Cell surface changes and Rous sarcoma virus gene expression in synchronized cells.

We have investigated whether cell surface changes associated with growth control and malignant transformation are linked to the cell cycle. Chicken embryo cells synchronized by double thymidine block were examined for cell-cycle-dependent alterations in membrane function (measured by transport of 2-deoxyglucose, uridine, thymidine, and mannitol), in cell surface morphology (examined by scanning electron microscopy), and in the ability of tumor virus gene expression to induce a transformation-specific change in membrane function. We reach the following conclusions: (a) The high rate of 2-deoxyglucose transport seen in transformed cells and the low rates of 2-deoxyglucose and uridine transport characteristic of density-inhibited cells do not occur in normal growing cells as they traverse the cell cycle. (b) Although there are cell cycle-dependent changes in surface morphology, they are not reflected in corresponding changes in membrane function. (c) Tumor virus gene expression can alter cell membrane function at any stage in the cell cycle and without progression through the cell cycle.

Determination of adhesive properties of variant metastatic melanoma cells to BALB/3T3 cells and their virus-transformed derivatives by a monolayer attachment assay.

The hypothesis that abnormalities in intercellular adhesion are a property of metastatic tumors was examined in vitro with B16 melanoma variants that were selected in vivo for increased metastatic behavior. The adhesive characteristics of low (B16-F1), intermediate (B16-F5), and high (B16-F10) metastatic lines were determined by quantitative adhesion assays that measured the rate and degree of attachment of single cells to confluent monolayers of melanoma, BALB/3T3, or virus-transformed 3T3 cells. Intercellular adhesions were monitored by loss of single cells from suspension and adherence of intraperitoneally grown 125I-5-iodo-2'-deoxyuridine-labeled cells to the monolayers, and were affected by time, temperature, and serum concentration. Although there was little difference in adhesive properties between the untransformed and transformed 3T3 cell lines, the more metastatic melanoma variants exhibited higher relative rates and extents of homotypic and heterotypic monolayer attachment compared with lower metastatic lines (B16-F10 greater than B16-F5 greater than B16-F1). The correlation between in vivo and in vitro tumor cell adhesive properties and metastasis was discussed.

Sequence dependence of administration of human recombinant tumor necrosis factor and interleukin-2 in murine tumor therapy.

Simultaneous administration of recombinant human tumor necrosis factor (rhTNF) and interleukin-2 (rhIL-2) has been shown to block tumor take in murine models. We investigated the effects of sequence and schedule of administration as a function of tumor burden with two tumor models (B16 and Meth A). rhTNF followed by rhIL-2 had extraordinary antitumor efficacy, but rhIL-2 followed by rhTNF was much less effective. Sequential rhTNF/rhIL-2 therapy resulted in complete tumor regression, whereas simultaneous therapy resulted in complete tumor regression, whereas simultaneous therapy resulted in only reduced growth rate. Experiments with genetically immunodeficient mice suggested that T cell factors may be required for synergistic antitumor activity.

Radioimmune assay and characteristics of antibodies to macromomycin (NSC 170105).

A radioimmune assay for the antitumor agent, macromomycin, using purified, radioiodine-labeled macromomycin and antisera raised in rabbits against a carbodiimide-catalyzed macromycin-Limulus polyphemus hemocyanin complex has been developed. Radiolabeled macromomycin was prepared by direct iodination of the polypeptide antibiotic with the use of iodine monochloride or solid-state lactoperoxidase. Antibody-bound drug was isolated from free macromomycin with dextran-coated, activated charcoal. The standard curve of the sequential saturation assay was linear on a logit-log plot and indicated a lower limit of sensitivity of approximately 100 pg macromomycin. The radioimmune assay was suitable for measuring macromomycin in the presence of other antitumor drugs, and detection of macromomycin was quantitative when it was added to normal human serum or urine. Drug binding to melanoma and mammary carcinoma cell surfaces could be inhibited by preincubating macromomycin with affinity-purified antimacromomycin antibodies. However, once the drug was bound to cell surfaces, addition of antimacromomycin antibodies did not result in removal of the drug from cell surfaces or in reversal of macromomycin-induced inhibition of thymidine incorporation into cellular DNA. Antimacromovide useful tools for developing pharmacokinetic and toxicity studies of macromomycin, as well as for analyzing the mechanism(s) of action of the drug.

