[Phenotyping of angiotensin-converting enzyme in the prostate in patients with prostate cancer and benign prostatic hyperplasia].

BACKGROUND: Angiotensin-converting enzyme (ACE) is expressed by all epithelial cells of the human body. Although the main proportion of ACE is synthesized by the lungs, in men, ACE is also secreted by the testes (testicular form), seminal vesicles and the prostate. In semen, the level of ACE is up to 50 times higher than in blood plasma. The substitution of highly specific epithelial cells of the prostate by tumor cells causes a dramatic decrease in ACE production by the prostate cells. AIM: To assess the possibility of using prostatic ACE as a new marker of prostate cancer (PCa). MATERIALS AND METHODS: ACE phenotyping in prostate of patients with PCa and benign prostatic hyperplasia (BPH) included measurement of the activity of two ACE substrates (HHL and ZPHL); calculation of the ratio of their hydrolysis rates (ZPHL/HHL ratio); quantitative assessment of the ACE immunoreactive protein, the ratio of the immunoreactive protein to the ACE activity, as well as the conformation of ACE using a panel of monoclonal antibodies (mAb) to different epitopes of ACE. RESULTS: ACE activity in tumor cells was markedly reduced and the ratio of immunoreactive ACE to its activity increased. The ratio of the hydrolysis rates of two substrates (ZPHL/HHL ratio) in patients with PCa increased compared to control group, while it was not observed in the vast majority of patients with BPH. There were several tissue samples with a histological diagnosis of BPH, but ACE phenotype was typical for PCa. DISCUSSION: Since a decrease in ACE activity was found in all patients with PCa, we suggest that it may serve as a reliable and early marker of the tumor development. Changes in the ACE phenotype, which are typical for PCa, but found in patients with BPH, may indicate earlier malignant changes in prostate cells, which are not visible on routine prostate biopsy. CONCLUSIONS: ACE activity and its conformation in prostatic biopsies has the potential to be an early biomarker or a differential criterion for PCa. In PCa, the ACE activity in the prostate is significantly reduced, and the ZPHL/HHL ratio is markedly increased in comparison to control group. However, there were no such changes in patients with BPH. In hyperplastic processes of the prostate (BPH, PCa), there is a change in ACE sialylation, which is accompanied by an increase in the binding of ACE to mAb 3F10 compared to the control group. Patients with negative biopsy result, but properties of prostate ACE, which are typical for PCa, require close follow-up, since they may have an increased risk of subsequent developing PCa. However, due to a small sample of patients, the diagnostic potential of prostate ACE for PCa and BPH requires to be validated in a larger number of patients to confirm its predictive accuracy.

[Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE)].

During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface-due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept - conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

Targeted gene delivery of BMPR2 attenuates pulmonary hypertension.

Pulmonary arterial hypertension (PAH) remains a fatal disease despite modern pharmacotherapy. Mutations in the gene for bone morphogenetic protein receptor type II (BMPR2) lead to reduced BMPR2 expression, which is causally linked to PAH. BMPR2 is predominantly expressed on pulmonary endothelium and has complex interactions with transforming growth factor (TGF)-ß signalling mechanisms. Our objectives were to assess the effect on PAH of upregulating BMPR2 by targeted adenoviral BMPR2 gene delivery to the pulmonary vascular endothelium. We used two established rat models of PAH: chronic hypoxia and monocrotaline (MCT). In both hypertensive models, those receiving BMPR2 had less right ventricular hypertrophy, less pulmonary vascular resistance, improved cardiac function and reduced vascular remodelling. In the MCT model, there was an increase in TGF-ß, which was prevented by BMPR2 treatment. In vitro, TGF-ß1-induced endothelial-mesenchymal transition (EndMT) in human pulmonary microvascular endothelial cells, which was associated with reduced BMPR2 expression. EndMT was partially ameliorated by stimulating BMPR2 signalling with appropriate ligands even in the ongoing presence of TGF-ß1. Collectively, these results indicate therapeutic potential for upregulation of the BMPR2 axis in PAH, which may be, in part, mediated by countering the remodelling effects of TGF-ß.

Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding.

