Biomedical subjects

S J Kennel

Publications and source records attributed to S J Kennel.

At least 19 recordsLinked to original sources

PubMed1995

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases.

The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N-terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses determined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E280 nm1% of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2 x 10(8) and 3.7 x 10(8) l/M/min at 37 degrees C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8 x 10(-4) M and 1.4 x 10(-3)/s for the human protein, 2.3 x 10(-4) M and 1.1 x 10(-3)/s for the wild-type mouse protein, and 2.1 x 10(-4) M and 1.4 x 10(-3)/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50 degrees C in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 micrograms/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins. Immunoprecipitation (69%) and affinity constant (19.4 nM) of human MGMT with a human-specific monoclonal antibody 4.A1 significantly discriminated the human protein from either of the mouse proteins.

Amino Acid Sequence↗

PubMed1994

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human lung carcinoma cells of different histotypes.

Structural and functional analyses of several integrin heterodimers were performed in non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines. The expression of beta 1, beta 3, beta 4, and beta 5 heterodimers was evaluated at protein and mRNA levels. By flow cytometry and immunoprecipitation experiments we demonstrate that NSCLC cells (A549 adenocarcinoma and DG 3 large cell carcinoma) coexpress integrin heterodimers composed of beta 1, beta 3, beta 4, and beta 5 subunits, whereas SCLC cells (AE2 and H69) express only beta 1 integrin heterodimers. Northern blot experiments confirmed immunochemical analysis: SCLC cells in contrast to NSCLC cells express only the mRNA coding for the beta 1 subunit. These data indicate that in lung carcinoma cells the diversity in the integrin repertoire depends upon differential gene expression. The functionality of integrin receptors has been studied using antibody blocking experiments. Data reported demonstrate that the alpha 6 beta 1 integrin is a laminin receptor in either SCLC or NSCLC cells. An antibody to the beta 4 subunit partially inhibits the adhesion of adenocarcinoma cells to lamin but does not block lamin adhesion of large cell carcinoma cells, even though alpha 6 beta 4 complexes are expressed on both cell types. Two antisera to vitronectin receptors inhibit the adhesion of NSCLC cels to both vitronectin and fibronectin. The same antisera inhibit the adhesion of SCLC cells only to laminin, indicating that the alpha v beta 1 integrin might function in these cells as laminin receptor.

Carcinoma, Non-Small-Cell Lung↗

PubMed1993

Production and immunodiagnostic applications of antihuman light chain monoclonal antibodies.

Hybridomas producing antihuman light chain monoclonal antibodies (MoAbs) were derived from fusion of SP2/O mouse myeloma cells with splenic lymphocytes from mice repeatedly immunized with purified kappa- and lambda-type Bence Jones proteins representative of the major V kappa (V kappa I, V kappa II, V kappa III, V kappa IV) and V lambda (V lambda I, V lambda II/V, V lambda III, V lambda IV, V lambda VI) subgroups or gene families. Monoclonal antibodies were obtained that had specificity for constant-region (CL) determinants common to all kappa or lambda light chains (C kappa and C lambda, respectively) as well as for variable-region (VL) epitopes unique to each of the V kappa or V lambda subgroups. The capability of these reagents to recognize CL and VL determinants on monoclonal immunoglobulin (Ig) molecules was demonstrated in fluid-phase antigen-capturing enzyme-linked immunosorbent assay (ELISA), solid-phase ELISA, and immunoblotting. In addition, these antilight chain MoAbs were used to establish immunocytochemically the kappa or lambda type and VL-subgroup nature of light chains expressed by the cytoplasmic Ig of monoclonal plasma cell and surface Ig of B-lymphocyte populations, respectively. These antibodies facilitated the immunohistochemical detection and characterization of light-chain-associated amyloid (AL amyloid) and other types of light-chain-related tissue deposits. Furthermore, the anti-CL-specific MoAbs were used to measure serum and urinary Ig kappa and Ig lambda concentrations. Quantification of Bence Jones protein excretion, even in the presence of other urinary proteins, was possible using the highly sensitive anti-C kappa and anti-C lambda MoAbs reactive only with free light chains. The ability to identify and characterize, through the use of these antihuman light chain MoAbs, light-chain-related epitopes at the protein, cellular, and tissue level has clinical importance in the diagnosis and treatment of patients with monoclonal plasma cell and related B-cell immunoproliferative diseases.