Synergistic effects of combination therapy with human recombinant interleukin-2 and tumor necrosis factor in murine tumor models.

Human recombinant interleukin 2 (IL-2) and tumor necrosis factor (TNF) were evaluated individually and in combination for their antitumor efficacy in vivo, using five s.c. murine tumors: L1210 leukemia, P815 mastocytoma, B16 melanoma, EL-4 lymphoma, and the methylcholanthrene-induced sarcoma, Meth A. While only the s.c. methylcholanthrene-induced tumor exhibited regression and/or cures in response to immunomodulatory therapy with either agent alone, the simultaneous administration of a maximally tolerated dose of TNF and IL-2 given daily from within 1 day (B16 melanoma), 3 days (L1210 leukemia and P815 mastocytoma) or 5 and 10 days (EL-4 lymphoma and methylcholanthrene-induced sarcoma) after tumor cell implant resulted in no tumor takes (growth). The TNF dose was apparently rate limiting in that reduction of the amount of TNF in the combination by 50% resulted in the loss of curative effects, while IL-2 doses could be reduced by 90% (depending upon tumor type) and still result in an efficacious combination. The synergy seen in combination IL-2 and TNF therapy appeared to be dependent upon tumor burden, but somewhat independent of tumor location. For example, no tumors were seen in the artificial pulmonary metastasis model of the B16 melanoma, and the percentage of extension of median lifetime (test versus control) greater than 150% was seen in the i.p. B16 melanoma, as well as several other i.p. models of the five tumor types. On the other hand, no significant extension of lifetime (greater than 150%) was seen with either lymphokine alone when administered i.p. at maximally tolerated dose for any of the five tumors tested here. Results are discussed in relation to potential immune modulatory events which may be occurring during combination treatments.

Immunological block to synthetic alpha-melanocyte-stimulating hormone: melanocyte interaction by antibodies isolated from cell-column immunoadsorbents.

Antibodies to formalin-fixed, syngeneic melanoma cells were prepared in mice, purified by immunoaffinity chromatography, and tested for binding activity to viable melanoma cells. The radiolabeled antibodies detected congruent to 9 X 10(6) melanoma antigenic sites/cell. The calculated average association constant (Ka) for the antibody population was 7 to 10 X 10(7) M-1. The antibody was shown to block the binding of melanocyte-stimulating hormone in competitive cell surface binding studies. Results are discussed conceptually in terms of the potentially important role that the humoral immune response may play in the phenomenon of tumor progression.

Schedule dependency of the antitumor activity and toxicity of polyethylene glycol-modified interleukin 2 in murine tumor models.

Modification of recombinant human interleukin 2 (rhIL-2) with monomethoxy polyethylene glycol has been shown to alter its pharmacokinetic properties. Therefore, we investigated the pharmacological parameters of schedule and dose in order to assess the impact on the in vivo antitumor activity of this modification. The antitumor efficacy, as well as the toxicity, of polyethylene glycol-interleukin 2 (PEG-IL-2) was compared to that of rhIL-2 in three transplantable syngeneic murine tumor models, Meth A fibrosarcoma, B16 melanoma, and Pan-02 pancreatic carcinoma. At equitoxic dose levels, the antitumor activity of PEG-IL-2 was far superior to that of rhIL-2 in all three tumor models. This efficacy of PEG-IL-2 was dose dependent and was greatest on a Q7D x 2 schedule in Meth A and B16. When the same total doses were further divided and delivered on any of several alternative schedules, either the efficacy was reduced or the toxicity of the treatments was increased. In Pan-02, a rhIL-2-resistant tumor, PEG-IL-2 treatment on either the Q7D x 2, Q4D x 3, or Q3D x 4 schedule resulted in approximately a 200% increase in lifespan; however, the toxicity of the treatment increased as the interval between doses was shortened. Simulations of the pharmacokinetic profiles of these various regimens suggested that the toxicity of PEG-IL-2 and rhIL-2 was related to the minimum plasma concentration that was obtained and the time interval between peak levels. The efficacy of the treatment was associated with the interleukin 2 plasma peak height, since a dose response was observed; however, peak plasma concentration did not appear to be the only parameter which determined efficacy. We hypothesize that this observed schedule dependence is also affected by the kinetics of the host's biological response to rhIL-2.

