Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro.

Inappropriate signalling through the EGFR and ErbB2/HER2 members of the epidermal growth factor family of receptor tyrosine kinases is well recognised as being causally linked to a variety of cancers. Consequently, monoclonal antibodies specific for these receptors have become increasingly important components of effective treatment strategies for cancer. Increasing evidence suggests that ErbB3 plays a critical role in cancer progression and resistance to therapy. We hypothesised that co-targeting the preferred ErbB2/ErbB3 heterodimer with a bispecific single-chain Fv (bs-scFv) antibody would promote increased targeting selectivity over antibodies specific for a single tumour-associated antigen (TAA). In addition, we hypothesised that targeting this important heterodimer could induce a therapeutic effect. Here, we describe the construction and evaluation of the A5-linker-ML3.9 bs-scFv (ALM), an anti-ErbB3/ErbB2 bs-scFv. The A5-linker-ML3.9 bs-scFv exhibits selective targeting of tumour cells in vitro and in vivo that co-express the two target antigens over tumour cells that express only one target antigen or normal cells that express low levels of both antigens. The A5-linker-ML3.9 bs-scFv also exhibits significantly greater in vivo targeting of ErbB2'+'/ErbB3'+' tumours than derivative molecules that contain only one functional arm targeting ErbB2 or ErbB3. Binding of ALM to ErbB2'+'/ErbB3'+' cells mediates inhibition of tumour cell growth in vitro by effectively targeting the therapeutic anti-ErbB3 A5 scFv. This suggests both that ALM could provide the basis for an effective therapeutic agent and that engineered antibodies selected to co-target critical functional pairs of TAAs can enhance the targeting specificity and efficacy of antibody-based cancer therapeutics.

Delivery of the alpha-emitting radioisotope bismuth-213 to solid tumors via single-chain Fv and diabody molecules.

Intravenously administered anti-tumor single-chain Fv (scFv) and diabody molecules exhibit rapid clearance kinetics and accumulation in tumors that express their cognate antigen. In an attempt to fit the rate of isotope decay to the timing of delivery and duration of tumor retention, anti-HER2/neu CHX-A" DTPA-C6.5K-A scFv and diabody conjugates were labeled with the alpha-particle emitter (213)Bi (t(1/2) = 47 min). Radioimmunotherapy studies employing 0.64, 0.35, or 0.15 microCi of (213)Bi-labeled C6.5K-A diabody or 1.1, 0.6, or 0. 3 microCi of (213)Bi-labeled C6.5K-A scFv were performed in nude mice bearing early, established SK-OV-3 tumors. Only the 0.3 microCi dose of (213)Bi-labeled C6.5K-A scFv resulted in both acceptable toxicity and a reduction in tumor growth rate. The specificity of the anti-tumor effects was determined by comparing the efficacy of treatment with 0.3 and 0.15 microCi doses of (213)Bi-labeled C6.5K-A scFv and (213)Bi-labeled NM3E2 (an irrelevant scFv) in nude mice bearing large established tumors. The 0.3 microCi dose of (213)Bi on both the C6.5K-A and NM3E2 scFvs resulted in similar anti-tumor effects (p = 0.46) indicating that antigen-specific targeting was not a factor. This suggests that the physical half-life of (213)Bi may be too brief to be effectively paired with systemically-administered diabody or scFv molecules.

Metabolism of phosphonium choline by rat-2 fibroblasts: effects of mitogenic stimulation studied using 31P NMR spectroscopy.

Phospholipid turnover increases with both mitogenic stimulation and oncogenic transformation (1-9). Recent 31P nuclear magnetic resonance (NMR) spectroscopy studies of human tumors, animal tumor models and cell systems have reported elevated phosphomonoesters with growth and oncogenic transformation, as well as changes in these levels associated with treatment (10). In order to gain insights into the mechanisms underlying these changes, we used a phosphonium analog of choline and 31P NMR spectroscopy to study choline metabolism in quiescent and mitogenically stimulated Rat-2 fibroblasts. Cell growth status of these cells has a significant effect on choline metabolism. While overall uptake of the analog was similar in both quiescent and growing cells, distribution among metabolite pools differed. Quiescent cells accumulate label in the phosphodiester pool, with little or none in the phosphomonoester pool. On the other hand, mitogenic stimulation resulted in a significant fraction of the label in the phosphomonoester pool.

Quantitation of resonances in biological 31P NMR spectra via principal component analysis: potential and limitations.