Amyloidosis↗

PubMed1992

Targeted therapy of athymic mice bearing GW-39 human colonic cancer micrometastases with 131I-labeled monoclonal antibodies.

The therapeutic potential of radiolabeled antibodies is usually evaluated in experimental animal models bearing s.c. xenografts. We have established a micrometastatic model of the GW-39 human colonic carcinoma in the nude mouse lung (J. Natl. Cancer Inst., 83: 627-632, 1991) and presented preliminary findings on the efficacy of a 131I-anticarcinoembryonic antigen (CEA) antibody in this model. We now extend our observations on the use of radioiodinated labeled monoclonal antibodies (MAbs) to treat multiple small tumor nodules. Biodistribution and dosimetry analysis was performed for intact and F(ab')2 of NP-4 anti-CEA IgG, Mu-9 anti-colon-specific antigen IgG, isotype-matched irrelevant anti-AFP IgG, and intact MAb 34A anti-lung endothelial IgG antibody. Comparisons were made for rad dose delivered to small s.c. tumors, normal lung, lung with tumor nodules, and isolated tumor nodules. Survival curves were generated for tumor-bearing animals treated 1, 7, or 14 days after tumor cell implantation with these antibodies using the maximal tolerated dose for intact antibodies (275 microCi) and for F(ab')2 fragments (1.2 mCi). The studies established the following observations: (a) in contrast to previous results in a bulky tumor model in hamsters, intact antibodies are more therapeutic than MAb fragments for both NP-4 and Mu-9; (b) tumor nodule size, even on the microscopic level, affects therapeutic outcome; antibodies were more effective when administered 7 days postimplantation (mean nodule diameter, 150 microns) compared with treatment 14 days postimplantation (mean nodule diameter, 750 microns); (c) administration of radioiodinated Mu-9 was exquisitely effective on single avascular tumor cells that had seeded in lung; irrelevant antibody was minimally radiotoxic; (d) as in the bulky disease model, the anti-colon-specific antigen p antibody delivers a higher rad dose than the anti-CEA antibody and is significantly more therapeutic in the micrometastasis model; (e) a higher affinity anti-CEA antibody (MN-14) recognizing the same epitope on CEA as NP-4 was equally therapeutic; (f) the use of MAb directed against the lung endothelium was not as therapeutic as a tumor-associated antibody; and (g) all tumor-associated antibodies were more efficacious than administration of the maximal tolerated dose of 5-fluorouracil and leucovorin in this human tumor-xenograft model. These results provide further support for the use of radioimmunotherapy in the handling of minimal disease, probably as part of an adjuvant treatment regimen.

Animals↗

PubMed1992

Thrombomodulin is preferentially expressed in Balb/c lung microvessels.

Previously, two rat monoclonal antibodies where developed which bind distinct epitopes on a murine glycoprotein, P112, which is expressed primarily in lung capillary endothelium. In this paper we show that P112 is identical to the endothelial anticoagulant protein, thrombomodulin (TM). Several lines of evidence support this conclusion. First, amino acid analysis of P112 shows a high degree of homology to TM, and both molecules exhibit the same mobility in gel electrophoresis. Second, P112 and TM share reactivity for two different monoclonal antibodies. Third, purified P112, like TM, acts as a cofactor for protein C activation. Finally, two cDNA clones identified with P112 polyclonal antiserum contain sequence identity with the known TM cDNA sequence. Quantitative analysis of TM (P112) expression using a two-site monoclonal antibody assay demonstrates that significantly higher levels of TM are found in lung in comparison with other highly vascularized organs, i.e. the kidney and liver. Quantitative Northern blot data coincides with the two-site assay data and demonstrates that the high level of TM expression in lung is not due to preferential binding of the monoclonal antibodies to lung TM but rather to increased production of TM mRNA in the lung relative to other highly vascularized organs. It is suggested that expression of TM is highest in cells from continuous endothelium.

