved in cell migration. In addition, BR96 was a potent inhibitor of cell migration in vitro. When stationary BR96 treated cells were exposed to fresh culture media, membrane ruffles and microspikes developed at the cell margin and migration resumed. Immunogold microscopy showed that BR96 antigens were enriched on these membrane protrusions. BR96 cell surface immunoprecipitation analysis of 3H-glucosamine labeled breast carcinoma cells identified antigens with approximate molecular weights of 135 kd (upper antigen) and 85 kd (lower antigen). A short amino terminal sequence (8 residues) of the upper antigen matched that of human lysosomal membrane glycoprotein 1 (LAMP-1). In addition, the upper antigen was detected on immunoblots probed with anti-LAMP-1, and within the intracellular compartment BR96 was found predominantly in endosomes and lysosomes. A soluble LAMP-1/immunoglobulin fusion protein (LAMP-1/Ig) was transiently expressed in both BR96 binding and nonbinding cell lines. Immunoblot analysis of LAMP-1/Ig's from the various cell lines showed that (a) acquisition of the BR96 epitope is probably controlled at the level of polylactosamine modification (e.g., fucosylation) rather than LAMP-1 gene expression; (b) alternate forms of LAMP-1/Ig comigrate with the lower BR96 antigen raising the possibility that it may be a degradation product of the upper antigen; and (c) LAMP-1/Ig expressed in 3396 breast carcinoma cells has approximately 30-fold more BR96 epitopes than LAMP-1/Ig from non-tumorigenic mammary epithelial cells. Together these data indicate that a major BR96 antigen, LAMP-1, is present on unique cell surface domains involved in cell locomotion as well as membranes of the endocytic compartment. Altered glycosylation of LAMP-1 expressed in transformed cells may contribute to their ability to disseminate.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of the gene for the melanoma-associated ME20 antigen.

Human melanoma cells, but not tumor cells of other histological origin, express a unique membrane-associated glycoprotein, designated ME20-M, and secrete a soluble glycoprotein, designated ME20-S, defined by monoclonal antibody ME20. Here we report the isolation and characterization of a cDNA clone that when transfected into COS cells directs the expression of ME20-M and ME20-S. This cDNA contains an open reading frame which encodes a 661-amino-acid-long precursor that contains a 23-amino-acid signal peptide and a 26-amino-acid transmembrane domain, separated by a hydrophilic region containing 5 potential Asn-linked and 14 predicted Pro-associated, Thr-linked glycosylation sites. The transmembrane domain is followed by a carboxy-terminal 45-amino-acid putative intracellular domain rich in Ser residues. Analysis of ME20-M by amino acid sequencing identified the proteolytic processing site. Signal peptide cleavage occurs at the Thr-24-Lys-25 peptide bond of the precursor and results in the 637-amino-acid ME20-M with a calculated molecular weight of 67,782. ME20-M is derived from a single 3.3- to 3.4-kb mRNA transcript that is expressed at varying levels in melanoma cell lines, correlating with immunofluorescence determination of protein expression. The amino acid sequence of the ME20 antigen deduced from the cDNA differs from the human neonatal melanocyte-specific Pmel 17 gene product by a single amino acid substitution and deletion of 7 amino acid residues, and it is 80% homologous with the bovine retinal pigment RPE1 cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Effect of blood-brain barrier disruption on intact and fragmented monoclonal antibody localization in intracerebral lung carcinoma xenografts.

UNLABELLED: These studies highlight several factors that affect monoclonal antibody (Mab) localization to a tumor in the brain, including tumor permeability, nonspecific and specific binding, plasma half-life, radiolabeled antibody stability and the blood-brain barrier. METHODS: A pancarcinoma Mab [L6 IgG, F(ab')2 and Fab] and an irrelevant isotype-matched antibody [P1.17 IgG and F(ab')2] were given with and without osmotic blood-brain barrier disruption in a LX-1 human small-cell lung carcinoma intracerebral xenograft model. RESULTS: Intracerebral tumor size and permeability to antibody increased with the selection of 10, 14 or 17 days postinoculation when antibody was administered. Barrier disruption increased the delivery, particularly at earlier time points, which was dependent on antibody-specific and nonspecific binding and tumor permeability. Dehalogenation and/or antibody binding stability also appeared to affect the percent delivery. CONCLUSION: These studies demonstrate important variables that should be considered when clinical trials are designed or Mab delivery and localization in intracerebral tumor models are evaluated.

Animals↗
PubMed1993

Metastatic conversion of cells by expression of human papillomavirus type 16 E6 and E7 genes.