This paper examines the potential and limitations of peak area quantitation of biological NMR spectra using principal component analysis (PCA), including its requirement for prior knowledge. The principles of the method are presented without in-depth mathematical treatment. PCA is illustrated for simulated data, 31P NMR spectra obtained consecutively over 1-2.5 days from perfused Rat-2 cells metabolizing the choline analogue phosphoniumcholine (Chop) and in vivo proton-decoupled, NOE-enhanced, three-dimensional CSI localized 31P NMR spectra of the liver of healthy volunteers. The results show that PCA can be used to quantitate strongly overlapping peaks without prior knowledge of the peak shapes or positions and to reconstruct spectra with significantly reduced noise variance. Two major limitations of PCA are presented: (1) PCA cannot separate peaks whose intensities are well correlated; (2) PCA is sensitive to differences in chemical shift and line-width of peaks between spectra. The discussion focuses on what knowledge of the biological and spectroscopic features of the samples and the principles of PCA is necessary for peak area quantitation via PCA.

Characterization of a phosphonium analog of choline as a probe in 31P NMR studies of phospholipid metabolism.

Tumors and transformed cells have been shown by 31P NMR to contain elevated concentrations of two phosphomonoesters, phosphorylcholine and phosphorylethanolamine, involved in phospholipid metabolism. In order to understand the biochemical basis for these phenomena new methods are needed to allow for analysis of the relevant metabolic pathways in intact cells. One such promising tool may be phosphonium-choline, a 31P NMR-visible analog of choline in which the trimethyl-ammonium group of choline has been replaced with a trimethyl-phosphonium moiety. As shown previously [Sim et al. Biochem. J. 154, 303 (1976)], this compound is non-toxic and readily metabolized by cultured cells into phospholipids. In this paper we describe in greater detail some of the chemical and NMR spectroscopic properties of this material. Most significantly we show here that the chemical shift of phosphonium-choline is sensitive to the phosphorylation state of the analog and that the phosphonium nucleus is NMR-visible even after its incorporation into phospholipid. The unique properties of this analog should make it possible to use high-field 31P NMR to follow the flux of phosphonium-choline through the Kennedy pathway in intact perfused cells cultures.

Inositol 1,4,5-triphosphate-induced granule secretion in platelets. Evidence that the activation of phospholipase C mediated by platelet thromboxane receptors involves a guanine nucleotide binding protein-dependent mechanism distinct from that of thrombin.

Phosphoinositide hydrolysis in platelets stimulated by thrombin is thought to be regulated by a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) referred to as Gp. The present studies examine the role of Gp in platelet responses to the thromboxane A2 analogue U46619 and in the pathway by which the phosphoinositide hydrolysis product inositol 1,4,5-triphosphate (IP3) causes secretion. In permeabilized platelets, U46619 caused phosphatidic acid formation and secretion, which were abolished by the G protein inhibitor, guanosine 5'-O-(2-thiophosphate) (GDP beta S). Unlike thrombin, however, U46619-induced phosphoinositide hydrolysis was unaffected by pertussis toxin, and U46619 was unable to inhibit the [32P]ADP ribosylation of the 42-kD pertussis toxin substrate in platelets. IP3-induced secretion, which is known to depend upon intracellular Ca release and subsequent arachidonic acid metabolism, was also inhibited by GDP beta S, as was Ca-induced secretion. These observations suggest that platelet thromboxane A2 (TxA2) receptors are coupled to a toxin-resistant form of Gp distinct from the one that is coupled to thrombin receptors, and that TxA2-stimulated phosphoinositide hydrolysis may serve as a feedback mechanism by which stimuli for arachidonic acid release, such as IP3 and Ca, amplify responses to agonists.

Self-exchange of sodium in human lymphocytes.

Self-exchanges of Na and K in human lymphocytes were measured by isotopic efflux techniques. In washed cells, K exchanged in a single slow exponential fraction, but the Na exchange had a marked curvature. It was shown that the curvature was not caused by simple bulk-phase diffusion, and it was resolved into three major fractions: fast (F) (half-time, t1/2 = 2-4 min), intermediate (I) (t1/2 = 12 min), and slow (S) (t1/2 = 125 min). Each of these appeared to follow an exponential function. The I fraction contained approximately 10 mmol Na/kg cells (25-30% of normal cellular Na), was not affected by manipulations that cause lymphocytes to gain Na, and had little or no temperature dependence. The S fraction of Na in normal cells (S1) contained approximately 10 mmol Na/kg cells, had only a slight temperature dependence, and the amount and rate of S1 were independent of external K concentration (Kex). Another slow fraction (S2) appeared when the cells underwent a net gain of Na in exchange for K, and was characterized by a steep temperature dependence and a peak rate around the transition point (the point at which half of cellular K is replaced by Na) at 0.4 mM Kex. The results are discussed within context of a theory that assigns the exchange of the major part of K in its slow exponential fraction and the Na exchange in S2 to interactions of these ions with fixed anionic sites, on intracellular macromolecules, which have been shown previously to interact cooperatively in their association with K and Na.