Antitumor activity of an immunotoxin in a nude mouse model of human ovarian cancer.

An immunotoxin composed of an antibody to the human transferrin receptor (454A12) and ricin A chain (RTA) was shown to inhibit the growth of NIH:OVCAR-3 tumors in a nude mouse model of human ovarian cancer. Inhibition of tumor growth by 454A12-RTA was related to the dose administered. The antitumor activity of the immunotoxin was blocked by coinjection of excess antibody with immunotoxin. An immunotoxin made using 454A12 and recombinant ricin A chain (rRTA) had an activity similar to that made with native RTA. The administration of 10 micrograms or greater of the immunotoxin 454A12-RTA/rRTA had significant antitumor activity. The injection of 30 micrograms of an irrelevant immunotoxin, MOPC21-RTA, or 30 to 500 micrograms of the 454A12 antibody had no antitumor activity.

Contrast-enhanced magnetic resonance imaging of tumor-bearing mice treated with human recombinant tumor necrosis factor alpha.

Pharmacological effects of recombinant human tumor necrosis factor alpha (TNF) were studied in a mouse fibrosarcoma model using magnetic resonance imaging enhanced with a macromolecular contrast agent, albumin(gadolinium-diethylenetriamine pentaacetic acid)35. TNF was administered i.v. in a dose of 150 micrograms/kg, 60 to 80 min prior to imaging. Contrast-enhanced and nonenhanced magnetic resonance images of TNF-treated (n = 10) and untreated (n = 8) Meth A fibrosarcomas were obtained at 2.0 Tesla using T1-weighted spin-echo pulse sequences. Serial images spanning an interval of 60 to 120 min after TNF administration showed that the TNF-treated tumors enhanced significantly more overall than did untreated tumors (43% versus 31%). The most marked differential tumor enhancement was observed in the tumor rim (59% versus 40%). Nontumorous tissue, including muscle and brain, revealed no significant enhancement differences between TNF-treated animals and controls. The observed tumor enhancement corresponded strongly with Evans blue staining; the TNF-treated tumors stained deep blue, while untreated tumors and normal tissues observed did not stain. The different enhancement and Evans blue staining patterns between TNF-treated tumors and untreated tumors are attributed to TNF-induced changes in tumor capillary integrity. The data indicate that TNF effects on tumors include an increased capillary permeability for macromolecules at early times after administration. The ability to detect changes in capillary permeability in vivo using contrast-enhanced magnetic resonance imaging may prove to be clinically useful to monitor tumor response to TNF.

Antigenic peptide binding to MHC class II molecules at increased peptide concentrations.