A panel of monoclonal antibodies (mAbs) raised against both the N and C domains of angiotensin-I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) have been extensively mapped and have facilitated the study of various aspects of ACE structure and biology. In this study, we characterize two mAbs, 9B9 and 3G8, that recognize the N domain of ACE and that influence shedding and dimerization. Fine epitope mapping was performed, which mapped the epitopes for these mAbs to the N terminal region of the N domain where they overlap to a large extent, despite having different effects on ACE processing. The mAb 3G8 epitope appears to be shielded by the C domain and to be carbohydrate dependent as binding increased significantly as a result of underglycosylation, whereas these factors did not influence mAb 9B9 recognition. Three mutations within the overlapping region of these two epitopes, Q18H, L19E, and Q22A, which decreased mAb 3G8 binding to the soluble N domain, were introduced into full-length somatic ACE (sACE) to determine their influence on ACE expression and processing. Increased ACE expression, cell surface expression, and basal shedding were observed with all three mutations. Furthermore, cross-linking and western blotting of Chinese hamster ovary (CHO) cell lysates detected two distinct ACE dimers, a native and cross-linked dimer. Increasing amounts of the cross-linked dimer were observed for the mutant sACEQ22A, further implicating the overlapping region of the mAb 9B9 and 3G8 epitopes in ACE processing.

[Leukemic dendritic cells in patients with acute myeloid leukemia].

AIM: To determine surface and intracellular expression of ACE antigen and Bip shaperon on leukemic dendritic cells (LDC); to study expression of ACE genes and Bip, Calnexin, calreticulin shaperons in LDC at diagnosis of acute myeloid leukemia (AML) under standard and stress cultivation. MATERIAL AND METHODS: Expression of ACE antigens and Bip was studied with immunophenotyping and flow cytometry using monoclonal antibodies to shaperon Bip and to CD143), expression of genes of ACE and shaperons Bip, Calnexin, Calreticulin--with polymerase chain reaction (RT-PCR). Dendritic cells (DC) were obtained by culturing of a monoclonal fraction of donor peripheral blood and AML patients in the presence of 180 ng/ml calcium ionophor A23187 (Sigma) for 4 days in parallel at 37 degrees C and 33 degrees C in the atmosphere of 5% CO2. The trial included 9 patients (5 males and 4 females) aged 39-53 years (median 43 years). The control group consisted of 8 healthy donors. RESULTS: Lowering of cultivation temperature did not increase ACE expression. Intracellular shaperon Bip rose insignificantly (1.3-fold) in DC of the controls. ACE and Bip shaperon expression on LDC membrane increased 15- and 11-fold, respectively, while the level of intracellular ACE and Bip decreased 11- and 2-fold, respectively. Expression of the genes was investigated in cultivation temperature lowering from 37 to 33 degrees C and was presented as a logarithmic scale. Changes in expression of the genes Bip, Calnexin, Calreticulin in LDc and DC of the controls were insignificant. ACE expression in LDC significantly differed from ACE gene expression in DC (p = 0.05). CONCLUSION: LCD and DC of healthy donors are cells which differ by genetic and functional characteristics. Therefore, LDC may response inadequately in development of antitumor immune response. The phenomenon of ACE antigen expression normalization on cell membrane in stress open new opportunities for regulating functional activity of LDC.

Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

[Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme].

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.

Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

Previous studies suggest that while lung angiotensin converting enzyme (ACE) activity is reduced during chronic hypoxia, inhibitors of ACE attenuate hypoxic pulmonary hypertension. In an attempt to explain this paradox we investigated the possibility that whole lung ACE activity may not reflect local pulmonary vascular ACE expression. The experimental approach combined in vivo hemodynamic studies in control and chronically hypoxic rats, measurement of whole lung ACE activity, and evaluation of local pulmonary vascular ACE expression by in situ hybridization and immunohistochemistry. Total lung ACE activity was reduced to 50% of control activity by 5 d of hypoxia and remained low for the duration of the study. Immunohistochemistry showed a marked reduction of ACE staining in alveolar capillary endothelium. However, an increase in ACE staining was observed in the walls of small newly muscularized pulmonary arteries at the level of alveolar ducts and walls. In situ hybridization studies showed increased signal for ACE mRNA in the same vessels. Inhibition of ACE by captopril during chronic hypoxia attenuated pulmonary hypertension and markedly reduced distal muscularization of small pulmonary arteries. In addition, we demonstrated marked longitudinal variation in ACE expression along the normal pulmonary vasculature with the highest levels found in small muscular arteries associated with terminal and respiratory bronchioles. We conclude that local ACE expression is increased in the walls of small pulmonary arteries during the development of hypoxic pulmonary hypertension, despite a generalized reduction in alveolar capillary ACE expression, and we speculate that local arteriolar ACE may play a role in the vascular remodeling associated with pulmonary hypertension.