The human papillomavirus type 16 (HPV-16) is a DNA tumor virus highly associated with cervical carcinoma. Viral DNA from HPV-16 is found in primary tumors and their metastatic lesions. To investigate the role of HPV-16 oncoproteins in the development of cancer metastasis, the E6 and E7 genes from HPV-16 were inserted into retrovirus and introduced into nonmetastatic mouse cell lines. Expression of either of the viral genes from HPV-16 made the cells metastatic in nude mice. In contrast, expression of the E6 and E7 genes of HPV type 6 (HPV-6b), which is frequently found in nonmalignant HPV-associated diseases, did not. The metastatic ability of cells transduced with viral genes of HPV-16 did not correlate with their growth rate or sensitivity to destruction by natural killer cells. Our results demonstrate that expression of oncogenic proteins of HPV-16 can cause tumor metastasis and implicate HPV-16 in an important role regarding the progression of HPV-associated human cancers.

Animals↗
PubMed1993

Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates.

Immunoconjugates (BR96-DOX) were prepared between chimeric monoclonal antibody BR96 and the anticancer drug doxorubicin. The monoclonal antibody binds an antigen related to Lewis Y that is abundantly expressed at the surface of cells from many human carcinomas; it has a high degree of tumor selectivity and is internalized after binding. BR96-DOX induced complete regressions and cures of xenografted human lung, breast, and colon carcinomas growing subcutaneously in athymic mice and cured 70 percent of mice bearing extensive metastases of a human lung carcinoma. Also, BR96-DOX cured 94 percent of athymic rats with subcutaneous human lung carcinoma, even though the rats, like humans and in contrast to mice, expressed the BR96 target antigen in normal tissues.

Animals↗
PubMed1993

Dexamethasone decreases the delivery of tumor-specific monoclonal antibody to both intracerebral and subcutaneous tumor xenografts.

The effect of dexamethasone on the delivery of monoclonal antibody L6 IgG to intracerebral and subcutaneous LX-1 small cell lung carcinoma xenografts was evaluated in nude rats (n = 157). Dexamethasone (0, 8, or 24 mg/m2) was given 18 hours before infusion of L6 IgG, with or without osmotic disruption of the blood-brain barrier. Compared with controls, the 8 mg/m2 dose decreased delivery of L6 IgG (12-37%) to all tissues, but the only significant decrease (P < 0.001) was in the subcutaneous tumor (37%). In the 24 mg/m2 group, L6 IgG delivery was significantly (P < 0.001) decreased to all tissues (37-60%). Dexamethasone had no effect on plasma levels. Barrier disruption significantly (P < 0.0001) increased L6 IgG delivery to intracranial tumor and surrounding brain, but not to subcutaneous tumor or plasma. The percentage of decremental effect of dexamethasone on L6 IgG delivery was the same with and without barrier disruption and was not associated with the time the animals were killed (P > 0.05). Compared with controls, the ratio of intracranial tumor to normal brain showed no change with dexamethasone, but the ratios of both intracranial and subcutaneous tumors to plasma significantly (P < 0.002) decreased with both doses. The in vitro cell binding capacity of L6 IgG to LX-1 cells remained unchanged after incubation of cells with dexamethasone over a 3-log concentration for 4 days, demonstrating no effect on antigen expression. This study suggests that dexamethasone has a clinically relevant generalized (i.e., central nervous system and systemic) vascular effect on permeability to L6 IgG monoclonal antibody.

Animals↗
PubMed1993

Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes.

BR96 is a monoclonal antibody (MAb) that recognizes many human carcinomas and can kill antigen-positive tumor cells in vitro. Using both gold and radiolabeled MAb, the distribution and cellular processing of BR96 during cytolysis has been determined. After a brief (< 3 minutes) MAb treatment, cells in suspension are stained by the nuclear viability dye propidium iodide. Whole MAb and F(ab')2 fragments are equally cytotoxic; monovalent F(ab) fragments, however, have no effect on dye uptake unless cross-linked with goat anti-mouse IgG. The level of toxicity is dependent on both MAb dose and on cell surface receptor density. Cell contact may regulate receptor expression. BR96 receptors are more abundant on cells migrating into the open areas of a scratch wounded confluent culture than on the adjacent contact-inhibited cells. BR96 can also inhibit the anchorage-independent growth of tumor cells in soft agar showing that its effects on propidium iodide staining are not due to transient changes in membrane permeability. Immunogold electron microscopy reveals that, after a 1-minute treatment, BR96 induces significant infolding of the plasma membrane and that internalized MAb is localized to these structures. Immediately thereafter, large cell surface and intracellular vesicles form, mitochondria are swollen, and membrane integrity is lost. Therefore, BR96 seems to cause morphological changes characteristic of necrosis rather than apoptosis. When bound to adherent carcinoma cells, BR96 is distributed uniformly on the apical surface of cells labeled at 4 C and is enriched at points of cell substratum contact. Upon warming of the cells to 37 C, BR96 localizes in small perinuclear clusters and the cell margin is now devoid of label. Immunogold electron microscopy reveals that BR96 undergoes receptor mediated internalization and is localized within the same coated pits, endosomes, and lysosomes as the transferrin receptor. Quantitative studies using iodinated BR96 show that after 6 hours of chase, a maximum of 53% of the radiolabel is located within the intracellular pool. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that 84% of this fraction is nondegraded. BR96 probably cycles between the medium and intracellular pools because the remainder of the radiolabel is in the medium as intact MAb. By 24 hours of chase, the intracellular fraction drops to 30%, while the remaining 70% is present in the culture medium, mostly as low molecular weight degradation products.