Affinity-purified major histocompatibility complex (MHC) class II molecules are known to bind antigenic peptides in vitro. The percentage of MHC class II molecules occupied with such peptides is usually very low and varies significantly depending upon the sequence and size of a given antigenic peptide. The present study describes a method by which complete saturation of affinity-purified MHC class II with antigenic peptide can be achieved by simply incubating purified MHC class II molecules at neutral pH in the presence of several 100-fold molar excess of antigenic peptide. Complexes of human HLA-DR2 and a peptide analog from human myelin basic protein MBP (83-102)Y83 were selected for this study. The on-rate kinetic results showed saturation of MHC class II occupancy at 300-500-fold molar excess peptide concentrations. The specificity of the MBP (83-102)Y83 peptide binding to HLA-DR2 at higher peptide concentration was demonstrated by incubating an equivalent amount of another epitope from myelin basic protein [MBP (1-14) peptide] as well as by competitive binding assays. The quantitation of bound peptide was carried out using biotinylated-MBP (83-102)Y83 peptide which showed 100-125% occupancy of HLA-DR2 with a recovery of 100%. The presence of a single peptide entity in purified complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid extracted supernatant and by mass spectrometry analysis. Two-dimensional gel electrophoresis (IEF/SDS) of purified HLA-DR2 and DR2.MBP (83-102)Y83 complexes showed the absence of various endogenous polypeptides in 100% loaded complexes. These results demonstrate that higher peptide concentrations can be useful in generating MHC class II-peptide complexes of defined composition. Such complexes of MHC class II occupied with a single peptide may have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of MHC-peptide-TCR interactions.

Acute phase (C-reactive) protein-like macromolecules from rainbow trout (Salmo gairdneri).

A protein which reacts with the Cx-polysaccharide of Streptococcus pneumoniae and is inhibited by phosphorylcholine was isolated from the serum of rainbow trout by affinity chromatography. The protein, which exists in monomeric and oligomeric forms in non-immune trout serum, is very similar with regard to specificity and size to the Cx-reactive protein from rabbits. A semi-quantitative analytical method for evaluating bacterial agglutination with an electronic particle counter and size distribution analyzer was developed to compare natural and acute serum levels of trout and rabbit Cx-reactive proteins. Results indicate that the poikilotherm has much higher concentrations in normal serum. The trout serum protein can also be rapidly induced to yet higher levels by both chemical and physical stress. The implications for such a protein in the teleost's natural defense system and overall homeostasis are discussed.

Contrast-enhanced MRI of tumors. Comparison of Gd-DTPA and a macromolecular agent.

The study aim was to define potential differences and advantages in magnetic resonance (MR) patterns of tumoral contrast enhancement using either a small molecular, extracellular fluid contrast enhancer [Gd-DTPA] or a macromolecular agent [albumin-(Gd-DTPA)20], designed for primary intravascular biodistribution. MR images of 25 mice with implanted fibrosarcomas were obtained before and repeatedly for up to 120 minutes after injection of either Gd-DTPA [0.2 mmol/kg, n = 11] or albumin-(Gd-DTPA) [0.0029 mmol/kg, n = 14]. Histologically, this hypovascular tumor contained zones of viable tissue and non-viable, necrotic tissue. Using either type of contrast media, the viable portions enhanced strongly, up to 152% and the necrotic portions enhanced poorly, less than 31%. However, the time-course of enhancement differed between contrast agents. Gd-DTPA tended to provide maximal enhancement soon after administration with no significant changes over two hours. Enhancement from albumin-(Gd-DTPA) was weak initially, corresponding to tumor hypovascularity, but over two hours the signal of the viable tumor zones progressively increased in intensity. This gradual tumoral accumulation of the macromolecular agent within the tumor was considered to reflect abnormal capillary permeability, associated with neovascularity. Thus, the increasing intensity within the neoplastic tissues over time, reflecting abnormal capillary permeability for macromolecules, may serve as a useful, albeit indirect, marker of neoplasia.

Effects of chemical modification of antibodies on their clearance from the circulation. Addition of simple aliphatic compounds by reductive alkylation and carbodiimide-promoted amide formation.