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo.

The promise of gene therapy for health care will not be realized until gene delivery systems are capable of achieving efficient, cell-specific gene delivery in vivo. Here we describe an adenoviral system for achieving cell-specific transgene expression in pulmonary endothelium. The combination of transductional targeting to a pulmonary endothelial marker (angiotensin-converting enzyme, ACE) and an endothelial-specific promoter (for vascular endothelial growth factor receptor type 1, flt-1) resulted in a synergistic, 300,000-fold improvement in the selectivity of transgene expression for lung versus the usual site of vector sequestration, the liver. This combined approach should be useful for the design of other gene delivery systems.

Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE).

During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface-due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept-conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

Affinity chromatography and some properties of the angiotensin-converting enzyme from human heart.

Human heart angiotensin-converting enzyme has been purified by affinity chromatography on immobilized N-[1(S)-carboxy-5-aminopentyl]-Gly-Gly. The isolation procedure permitted a 1650-fold-purified enzyme to be obtained. The specific activity of angiotensin-converting enzyme was 38 units per mg protein. The molecular weight of enzyme determined by polyacrylamide gel electrophoresis in denaturing conditions was 150,000. The isoelectric point (5.3) of the enzyme was determined by chromatofocusing. The Km values of the enzyme for Bz-Gly-His-Leu and angiotensin I are 1.2 mM and 10 microM, respectively. Substrate inhibition of heart angiotensin-converting enzyme with a K's of 14 mM has been shown. The human heart enzyme is inhibited by SQ 20881 (IC50 = 40 nM). It was shown that NaCl, CaCl2 as well as Na2SO4 in the absence of Cl- are activators of the heart angiotensin-converting enzyme, whereas CH3COONa and NaNO3 have no effect on a catalytic activity of the enzyme.

Protection of cultured endothelial cells from hydrogen peroxide-induced injury by antibody-conjugated catalase.

The cytoprotective features of catalase-antibody conjugate prepared by covalent conjugation of catalase to rabbit antibody against mouse IgG is described. The bifunctional cross-linking agent m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) was used for conjugation. Functionally active conjugate binds specifically to the plastic-adsorbed mouse IgG and to the surface of live human endothelial cells treated with mouse antiserum against human endothelial cells. Up to 4 units of catalase activity can bind to 1 cm2 of the endothelial monolayer. The targeted catalase protects endothelial cells from cytotoxic action of hydrogen peroxide: the minimal cytotoxic concentration of H2O2 for protected cells is 80-times higher than for intact cells. This effect is attributed partly to local reduction of H2O2 concentration in the cell microenvironment. Targeted catalase was estimated to reduce H2O2 concentration 8-fold near the cell surface with respect to average total concentration.

Point mutation in the stalk of angiotensin-converting enzyme causes a dramatic increase in serum angiotensin-converting enzyme but no cardiovascular disease.

BACKGROUND: Angiotensin-converting enzyme (ACE) metabolizes many small peptides and plays a key role in blood pressure regulation. Elevated serum ACE is claimed to be associated with an increased risk for cardiovascular disease. Previously, two families with dramatically increased serum ACE were described, but no systematic survey of affected individuals was performed, and the molecular background of this trait is unknown. METHODS AND RESULTS: Eight families were identified with autosomal dominant inheritance of a dramatic (5-fold) increase of serum ACE activity. Strikingly, no clinical abnormalities were apparent in the affected subjects. Isolated blood cells were used for genetic and biochemical analysis. The level of ACE expression on the blood leukocytes and dendritic cells and total cell-associated ACE of the affected individuals was similar to that in nonaffected relatives; however membrane-bound mutant ACE was much more efficiently clipped from the cell surface compared with its wild-type counterpart. A point mutation causing Pro1199Leu in the stalk region of the ACE molecule cosegregates with the increase in serum ACE (LOD score, 6.63). CONCLUSIONS: A point mutation in the stalk region of the ACE protein causes increased shedding, leading to increased serum ACE, whereas cell-bound ACE is unaltered, and affected individuals exhibit no clinical abnormalities. These findings qualify the importance of serum ACE and establish a new determinant of ACE solubilization.