Antibodies, Monoclonal↗
PubMed1992

Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4.

Interaction of the B7 molecule on antigen-presenting cells with its receptors CD28 and CTLA-4 on T cells provides costimulatory signals for T cell activation. We have studied the effects of B7 on antitumor immunity to a murine melanoma that expresses a rejection antigen associated with the E7 gene product of human papillomavirus 16. While this E7+ tumor grows progressively in immunocompetent hosts, cotransfection of its cells with B7 led to tumor regression by a B7-dependent immune response mediated by CD8+ cytolytic T lymphocytes. The immune response induced by E7+B7+ tumor cells also caused regression of E7+B7- tumors at distant sites and was curative for established E7+B7- micrometastases. Our findings suggest that increasing T cell costimulation through the CD28 and CTLA-4 receptors may have therapeutic usefulness for generating immunity against tumors expressing viral antigens.

Abatacept↗
PubMed1992

Application of a filamentous phage pVIII fusion protein system suitable for efficient production, screening, and mutagenesis of F(ab) antibody fragments.

We describe the application of a novel filamentous phage vector system suitable for efficient screening and production of F(ab) antibody fragments. The vector system can concurrently produce free F(ab) fragments and F(ab) displayed on the surface of M13 bacteriophage via a VHCH1-pVIII fusion protein. When expressed in a supO (nonsuppressor) strain of Escherichia coli free F(ab) can be produced. Antibody F(ab) fragments are secreted into culture medium at concentrations up to 0.3 mg/liter and conveniently subjected to detailed analysis with little or no purification. Higher concentrations of F(ab) (approximately 10 mg/liter) were found to accumulate in the periplasmic space. In this report the vector system is shown to produce correctly folded and assembled F(ab) fragments of chimeric L6, a mAb against a tumor-associated Ag expressed by many human carcinomas.

Antibodies, Monoclonal↗
PubMed1992

Chimeric L6 anti-tumor antibody. Genomic construction, expression, and characterization of the antigen binding site.

We report the cloning of the genomic variable region genes of the human carcinoma reactive murine monoclonal antibody L6 and their genetic linkage to human constant region exons to encode a human IgG1/kappa chimeric antibody. The chimeric protein was produced at levels greater than 20 micrograms/ml (enabling the initiation of clinical trials) and was found to have binding properties identical with that of the murine parent. The nucleic acid sequence of the variable regions was determined and found to be different than that previously reported (1). The deduced amino acid sequence was then used to generate a structural homology based three-dimensional model of the antibody binding site, which was found to share features with antibodies known to interact with a protein surface, but distinct from those that bind to carbohydrate epitopes. Biochemical analysis of binding between antibody and the in vitro-translated product of a cDNA clone that confers L6 immunoreactivity demonstrates that the antibody recognizes a protein epitope encoded by this transcript which requires the presence of membranes, but is unaffected by the removal of carbohydrate side chains.

Amino Acid Sequence↗
PubMed1992

An unmodified anticarcinoma antibody, BR96, localizes to and inhibits the outgrowth of human tumors in nude mice.

The antitumor effects of an unmodified murine monoclonal antibody, BR96, were examined in nude mice bearing human lung adenocarcinoma xenografts. BR96, a murine IgG3 that internalizes and is cytotoxic to cells expressing the antigen in vitro, also elicits strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity effector functions. Its in vivo antitumor effects were compared with those of its F(ab')2 fragments, a mouse-human chimeric form, and an IgG1 class switched variant of the original (IgG3) BR96. Antitumor effects were observed with antigen-positive tumor lines (but not with tumors which did not bind with BR96) and correlated with the levels of antigen expression as detected in vitro. The chimeric form of BR96 gave the strongest antitumor effects, followed by the murine IgG3, while limited effects were seen with the IgG1 and with F(ab')2 fragments of BR96, indicating that Fc-dependent host effector functions are primarily responsible for its in vivo activity. The antitumor effects observed were modest unless the antibody treatment was started on the day following tumor grafting.