Development of a protocol for photoradiation therapy of malignant brain tumors: part 1. Photosensitization of normal brain tissue with hematoporphyrin derivative.

The successful application of phototherapy to subcutaneous tumors has suggested that a similar procedure should be developed for treating gliomas. As a result, attempts are being made to determine a set of conditions that would optimize the destruction of tumor cells while minimizing injury to surrounding brain tissue. To initiate this task, we developed a novel assay method to assess the amount of phototoxicity induced in normal brain by light exposure of mice treated with hematoporphyrin derivative (HPD). The application of this procedure demonstrated that a sufficient amount of HPD was retained in brain tissue, even 72 hours after injection, to cause severe cerebral damage in light-treated mice.

Effects of valinomycin on lymphocytes independent of potassium permeability.

10(-7) M valinomycin affects human lymphocytes in the following manner: (1) it is non-toxic; (2) it inhibits mitogenesis; (3) it causes a reduction in cell ATP; and (4) it causes a marked increase in steady-state Na+ exchange. However, it has a minimal effect on cell ion (K+, Na+, Ca2+, Mg2+) contents and no effect whatever on K+ exchange. Neither the fast nor the slow fraction of steady-state K+ exchange is affected by 10(-7) M valinomycin. The various reported effects of valinomycin on lymphocyte functions cannot be assumed to be due to changes in plasma membrane K+ permeability. The mechanism of the increase in steady-state Na+ exchange, and whether or not it is related to inhibition of mitogenesis, are unsettled issues.

The effect of metabolic inhibition on ion contents and sodium exchange in human lymphocytes.

Lymphocytes depleted of ATP by incubation in iodoacetate (IAA) and nitrogen (N2) lost K and gained Na. Isotopic Na exchange showed a fast fraction and a slower exponential fraction, the latter conventionally assumed to reflect surface membrane properties. The gain of cell Na was not accounted for by a decrease in 22Na efflux in either the slow or the fast fraction. After 3-5 hours, Na efflux increased. These results led us to question the concept that normal cell ion levels are maintained by an ATPase pump and could not be explained by exchange diffusion, co-transport, countertransport, or other inherently dissipative mechanisms. The data are, on the other hand, consistent with the concept that cell ion contents are determined by their relative exclusion from cell water coupled with selective adsorption onto fixed macromolecular anionic sites within the cell. In this view, the IAA,N2-induced rise in cell Na is due to the occupancy of adsorption sites losing K, while the increased isotopic exchange is due to a decreased activation energy for ion-site interaction.

Temperature-dependence of ATP level, organic phosphate production and Na,K-ATPase in human lymphocytes.

It was previously shown that human lymphocytes maintain a normal accumulation of K+ and exclusion of Na+ between 37 degrees and 10 degrees C., and a significant net accumulation of K+ and exclusion of Na+ at even lower temperatures. The studies reported here show that the level of ATP is near-normal for at least 24 hours between 37 degrees and 10 degrees C., but that ATP synthesis and utilization are progressively and markedly decreased with decreasing temperatures below 37 degrees C. The activities of the membrane Na+- and K+-activated ATPases have typical marked temperature-dependences. Therefore, the normal accumulation of K+ and exclusion of Na+ between 37 degrees and 10 degrees C., and the normal rate of Na+ efflux at these temperatures, do not correlate with properties of the Na+,K+-ATPase or with rates of synthesis and utilization of ATP.

Simultaneous net accumulation of both K+ and Na+ by lymphocytes at 0 degrees C.

Human lymphocytes at 0 degrees C in low Na+ medium accumulate both K+ and Na+ to levels higher than in the external medium. This is not due to an impermeable compartment or a Donnan equilibrium, and is incompatible with the membrane Na+-pump concept. In contrast, it supports prior evidence that ion exchange in lymphocytes is mediated by adsorption onto and desorption from fixed anionic sites within the cell. Additional aspects of ion and water contents of cells in low Na+ medium are described and are explained by this concept.

Multiple fractions of sodium exchange in human lymphocytes.

Human lymphocytes contain a large, saturable fraction of K+ that exchanges slowly with K+ in the external medium, and a small non-saturable fraction that exchanges rapidly. We determined whether or not Na+ exchanges in a similar manner with external Na+. Cells were pre-equilibrated to ensure absence of net ion movements. Efflux was studied by loading with 22Na and transferring without washing to a non-labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na+ exchange, with half-times of 2, 14, and 120 minutes. At normal external K+, most cells Na+ exchanges rapidly, while at lower external K+ the Na+ that replaces cell K+ exchanges slowly. Parellel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous 42K and 22Na fluxes and by a quantitative analysis of the combined K+ and Na+ content and flux data over a range of external K+ and Na+ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclear exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K+ and Na+ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate-limited by adsorption onto and desorption from cellular macromolecules. The data support the association-induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.

