Arnold S. Dion

Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2☆

The murine monoclonal antibody, LL2, is a B-cell (CD22)-specific IgG2a which has been demonstrated to be clinically significant in the radioimmunodetection of non-Hodgkins B-cell lymphoma. The antibody carries a variable region-appended glycosylation site in the light chain and is rapidly internalized upon binding to Raji target cells. Humanization of LL2 was carried out in order to develop LL2 as a diagnostic and immunotherapeutic suitable for repeated administration. Based on the extent of sequence homology, and with the aid of computer modeling, we selected the EU framework regions (FR) 1, 2 and 3, and the NEWM FR4 as the scaffold for grafting the heavy chain complementarity determining regions (CDRs), and REI FRs for that of light chains. The light chain glycosylation site, however, was not included. Construction of the CDR-grafted variable regions was accomplished by a rapid and simplified method that involved long DNA oligonucleotide synthesis and the polymerase chain reaction (PCR). The humanized LL2 (hLL2), lacking light chain variable region glycosylation, exhibited immunoreactivities that were comparable to that of chimeric LL2 (cLL2), which was shown previously to have antigen-binding properties similar to its murine counterpart, suggesting that the VK-appended oligosaccharides found in mLL2 are not necessary for antigen binding. Moreover, the hLL2 retained its ability to be internalized into Raji cells at a rate similar to its murine and chimeric counterparts.

Polypeptides of the mouse mammary tumor virus: I. Characterization of two group-specific antigens

Abstract Polyacrylamide-gel electrophoresis of the mouse mammary tumor virus (MuMTV) reproducibly resolves at least 11 polypeptides. The estimated molecular weights of the five major polypeptides are 55,000 (p55), 34,000 (p34), 28,000 (p28), 18,000 (p18) and 12,000 (p12). Two of these polypeptides, p55 and p34, have been characterized as glycoproteins on the basis of staining with periodic acid-Schiff reagent and incorporation of radioactively labeled glucosamine. Intracytoplasmic A particles have been found to contain mainly five polypeptides; the molecular weights of these are 52,000 (Ap52), 43,000 (Ap43), 37,000 (Ap37), 29,000 (Ap29) and 15,000 (Ap15). Antisera prepared against purified p55 have been used for ferritin labeling by indirect immunoelectron microscopy with hybrid antibody (anti-rat-λg/anti-ferritin) and ferritin. Anti-p55 serum resulted in labeling the viral surface with ferritin; it also precipitated intact virus. These results unambiguously established that polypeptide p55 is a component of the viral surface. Antigen p55 is the most prominent viral protein and is common to viruses isolated from RIII, C3H, GR and A mouse strains. Therefore, p55 may be considered as a group-specific, surface antigen (gs-1) of MuMTV. In contrast, polypeptide p28 is not a glycoprotein; antisera against purified p28 does not react with the viral surface. Immunodiffusion results show that p55 and p28 are immunologically unique and that p28 shares a common antigenic determinant with intracytoplasmic A particles, probably with the polypeptide Ap29. Furthermore, p28 is found to be a common antigen in RIII, C3H, GR and A viruses. These data indicate that p28 is most likely a major group-specific internal antigen (gs-2) of MuMTV.

Replication of mouse mammary tumor virus in tissue culture: I. Establishment of a mouse mammary tumor cell line, virus characterization, and quantitation of virus production

Abstract The establishment of an epithelial cell line (MuMT-73) derived from spontaneous mammary tumors of BALB/cfC3H mice as a source for the continuous production of mouse mammary tumor virus (MuMTV or B particles) is described. Morphological, immunological, and biochemical techniques were used to characterize the virus. It was found that, under conditions of normal cellular growth, the cells produced only type-B particles; no concomitant synthesis of Type-C particles was detected. A unique feature of this cell line is that the cells do not produce detectable amounts of intracytoplasmic type-A particles, but many B particles were found to assemble at the cell surface with gradual development of the nucleocapsid. The kinetics of virus release into tissue culture medium after transfer of cells was studied quantitatively by particle counting and viral reverse transcriptase (RDDP) measurements. When the medium was changed every 24 hr, the cells continuously produced virus at a slow rate for up to 6 to 8 days and thereafter at an increased rate for up to 12 days. After this period, virus production gradually declined but did not decrease to the basal level. During the period of high level virus production, about 30–40% of the cells were found to produce MuMTV antigens. By estimating the total number of virus particles in the culture medium and by counting the number of antigen-positive cells, it was found that over a 20-hr period during optimum virus production a cell would release approximately 800 virus particles. Treatment of the cells with either hydrocortisone or dexamethasone resulted in an increased production of type-B particles. An additional increase in virus production was obtained when the cells were treated with insulin together with either hydrocortisone or dexamethasone.