Animals↗

PubMed1992

Effects of target antigen competition on distribution of monoclonal antibody to solid tumors.

Monoclonal antibody (MoAb) 11A and MoAb 13A recognize normal murine cell surface glycoproteins, which are also expressed in high concentrations on Line 1 lung carcinoma. Studies were initiated to examine the competition in vivo for radiolabeled MoAb between sites on the tumor versus sites on normal tissue. Quantitative 2-site assay of the alpha 6 beta 4 integrin recognized by MoAb 11A showed that major sites of expression are tumor, intestine, and skin. Microdistribution studies show that at doses of 125I-labeled MoAb 11A less than the total body antigen load, the MoAb bound to beta 4 endothelial cells with little extravasation to epithelial sites. As the MoAb dose was increased, and endothelial sites became saturated, deposition at epithelial sites including skin and tumor became apparent. Quantitative radioimmunoassay with MoAb 13A, recognizing CD44 (P100), demonstrated major sites of expression as tumor, intestine, liver and, to a lesser extent, spleen and skin. Microdistribution studies at low doses of 125I-labeled MoAb showed deposition mainly in the liver and spleen sinusoids, whereas higher doses were necessary to maximize MoAb accumulation in tumor. The rapid access of MoAb to target antigen is the most important parameter in efficient localization of MoAb. Access of antigen outside the vascular space is regulated at least in part by the permeability of the endothelial barrier. Of the organs studied, antigen accessibility increases in the following order: lung epithelium less than skin epithelium less than intestine and uterus epithelium less than epithelial tumors less than liver and spleen sinusoids less than intervascular sites. Antigens in easily accessible sites interact with MoAb first and must be saturated before MoAb will penetrate to less accessible areas. Thus, the extent of competition of antigen for available MoAb depends not only on the amount of antigen, but also on the site at which it is expressed. Doses that achieve maximum binding to tumor sites can be predicted if accurate target antigen quantities and sites of expression are known.

Animals↗

PubMed1992

A rat monoclonal antibody specific for murine type 1 pneumocytes.

A rat monoclonal antibody (MAb), 411-52, that binds specifically to murine pulmonary alveolar type 1 cells was developed. The cell-binding specificity of MAb 411-52 was assessed by light microscopy on immunoperoxidase-labeled tissue sections, electron microscopy on immunogold-labeled tissue blocks, and by flow cytometric analysis and fluorescence-activated cell sorting of immunofluorescently labeled cells enzymatically dissociated from murine lungs. The epitope recognized by MAb 411-52 was first detected in immunoperoxidase-stained sections of neonatal lungs of mice approximately 3 weeks after birth. In adult mice, the MAb 411-52-directed, immunoperoxidase-staining pattern was uniform throughout the lung parenchyma, was restricted to the luminal surfaces of alveoli, and was absent from type 2, endothelial, and interstitial cells, as well as from the epithelial cells of conducting airways. Electron microscopic analysis of immunogold-labeled lung tissue confirmed the type 1 cell binding specificity of MAb 411-52. Analysis by multiparameter, laser flow cytometry indicated that MAb 411-52 binds to 4.6 +/- 0.5% (mean +/- SD) of enzymatically dissociated cells from the lungs of normal adult mice. The absence of immunogold-labeling of type 2 cells suggested that the epitope recognized by MAb 411-52 might be a differentiation marker for the type 1 cell phenotype. With this MAb and standard immunohistochemical techniques, it is possible to visualize directly type 1 cells in paraffin sections.