Right ventricular angiotensin converting enzyme activity and expression is increased during hypoxic pulmonary hypertension.

OBJECTIVE: To determine whether local cardiac angiotensin converting enzyme (ACE) expression is upregulated during the development of hypoxia-induced right ventricular hypertrophy. METHODS: ACE activity was measured in membrane preparations from the right ventricle and left ventricle plus septum in normoxic rats and animals exposed to chronic hypoxia for 8 and 14 days. Local cardiac ACE expression was studied by immunohistochemistry using a monoclonal antibody to ACE (9B9). RESULTS: In the normal rat heart, ACE expression was confined to vascular endothelium, the valvular endocardium, and localized regions of parietal endocardium. We found that the development of pulmonary hypertension and right ventricular hypertrophy were associated with 2.6- and 3.4-fold increases in membrane-bound right ventricular ACE activity by 8 and 14 days of hypoxia, respectively. Right ventricular ACE activity was positively correlated with the degree of right ventricular hypertrophy (r = 0.83, P < 0.001). In contrast, left ventricular plus septal ACE activity was significantly reduced by approximately 40 and 60% by 8 and 14 days of hypoxia, respectively, compared to controls. In the right ventricle of chronically hypoxic rats, immunohistochemistry demonstrated increased ACE expression in areas of myocardial fibrosis. Interestingly, increased ACE expression was noted in the right ventricular epicardium in chronically hypoxic rats. In the free wall of the left ventricle there was a significant reduction in the number of myocardial capillaries which expressed ACE in chronically hypoxic rats. CONCLUSION: Chronic hypoxia has a differential effect on left and right ventricular ACE activity and that the sites of altered ACE expression are highly localized. We speculate that locally increased right ventricular ACE activity and expression may play a role in the pathogenesis of right ventricular hypertrophy secondary to hypoxic pulmonary hypertension.

Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

CD143 in the development of atherosclerosis.

The expression of CD143 (angiotensin-I-converting enzyme, ACE) in cardiovascular diseases may be an important determinant of local angiotensin and kinin concentrations. Much of the experimental and clinical evidence suggests a crucial role for Ang II in fibrogenesis and the development of atherosclerosis. Therefore, we have studied the distribution of CD143 in atherosclerotic and non-atherosclerotic segments isolated from different parts of the human vascular tree, including aorta and coronary, carotid, brachial, renal, iliac and femoral arteries, and staged according to the AHA. Two hundred and thirty native and formalin-fixed specimens of 80 patients were analysed by sensitive APAAP-technique using ten different monoclonal and polyclonal antibodies to human CD143 and several controls. In non-atherosclerotic segments or intimal thickening, CD143 was found almost restricted to the endothelial cells of adventitial arterioles and small muscular arteries. In contrast, a striking accumulation of CD143 was detected in all early and advanced atherosclerotic lesions. This de-novo occurrence of CD143 within the intimal vascular wall was caused by spindle-shaped subendothelial cells with macrophagic/histocytic features, activated macrophages and foam cells. In addition, advanced lesions of atherosclerosis showed a marked neo-expression of CD143 in newly formed intimal microvessels. Hypocellular fibrotic plaques depleted in microvessels and macrophages showed only little CD143. The de-novo occurrence of CD143 was dependent on the stage of atherosclerosis but not on its particular localisation within the vascular system. This early and obligatory CD143 expression at an unusual vascular site may contribute to unusual tissue levels of angiotensins as indicated by co-localisation of immunoreactive Ang II. Thus, it may be an important pathogenetic step in the development of atherosclerosis and an established target for pharmacological prevention.

Immunotargeting of streptavidin to the pulmonary endothelium.