Polypeptides of the mouse mammary tumor virus: II. Identification of two major glycoproteins with the viral structure

Abstract In order to localize the major structural polypeptides of the murine mammary tumor virus (MuMTV), three subviral fractions were prepared, examined by electron microscopy, and analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. Treatment of intact virus with 0.05 N HCl at 4° for 20 min resulted in the removal of the characteristic surface projections of the virus. Viral membrane fragments with attached projections (rosettes), consisting of stalks and knobs, were prepared by treating MuMTV with Tween-80 and ether. The buoyant density (in sucrose gradients) of the particles after removal of the projections was 1.181 g/cm 3 , as compared with 1.171 g/cm 3 for the intact virus. PAGE analysis of the viral material released by the action of acid (for 20 min at 4°) revealed that it contained mainly two glycoproteins of 55,000 (gp55) and 68,000 (gp68) molecular weight (MW), gp55 being the major species. The polypeptide composition of the projectionless particles was found to be complex; the major component was a glycoprotein of 34,000 molecular weight (gp34). Although intact virus contained only one glycoprotein (gp55) in the 50,000–60,000-MW range, the projectionless particles were found to contain two minor components within the same MW range, only one of which was a glycoprotein. The nonglycopeptide component with an apparent MW of 51,000 (p51) appears to be a normal minor protein constituent of MuMTV. The viral rosettes were found to contain gp34, gp55, gp68, and an additional glycoprotein of MW 90,000 (gp90). A comparison of the morphology and polypeptide composition of the intact virus and subviral components suggests that the knobs of the viral projections are composed of gp55. The gp34 component is associated with the viral membrane and may be situated within the membrane bilayer from which the projections protrude.

The major structural proteins of murine mammary tumor virus. Techniques for isolation.

Abstract We have utilized a variety of techniques of virus solubilization and protein separation for the isolation of the major structural polypeptides of murine mammary tumor virus, a B-type oncornavirus. Of these techniques, two isolation protocols proved to be effective and are reported here. In the first method, allowing the simultaneous purification of RNA-directed DNA polymerase and four major structural polypeptides (gp68, gp55, p28, and p12), the virions are disrupted with a nonionic detergent, and the solubilized proteins are separated by ion-exchange chromatography on DEAE- and phosphocellulose; final purification is achieved by molecular sieving chromatography. The second technique involves the solubilization of viral proteins with sodium dodecyl sulfate and chromatography on Sephadex G-200. The latter method is efficacious for the isolation of gp55, gp34, p28, and p12 after removal of the ionic detergent on Dowex columns, followed by final purification by molecular sieving chromatography.

VIRUS ENVELOPE-BASED PEPTIDE VACCINES AGAINST VIRUS-INDUCED MAMMARY TUMORS

Previous studies by us and others established that mammary tumors induced by murine mammary tumor virus (MuMTV) could be prevented to various extents by prior vaccination with MuMTV-containing or subviral component immunogens. In this report, four predicted surface-accessible peptide regions (EP-1 to EP-4) of the major viral envelope glycoprotein (gp52) of C3H-MuMTV were tested as carrier-conjugated vaccines for the protection of Balb/c mice against a live virus challenge. With tumor incidence as an endpoint, vaccination with one of these synthetic peptides (EP-3) resulted in a significant reduction in the frequency of early onset tumors and 67% of the test animals remained tumor-free for the entire observation period (16 months). In contrast, only marginal protection was obtained by immunization with the intact glycoprotein (gp52). Immunologic interference may explain the lower protective efficacy of gp52, as compared to EP-3.