Anti-hapten antibodies from the ascitic fluid of inbred mice were purified by immunoadsorption and characterized immunochemically for in vivo studies of their plasma clearance rates and organ distributions after chemical modification. Following sodium borohydride-promoted reductive methylation and carbodiimide-promoted amide linkage of glycine and several other simple aliphatic compounds, the antibody populations were recharacterized, radiolabeled, and introduced intravenously into syngeneic animals. Using double radioiodine labels, it was possible to show that stoichiometric combinations of additive and chemical reactant did not alter antibody survival time in the circulation or antigen-binding capabilities. However, modifications involving excess reagent (carbodiimide or sodium borohydride) resulted in significant decreases in both circulatory longevity and ligand binding capacities. Excess carbodiimide treatments resulted in immunoglobulin cross-linkage which could be detected by molecular sieve chromatography. Increased kidney localization found with carbodiimide-treated antibodies was apparently due to trapping of the cross-linked aggregates. Elevated clearance of highly methylated antibodies could not be attributed to a single organ. However, sodium borohydride-catalyzed reductive methylation resulted in antibody populations which were localized primarily in the liver and spleen. Results are evaluated in terms of the concept, developed in this paper, of essential groups for the circulatory longevity of glycoproteins.

Fc:Fc interactions revealed by spin-labeled IgG heterosaccharides in model immune complexes.

Dynamic properties of spin-labelled heterosaccharides in the Fc-region of murine monoclonal antihapten immunoglobulin G were studied in model immune complexes (IC) as a function of the IC size. Model IC dimers, trimers and oligomers were formed using bivalent photoaffinity antigens. The ESR spectrum exhibits two components. The rotational correlation time of the less-immobilized species is shorter than 10(-10) sec, and that of the more-immobilized component is in the order to 10(-9) approximately 10(-8) sec depending on the IC size. Fraction of the more-immobilized spin labels increases, and the mobility of this component decreases with increase in IC size (i.e., mobility: monomers approximately equal to dimers greater than trimers much greater than immune-complex precipitates). These data strongly suggest the existence of Fc:Fc interactions in IC, and provide the basis for a model in which such interactions underlie the initial mechanism by which the information of antigen binding to Fab region is transferred into organized Fc:Fc association structure for IgG effector activities.

Studies on the mechanism of covalent incorporation of a lysergyl derivative to immunoglobulin peptides in vitro.

In vitro incubation of hyperimmune rabbit lymphoid cells with the hallucinogenic indole alkaloid, d-lysergic acid diethylamide (LSD), results in biosynthesis of modified, secretable immunoglobulin peptides. Modification involves covalent attachment of the lysergyl moiety to COOH-terminal portions of the peptides. Aalogous effects occur when cells are incubated in vitro in the presence of the non-hallucinogenic LSD analogue, d-lysergic acid, and N-[3H]-carboxymethyl-d-lysergamide. The phenomenon is reversed by tryptophan and is inhibited by puromycin and cycloheximide. In vitro attachment of the lysergyl moiety occurs in the presence of actinomycin D at levels which inhibit RNA synthesis. While LSD is not attached to intracellular tRNA, the drug binds to 80 S ribosomes from hyperimmune lymphoid cells with high affinity (K A equals 3.5 times 10-8 M-1). Similar binding occurs to nonimmune splenic ribosomes. Implications of these findings are discussed with respect to the degree of involvement of cellular protein translational mechanisms in the covalent attachment of the lysergyl moiety to low molecular weight immunoglobulin peptides.

Mechanism of lysergic acid diethylamide interference with rabbit antibody biosynthesis.

Lymphoid cells from hyperimmune rabbits producing antibodies to a hapten, incubated in the presence of d-lysergic acid diethylamide, continued to synthesize protein at a normal rate. Isoelectric focusing analysis of the low-molecular-weight protein secreted by the cells incubated with lysergic acid diethylamide indicated two components, with pI's of 4.9 and 5.2. Immune cells not exposed to lysergic acid diethylamide secreted only 7S IgG molecules with an average pI of approximately 7.0.