UNLABELLED: We have observed previously that monoclonal antibody to angiotensin-converting enzyme (Mab 9B9) accumulates selectively in the lung after intravenous injection. The objective of the present work is the development of a universal system for targeting of drug or radiolabel to the lung, using biotinylated Mab 9B9 and streptavidin. METHODS: Mab 9B9 was biotinylated with biotin succinimide ester (b-Mab 9B9), while streptavidin (SA) was radiolabeled with 125I. Interaction between b-Mab 9B9 and SA has been estimated in solid-phase radioassay. Radiolabeled SA was conjugated with b-Mab 9B9 or with b-IgG and injected intravenously in rats or perfused in isolated rat lungs. RESULTS: Radiolabeled b-Mab 9B9 biotinylated at biotin-to-antibody molar ratio 10 (b-Mab 9B9) retains its ability to accumulate in rat lungs after intravenous injection. Radiolabeled SA conjugated with b-Mab 9B9 accumulates in the lung tissue in perfused isolated rat lungs. About 20% of injected SA accumulates in the rat lung 1 hr after intravenous injection (localization ratio is 20, immunospecificity of the conjugate pulmonary uptake is 70). As compared with conjugate injection, stepwise intravenous injection of b-Mab 9B9 and radiolabeled SA leads to a marked reduction of SA pulmonary uptake. Maximal pulmonary uptake of Mab 9B9 has been observed 2-3 hr after intravenous injection, while 24 hr later, radioactivity in the lung was markedly reduced. In contrast to radiolabeled Mab 9B9 alone, radiolabeled SA conjugated with b-Mab 9B9 was retained in the lung for at least 48 hr. In concert with effective blood clearance of the conjugate, its prolonged lung retention leads to a marked increase in its lung-to-blood ratio: 80 for SA-b-Mab 9B9 versus 15-20 for Mab 9B9. CONCLUSION: Conjugation of Mab 9B9 with streptavidin enhances selective pulmonary uptake of the preparation, providing a background for intrapulmonary immunotargeting of various biotinylated agents.

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without any change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.

Blood clearance of radiolabeled antibody: enhancement by lactosamination and treatment with biotin-avidin or anti-mouse IgG antibodies.

Methods of rapid blood clearance of 111In-labeled mouse monoclonal antibody 9B9 against angiotensin-converting enzyme were studied. Indium-111-9B9 is specifically accumulated in rat lung, but its blood clearance is relatively slow and target-to-blood radioactivity ratio/g tissue (localization ratio) increases from 11 to 30 only 48 hr postinjection. Injection of second (anti-mouse immunoglobulin) antibodies results in slight (1.8-fold) increase of 9B9 localization ratio. Chemical modification of 9B9 aminogroups with lactose results in enhanced liver uptake and rapid blood clearance of antibody. Blood radioactivity level decreases tenfold, and as a result localization ratio increases threefold (up to 38 in 30 min). Injection of avidin following the injection of biotinylated 9B9 results in rapid clearance of blood radioactivity with increased uptake in liver and spleen. Lung uptake is not changed. Localization ratio increases fivefold over the avidin-untreated animal value. Implications of these approaches for various applications in immunoimaging are discussed.

Radioimmunoimaging of lung vessels: an approach using indium-111-labeled monoclonal antibody to angiotensin-converting enzyme.

A murine monoclonal antibody against human angiotensin-converting enzyme was radiolabeled with 111In via diethylenetriaminepentaacetic acid without substantial loss of antigen-binding capacity. This monoclonal antibody designated 9B9 cross-reacted with rat and monkey angiotensin-converting enzyme. Indium-111-labeled 9B9 selectively accumulated 10-20 times greater in the lung than in blood or other organs following intravenous administration in rats. Kinetics of lung accumulation and blood clearance were studied for 111In-9B9-antibody and compared to that of 125I-labeled 9B9 in rat. Highly specific accumulation of 111In-9B9-antibody in the lung of Macaca Rhesus monkeys after intravenous injection was monitored by gamma-imaging. Images of 111In-labeled antibody 9B9 biodistribution in monkey lung noticeably differ from the images of biodistribution of 99mTc-labeled albumin microspheres. This difference may provide information concerning the state of the endothelium of lung capillaries, which is different from the blood flow characteristics determined with routine microsphere technique.