Vicinal relationships between the major structural proteins of murine mammary tumor virus

Abstract Spatial relationships between the major structural proteins of murine mammary tumor virus (MuMTV), a type-13 RNA tumor virus, were investigated. Untreated virions, when analyzed by diagonal gel electrophoresis, contained two major species of disulfide-linked protein oligomers, i.e., a high-molecular-weight aggregate of p18 and a dimer composed of gp34 and p28. These complexes are termed “native crosslinked oligomers.” In addition, induced crosslinking by dithiobis succinimidyl propionate (DTSP) of vicinal proteins of whole virions or rosettes (viral membranes with attached spikes or projections) was also investigated. Diagonal gel electrophoretic analyses of DTSP-crosslinked virions revealed dimers of gp55 · gp55 and gp55 · gp34 as well as the native crosslinked oligomers. No DTSP-induced crosslinking of internal viral proteins was observed. Similar studies with rosettes indicated the formation of DTSP-induced crosslinked dimers consisting of gp55·gp55 and gp34 · gp34 in addition to gp34 · p28. From these studies, we conclude that the MuMTV projection is a homooligomer of gp55 which is directly apposed to the transmembrane protein, gp34, on the external surface. In addition, a portion of the gp34 transmembrane protein interacts internally with the major core shell protein (p28) as a native crosslinked oligomer. The latter observation most probably accounts for the eccentric location of the MuMTV nucleoid. A model which derives from these studies is discussed vis-a-vis virus assembly and previous studies of MuMTV structure.

Virus-Like Particles and Macromolecules in Human Milk and Breast Tumors

Relevant data pertaining to present evidence for virus-like particles and virus-related macromolecules in human milk and breast tumors are presented. A critical review and discussion of reported observations concerning virus-related macromolecules will include RNA-directed DNA polymerase, viral antigens, and RNA related to murine mammary tumor virus and/or Mason-Pfizer monkey virus. From the standpoint of clinical applications, the finding of viral-related antigens in human breast tumors and evidence for specific host immune responses to one or more of these antigens may be especially pertinent. The latter data, therefore, will be discussed in depth as to possible employment of these parameters in diagnosis, prognosis and possible management of the human disease.

A fluorometric assay for the quantitation of aminosugars

Abstract A method is presented for the quantitative determination of aminosugars in glycoproteins. Glycoproteins are acid hydrolyzed and reacted with the fluorogenic reagent 1-dimethylaminonaphthalene-5-sulfonyl chloride. Following cellulose thin layer electrophoresis in pyridine/acetic acid (pH 4.4), 5 h at 500 V, appropriate areas of the plate are scraped and the dansyl hexosamines are eluted with 95% ethanol. Aliquots are then applied to a silica gel tlc plate and subjected to ascending thin layer chromatography in cyclohexane/ethylacetate/ethanol (6/4/3). After spraying the plate with triethanolamine/isopropanol ( 1 4 ), fluorescent intensities are measured by in situ scanning. The aminosugar content of the glycoprotein is determined from a curve generated from a series of standards run concurrently on each plate. The method clearly resolves glucosamine from galactosamine, and is sensitive for the detection of aminosugars in the subnanomole range.

Enhanced Expression and Secretion of an Epithelial Membrane Antigen (MA5) in a Human Mucinous Breast Tumor Line (BT549)

The mouse monoclonal antibody MA5, generated versus a membrane-enriched extract of breast cancer metastatic to liver, detects one or two high molecular weight species (greater than 200 kD) in breast tumor membranes, human milk fat globule membranes, and various breast tumor cell lines. From comparative studies of five breast carcinoma lines (BT20, BT549, MCF-7, T47D, and ZR75-1), as well as an epithelial line established from milk (HBL-100), we report the stimulation of expression of MA5-reactive antigen in a mucinous breast tumor cell line (BT549) through the use of a culture medium supplemented with charcoal-absorbed fetal calf serum, insulin, and hydrocortisone. Large amounts of aggregated MA5-reactive antigen are secreted into the culture medium and can be recovered from the media for further purification by centrifugation. These findings suggest that BT549 cells, grown in the special nutritive medium, may be useful in providing an ample source of epithelial membrane antigen (also termed polymorphic epithelial mucin) for standardization of clinical assay protocols, as well as provide a model system for studies of the regulation of expression for this class of antigens in breast carcinoma.