Animals↗

PubMed1992

A monoclonal antibody produced against a rat esophageal carcinoma cell line reacts with an integrin-like molecule expressed by rat epithelial cells.

A monoclonal antibody, designated MAb-5A IIgG1), was generated against a tumorigenic rat esophageal epithelial cell line, B-2T. MAb-5A reacted with a series of non-tumorigenic and tumorigenic epithelial cell lines derived from F-344 rat esophagi or tracheas. However, the highest level of antigen expression was detected on tumorigenic rat epithelial cell lines. A trace amount of antigen was detected in primary cultures of normal rat epithelial cells derived from either esophagi or tracheas. MAb-5A did not react with rat fibroblasts. MAb-5A reacted with B-2T derived tumor tissues propagated in vivo but showed only slight reactivity with normal rat esophageal epithelial tissues. Cell surface radioiodination, extraction and immunoprecipitation experiments were conducted to characterize the antigen molecule. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed two protein bands with mobilities corresponding to approximately 140 and 120 kDa, respectively. Under reducing conditions the 140 kDa band shifted to approximately 120 kDa whereas the 120 kDa band shifted upward to approximately 130 kDa. The non-reduced/reduced mobilities of these bands suggest that they may be members of the integrin family of matrix receptors. A polyclonal antibody to the beta 1 subunit immunoprecipitated two similar bands detected by MAb-5A, further suggesting that the antigen complex may be related to members of the integrin superfamily.

Animals↗

PubMed1992

The beta 4 subunit of the integrin family is displayed on a restricted subset of endothelium in mice.

More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The alpha 6 beta 4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the beta 4 subunit in mice. Sequence analysis of a cDNA clone coding for this epitope localizes reaction of the mAb to a portion about half way through the extracellular domain at the beginning of the cysteine-rich region. Sites of beta 4 expression in mice were detected by autoradiographic analysis of tissues collected from mice 24 or 48 h after intravenous injection of 125I-labeled mAb 346-11A. This non-quantitative technique emphasizes detection of antigen exposed in the vascular space. These data show that in addition to the epithelia of several organs, the endothelia of intermediate vessels throughout the body are sites of beta 4 expression. In particular, endothelium of larger vessels but not capillaries in lung, vessels in thymus, spleen and Peyer's patches, and portal vessels but not the central veins of the liver, are positive. The expression of the beta 4 integrin in blood vessels may indicate a specialized function for beta 4 at these sites that is distinct from its role in hemidesmosome-mediated attachment.

Animals↗

PubMed1991

Microdistribution of specific rat monoclonal antibodies to mouse tissues and human tumor xenografts.

Detailed evaluations of the microdistribution of 125I-labeled monoclonal antibodies (MoAbs) to normal tissue antigens were conducted in BALB/c mice. MoAb 273-34A, which binds to a target molecule on the lumenal surface of lung endothelial cells, localizes quickly and efficiently throughout the lung vasculature. MoAb 133-13A, which binds to an antigen on macrophage-like cells expressed in nearly equal amounts in lung, liver, and spleen, localizes most efficiently to spleen and less well to liver and lung. The microdistribution of MoAb 133-13A in liver and spleen is consistent with the antigen distribution in these organs, but in the lung a more diffuse microdistribution is observed, indicating poor access of MoAb to the antigen-positive alveolar macrophages. These findings are consistent with the hypothesis that tight endothelium (lung) represents a significant barrier to extravasation of MoAb into tissue while fenestrated (spleen) and sinusoidal (liver) endothelium are more easily penetrated. In human tumor bearing nu/nu mice, the microdistribution of MoAb to the beta 4 and alpha 6 subunits of integrin was studied. These MoAbs do not cross-react with murine integrins and thus are tumor-specific in the nu/nu mouse model. Localization of 125I-labeled MoAb 450-11A, which reacts with an intercellular domain of beta 4 integrin, is very weak and diffuse. All MoAbs to extracellular domains (mouse 450-9D, 450-30A1, and rat 439-9B) localize well to the tumor. Microdistribution of these MoAbs in the 3 different tumors is nonuniform with heavy distribution near the blood vessels, whereas antigen distribution as determined by immunoperoxidase shows a much more uniform pattern throughout the tumors. In experiments with 125I-labeled MoAb 439-9B F(ab')2, the nonuniform pattern of distribution was not changed. Gross and microdistribution of different doses of 125I-labeled MoAb 439-9B were studied. The percent of injected dose per g of MoAb in the tumor at 48 h did not vary significantly (P greater than 0.1) up to a dose of 500 micrograms/mouse, and active MoAb was recovered in comparable amounts in the serum from animals in all doses. In contrast, the microdistribution of MoAb at the high dose was different than that at low doses. At doses up to 100 micrograms/mouse, a perivascular pattern was obtained, whereas at 500 micrograms/mouse the 125I-labeled MoAb was distributed nearly evenly throughout the tumor. These data indicate that high doses of MoAb penetrate deeply into portions of the tumor that are distant from blood vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.