A critical temperature transition of K+-Na+ exchange in human lymphocytes.

Human lymphocytes were equilibrated for 48 hours at 5-6 mM K+ ex over a range of temperatures between 0 and 37 degrees C, and at 5 degrees C over a range of external K+ levels between 0 and 32 mM. Cell K+ and Na+ contents are normal between 37 and 10 decrees. Below 10 degrees there is a critical thermal transition in ion content centering around 3 degrees C. This and the steep sigmoidal isotherms of K+ and Na+ at 5 degrees C confirm the cooperative nature of ion exchange. At 0 degrees, cell K+ is maintained at a concentration that is seven to eight times that of the external medium. Isotopic K+ influx shows smaller, rapidly-exchanging, and larger, slowly-exchanging fractions. The latter, which correspond to the saturable, sigmoidal components of cell K+, are slowed by decreasing temperature. Although there is a critical temperature transition of K+-Na+ exchange, there is no corresponding transition for isotopic K+ exchange, which has an activation energy of 11.6 kcal/mole. The combined ion content and flux data are readily understood by reference to two major concepts of the association-induction hypothesis: that of rapid solute exclusion from cell water existing in a state of polarized multilayers, and that of solute accumulation limited by adsorption onto and desorption from fixed anionic sites that interact with one another in a critical, cooperative fashion.

Hereditary spherocytosis with normal osmotic fragility after incubation. Is the autohemolysis test really obsolete?

Two patients in two families had hereditary spherocytosis but lacked a population of RBCs with increased osmotic fragility after incubation. The diagnosis in each patient was confirmed by the presence of splenomegaly, spherocytosis, reticulocytosis, and abnormal autohemolysis corrected by glucose. Sodium flux studies showed increased sodium permeability of the RBC membrane in one patient and normal permeability in another. Hereditary spherocytosis was also present in three other family members of patient 2. The autohemolysis test is of value in confirming the diagnosis in patients with hereditary spherocytosis and normal incubated osmotic fragility.

Fast and slow fractions of K+ flux in human lymphocytes.

Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, and half-times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non-saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association-induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K+ex, then a trough around 4mM K+ex, and then a gradual rise. This relation was readily explained, in terms of the association-induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that adsorb K+ or Na+.

Potassium-sodium distribution in human lymphocytes: description by the association-induction hypothesis.

Human lymphocytes were equilibrated for 48 hours over a wide range of external potassium levels, and their contents of potassium, sodium, and water determined. As external potassium rose from zero, cell potassium rose steeply in a sigmoidal fashion, reached half-saturation at 0.4 mM esternal potassium, and then saturated at 129 mmoles/kg cells. The saturable cell potassium exchanged mole-for-mole with sodium. Analysis of the saturable components by a statistical-mechanical adsorption model demonstrated a cooperative intraction between sites determining equilibrium potassium-sodium distribution. Superimposed upon the saturable fraction of cell potassium was a smaller one that was non-saturable with increasing external potassium to at least 64 mM, and that, when expressed as mmoles/liter cell water, existed in a ratio to external potassium of 0.6. The results strongly support the association-induction hypothesis, which predicts a small non-saturable component of ions determined by exclusion from oriented cell water and a cooperative interaction between sites throughout the cell that associate with potassium or sodium.

Selective loss of calcium permeability on maturation of reticulocytes.

Calcium and sodium permeability of human reticulocytes have been studied and compared to mature erythrocytes. Mature erythrocytes had extremely low Ca2+ permeability which was less than 0.1% of values published for squid axon or HeLa cells. Calcium entry was markedly increased in reticulocyte-rich suspensions and the uptake was linearly related to the percentage of reticulocytes present. The data suggest that reticulocytes are 43-fold more permeable to Ca2+ than mature cells although their Ca2+ concentration is not increased. Sodium influx into reticulocyte-rich suspensions was also increased in direct proportion to the percent of reticulocytes present. Reticulocytes are sixfold more permeable to Na+ than mature cells so the ratio of Ca2+:Na+ permeability falls by sevenfold as the reticulocyte changes to an erythrocyte. [3H]Ouabain binding was increased in reticulocyte-rich cell suspensions and the correlation suggested a value of about 4,000 sites per reticulocyte compared with 362+/-69 per mature cell. Maturation of the human reticulocyte produces disproportionate changes in cation permeability and in particular a selective loss of Ca2+ permeability.