Aglycosylantibody. Effects of exoglycosidase treatments on autochthonous antibody survival time in the circulation.

Rabbit anti-hapten antibodies were purified by affinity chromatography and characterized immunochemical for in vivo studies of their blood clearance rate and organ distribution after treatment with various glycosidases. Following sequential removal of sialic acid, galactose, and N-acetylglucosamine with the appropriate cellulose-immobilized exoglycosidases, the antibody populations were recharacterized, radiolabeled, and introduced intravenously into the original animals. Using double radioiodine lables it was possible to demonstrate alterations in purified antibody survival times in the circulation and altered organ distribution after glycolytic cleavage. Removal of terminal sialic acid resulted in rapid blood clearance and enhanced localization of asialoantibody in the liver. Subsequent removal of penultimate galactose residues returned both antibody survival time in the circulation and organ distribution to near normal. Removal of subpenultimate N-acetylglucosamine moieties resulted in aglycosylantibody survival values which were intermediate between asialo- and asialoagalactoantibodies. Removal of the three saccharide also increased kidney localization. The results are evaluated based on current concepts of the biological roles of protein-linked carbohydrate and plasma glycoprotein survival time in the circulation.

Bactericidal activity of macromomycin, a antitumor antibiotic.

Susceptibility testing by the broth dilution method showed that all the gram-positive but only some of the gram-negative bacteria tested were susceptible to the antitumor antibiotic, macromomycin (MCR; NSC 170105). The minimal inhibitory concentration for the susceptible organisms was less than 3 mug/ml. The action of MCR was bactericidal; however, at very high concentrations (50 mug/ml and above) some organisms occasionally escaped death. None of the escaped organisms was resistant to MCR. In combination with other commonly used antibiotics, MCR displayed partial synergy for Pseudomonas aeruginosa (from a minimal inhibitory concentration of >100 to 12.5 mug/ml with 100 mug of chloramphenicol per ml) and for Bacillus pumilus and Staphylococcus aureus (from 1.6 to 0.4 mug/ml and below) with polymyxin B. As with mammalian cells, (125)I-labeled MCR was irreversibly bound to both gram-positive and -negative bacteria. Treatment with trypsin of the (125)I-labeled MCR-exposed cells did not release the bound MCR or reverse its lethal effect. When in solution in a protective buffer at 4 degrees C, MCR was stable for up to 45 days; at 37 degrees C, however, 25% of its bactericidal activity was lost in 72 h. Loss of activity was enhanced 16-fold in the presence of both heated and unheated pooled human sera. Urine had no effect on the activity of MCR.

Human aglycosyl-IgG exhibits increased hydrophobicity. Binding/fluorescence studies with 8-anilinonaphthalene-1-sulfonic acid (ANS).

Human monoclonal, aglycosyl-IgG produced in vitro in the presence of tunicamycin, was compared with its native and acid pH-altered counterparts for their respective abilities to bind the fluorescent hydrophobicity probe, 8-anilinonaphthalene sulfonate. A novel technique based on continuous-flow dynamic dialysis (Sparrow et al., 1982, Anal. Biochem. 123:255-264) allowed binding studies under non-equilibrium conditions. While the native IgG conformation exhibits two, weak ANS binding sites (ca. 10(3) l/mol), aglycosyl-IgG has one weak and one moderate affinity (least squares average Ka = 2 X 10(4) l/mol) site, and the acid conformer binds yet another two ANS molecules with moderate affinity (4 X 10(4) l/mol). Increases in affinity and in the number of sites correlate roughly with increased relative percent fluorescence by conventional fluorimetry. The fluorescence lifetime of ANS bound to altered IgGs is about 10% longer (T2 = 15 nsec by time-resolved fluorimetry) than that for native IgG. All populations also exhibit a rapid decay component (T1 = 3 nsec) analogous to that seen for ANS in 50% aqueous dioxane. Results are discussed in relation to structural role(s) for IgG-linked heterosaccharides.