Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

Interaction of gastrin with transferrin: effects of ferric ions

The sedimentation behavior of 125I-labeled gastrin has been studied as a function of Fe3+ ion concentration and pH. Both sedimentation velocity and sedimentation equilibrium experiments indicated that high-molecular-weight Fe3+-gastrin complexes were formed at pH 5.0 and pH 7.4. Self-association of gastrin alone was observed at pH values below 5.0. 125I-labeled gastrin bound to human serum apotransferrin at pH 7.4. Scatchard analysis of the gastrin-apotransferrin complex gave a Kd of approximately 6.4 microM at 37 degrees C, with two binding sites per molecule of apotransferrin. No significant binding of gastrin to diferric transferrin was observed under the same conditions. The binding of gastrin to apotransferrin was inhibited by NaCl. The results are consistent with the hypothesis that gastrin and transferrin act synergistically in the uptake of dietary iron by the gastrointestinal tract.     

Fluorometric detection of the bilayer-to-hexagonal phase transition in liposomes

We have used the fluorescent probe N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) to detect the bilayer-to-hexagonal phase transition. The fluorescence intensity of the probe was found to increase during the bilayer-to-hexagonal transition. The bilayer-to-hexagonal transitions of various types of phosphatidylethanolamine or cardiolipin measured by this method are consistent with results obtained by differential scanning calorimetry. To establish this method for wider use, agents known to alter the bilayer-to-hexagonal transition were examined, and the results are comparable with the published data. The added advantage of this fluorometric method over other currently available techniques is that it is applicable not only for aggregated lipid samples but also for dilute liposome suspensions. This is especially important when one of the components of the system under study can partition between lipid and aqueous phase. Since NBD is located near the headgroup region of the bilayer, it most likely detects the change of the environment surrounding that region. On the basis of our present study, it appears that NBD-PE is sufficiently sensitive to detect bilayer-to-hexagonal phase transition.     

Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.     

Pseudomonas exotoxin A. Membrane binding, insertion, and traversal

Using vesicle targets composed of phosphatidylcholine and cholesterol (1:1 molar ratio), we found that Pseudomonas aeruginosa exotoxin A (PTx) binding and insertion are not only dependent on pH (Zalman, L.S., and Wisnieski, B.J. (1985) Infect. Immun. 50, 630-635) but also on ionic strength, reaching a maximum in pH 4 buffer that contains 150-200 mM NaCl. Insertion was monitored by photolabeling with an intramembranous probe. Higher levels of binding and insertion were attained with vesicles that contained 2.5 mol% dicetylphosphate than with neutral vesicles. Positively charged vesicles (2.7 mol% stearylamine) were the least effective targets. At pH 7.4, all binding levels were depressed. While PTx binding increased with increasing temperature, the relative proportion of the vesicle-associated toxin that was photolabeled decreased. The most likely explanation for the decrease is that the bilayer translocation rates increased with increasing temperature, and hence fewer PTx molecules were accessible at the time of photolabeling. At 37 degrees C, binding and insertion both plateaued within 10 min of lowering the pH to 4. After 10 min, the amount of bound toxin decreased slightly with time but there was a dramatic decrease in photolabeling, indicating that inserted PTx had begun to cross the bilayer. This was verified by the finding that when PTx was incubated with vesicles that contained trypsin, cleavage occurred only in those samples in which the pH was shifted down to pH 4. Entry is triggered by an acid-induced conformational change that promotes productive binding and insertion. After insertion, the kinetics of membrane traversal appear to be regulated by the physical properties of the bilayer.     