Animals↗
PubMed1992

Cloning and expression of the tumor-associated antigen L6.

The L6 cell surface antigen, which is highly expressed on lung, breast, colon, and ovarian carcinomas, has attracted attention as a therapeutic target for murine monoclonal antibodies and their humanized counterparts. Its molecular nature has, however, remained elusive. Here we describe the expression cloning of a cDNA encoding the L6 antigen. COS cells transfected with this cDNA direct the expression of an approximately 24-kDa surface protein that reacts with the two anti-L6 monoclonal antibodies available. The predicted L6 peptide sequence is 202 amino acids long and contains three predicted NH2-terminal hydrophobic transmembrane regions, which are followed by a hydrophilic region containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. The L6 antigen is related to a number of cell surface proteins with similar predicted membrane topology that have been implicated in cell growth. Two other members of this family of proteins, CD63 (ME491) and CO-029, are also highly expressed on tumor cells. The present findings should make it possible to further study the role of the L6-defined antigen in normal and neoplastic cells and to construct animal models for development of improved agents for active and passive cancer immunotherapy.

Amino Acid Sequence↗
PubMed1992

Induction of cytotoxic T lymphocytes specific for a syngeneic tumor expressing the E6 oncoprotein of human papillomavirus type 16.

Human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical carcinomas, but it is unknown whether HPV-specific immunity can function in controlling the growth of HPV-associated carcinomas. We previously demonstrated that CD8+ T lymphocytes can inhibit the in vivo outgrowth of murine tumor cells transfected with the HPV-16 E7 gene and have now established a murine model to study the CTL responses to the E6 oncoprotein of HPV-16. Immunization of C3H/HeN mice with syngeneic fibroblasts expressing a transfected HPV-16 E6 gene induced regression of transplanted tumors expressing this gene. Populations of CTL isolated from the spleens of mice whose E6+ tumors had regressed were shown to specifically lyse E6+ target cells. The cytolytic activity was mediated by CD8+ CTL in a MHC restricted pattern. These data and our previous findings with transfected tumor cells expressing the E7 gene, support the conclusion that tumor cells associated with HPV-16 can be inhibited by CTL specific for molecules encoded by the HPV-16 E6 and E7 genes.

Animals↗
PubMed1992

Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines.

The glycoproteins granule membrane protein 140 (GMP140), endothelial-leukocyte adhesion molecule 1 (ELAM-1), and Leu-8 are members of a family of glycoprotein receptors (selectins or LEC-CAMs) that play an important role in adhesive interactions between circulating leukocytes and vascular endothelium. Recently it has been reported that ELAM-1 is able to mediate the binding of the colon carcinoma cell line HT-29 to cytokine-activated vascular endothelium, suggesting that tumor cell adhesion to vascular endothelium, a prerequisite for tumor extravasation and metastasis, is in part the result of adhesive interactions between blood-borne tumor cells and cell surface proteins expressed by vascular endothelium. Here, using an approach in which soluble immunoglobulin chimeras of the GMP140 and ELAM-1 receptors were prepared and used to carry out immunohistological studies, we establish that GMP140 binds to tumor cells in a variety of human carcinoma tissue sections (colon, lung, and breast), whereas ELAM-1 binds exclusively to tumor cells in colon carcinoma tissue sections. In addition, GMP140 was found to bind to the cell surface of a number of cell lines derived from various carcinomas but not from melanomas, whereas ELAM-1 bound only colon carcinoma cell lines. We further investigated the nature of the ligands of GMP140 and ELAM-1 on the surface of the carcinoma cells and found that the GMP140 ligand on the surface of tumor cells appears to be distinct from that expressed on the myeloid cell line HL-60. Neuraminidase treatment of a breast carcinoma cell line does not affect, or in some instances increases, GMP140 binding, whereas it completely abolishes GMP140 binding to HL-60 cells. On the other hand, the ligand of ELAM-1 on both the colon carcinoma and HL-60 cells is neuraminidase sensitive in accord with its identification as sialyl-CD15. Parallel results were obtained with neuraminidase-treated frozen carcinoma tissue sections. The present findings form the basis for investigating the role of GMP140 in tumor invasiveness and metastasis.

Antibodies, Monoclonal↗
PubMed1992

Human melanotransferrin (p97) has only one functional iron-binding site.

The iron-binding properties of melanotransferrin, the tumour-associated antigen also known as p97, have been investigated by UV/visible and fluorescence spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other transferrins, melanotransferrin binds only one Fe3+ ion per molecule. The binding properties of its N-terminal site are similar to other transferrins, but its C-terminal site does not bind iron at all. The differences can be related to specific amino acid changes in the C-terminal site.

Antigens, Neoplasm↗