Antitumor properties of vindesine-monoclonal antibody conjugates

Summary The anticancer alkaloid vindesine (VDS) was conjugated to four mouse monoclonal antibodies recognizing human tumor-associated antigens. The antibodies were 96.5 (antimelanoma, IgG2a); 791T/36 (antiosteogenic sarcoma, IgG2b); 11.285.14, and 14.95.55 (anticarcinoembryonic antigen, IgG1 and IgG2a respectively). Conjugates VDS-96.5 and VDS-791T/36 were tested in vitro and shown to be specifically cytotoxic for target cells expressing the appropriate antigen. The in vivo effects of the antibodies and conjugates were tested against human tumor xenografts in athymic or immunodeprived mice using multiple treatments. Conjugate VDS-96.5 retarded the initial growth of a melanoma xenograft, whereas free antibody was without effect. Similarly, VDS-791T/36 but not free antibody retarded the growth of osteogenic sarcoma 791T. The most marked antitumor effects observed were those obtained with VDS conjugates of the anti-CEA antibodies against a colorectal tumor xenograft. Antibody 14.95.55 suppressed tumor growth both alone and as a VDS conjugate, whereas 11.285.14 produced only a slight effect alone but an almost complete and lasting suppression of tumor growth as a VDS conjugate. Free VDS had little effect at nontoxic levels. Acute studies showed that VDS-11.285.14 conjugate was considerably less toxic than free VDS in Balb/c mice.     

Buoyant density variation during the cell cycle of Saccharomyces cerevisiae

Cell buoyant densities of the budding yeast Saccharomyces cerevisiae were determined for rapidly growing asynchronous and synchronous cultures by equilibrium sedimentation in Percoll gradients. The average cell density in exponentially growing cultures was 1.1126 g/ml, with a range of density variation of 0.010 g/ml. Densities were highest for cells with buds about one-fourth the diameter of their mother cells and lowest when bud diameters were about the same as their mother cells. In synchronous cultures inoculated from the least-dense cells, there was no observable perturbation of cell growth: cell numbers increased without lag, and the doubling time (66 min) was the same as that for the parent culture. Starting from a low value at the beginning of the cycle, cell buoyant density oscillated between a maximum density near midcycle (0.4 generations) and a minimum near the end of the cycle (0.9 generations). The pattern of cyclic variation of buoyant density was quantitatively determined from density measurements for five cell classes, which were categorized by bud diameter. The observed variation in buoyant density during the cell cycle of S. cerevisiae contrasts sharply with the constancy in buoyant density observed for cells of Escherichia coli, Chinese hamster cells, and three murine cell lines.     

Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.     

Subunit composition of rodent isomyosins and their distribution in hindlimb skeletal muscles

Three adult skeletal muscle sarcomeric myosin heavy chain (MHC) genes have been identified in the rat, suggesting that the expressed native myosin isoforms can be differentiated, in part, on the basis of their MHC composition. This study was undertaken to ascertain whether the five major native isomyosins [3 fast (Fm1, Fm2, Fm3), 1 slow (Sm), and 1 intermediate (Im)], typically expressed in the spectrum of adult rat skeletal muscles comprising the hindlimb, could be further differentiated on the basis of their MHC profiles in addition to their light chain composition. Results show that in muscles comprised exclusively of fast-twitch glycolytic (FG) fibers and consisting of Fm1, Fm2, and Fm3, such as the tensor fasciae latae, only one MHC, designated as fast type IIb, could be resolved. In soleus muscle, comprised of both slow-twitch oxidative and fast-twitch oxidative-glycolytic fibers and expressing Sm and Im, two MHC bands were resolved and designated as slow/cardiac beta-MHC and fast type IIa MHC. In muscles expressing a mixture of all three fiber types and a full complement of isomyosins, as seen in the plantaris, the MHC could be resolved into three bands. Light chain profiles were characterized for each muscle type, as well as for the purified isomyosins. These data suggest that Im (IIa) consists of a mixture of fast and slow light chains, whereas Fm (IIb) and Sm (beta) isoforms consist solely of fast- and slow-type light chains, respectively. Polypeptide mapping of denatured myosin extracted from muscles expressing contrasting isoform phenotypes suggests differences in the MHC primary structure between slow, intermediate, and fast myosin isotypes. These findings demonstrate that 1) Fm, Im, and Sm isoforms are differentiated on the bases of both their heavy and light chain components and 2) each isomyosin is distributed in a characteristic fashion among rat hindlimb skeletal muscles. Furthermore, these data suggest that the ratio of isomyosins in a given muscle or muscle region is of physiological importance to the function of that muscle during muscular activity.