Animals↗

PubMed1990

Integrin (alpha 6/beta 4) expression in human lung cancer as monitored by specific monoclonal antibodies.

In this study, the expression of the alpha 6/beta 4 integrin complex was analyzed in human lung carcinomas both in vitro and in vivo, using two monoclonal antibodies which recognize the integrin subunits alpha 6 (Mab 135-13C) and beta 4 (Mab 439-9B). Immunoprecipitation patterns obtained from established human lung carcinoma cell lines demonstrated that the alpha 6 and the beta 4 subunits were differentially expressed in carcinomas of different types. The alpha 6 subunit was expressed in all the cell lines tested (squamous cell carcinoma A431, adenocarcinoma A549, large cell carcinoma DG3, and small cell carcinoma AE2). The beta 4 subunit was expressed in non-small cell cancer lines but was not detectable in the small cell cancer line tested. Using a quantitative two-site assay, we measured the concentration of the alpha 6/beta 4 integrin in matched biopsies from primary lung tumors and from normal lung. These studies confirmed that the complex was differentially expressed in non-small versus small cell lung cancers and that it was also detectable in lysates from normal lung at low levels. The highest levels of alpha 6/beta 4 were found in moderately differentiated squamous cell carcinomas. By immunohistochemistry, the beta 4 subunit was detectable in all the squamous cell carcinoma and adenocarcinomas tested (a total of 59), but not in 10 small cell cancers. The patterns of immunoreactivity were consistent with the expected distribution of membrane glycoproteins and, in some squamous cell carcinomas, were suggestive of the localization displayed by molecules involved in carcinoma-stroma interaction. Immunohistochemical staining indicated that beta 4 was also expressed in specific types of nonrespiratory pulmonary epithelial cells.

Adenocarcinoma↗

PubMed1990

Characterization of in vivo immunoliposome targeting to pulmonary endothelium.

Two rat monoclonal antibodies, 34A and 201B, which specifically bind to a surface glycoprotein (gp112) of the pulmonary endothelial cell surface, have been coupled to unilamellar liposomes of approximately 0.25 microns in diameter. The 34A- and 201B-liposomes (monoclonal antibodies 273-34A and 411-201B, respectively), but not antibody-free liposomes and liposomes coupled to 14, a nonspecific monoclonal antibody, accumulate efficiently (approximately 30% injected dose) in the lung of mice which have been injected via the tail vein. Immunoliposome targeting to lung is demonstrated both by using a 125I-labeled lipid marker and an entrapped water-soluble marker. Lung accumulation of 34A-liposomes is completely blocked by a preincubation of free antibody 34A, but not antibody 14, indicating that the immunoliposome accumulation at the target site is immunospecific. Time course studies have revealed that 34A-liposomes bind to lung antigens within 1 min after injection, indicating that the target binding takes place during the first few passages of immunoliposomes through the lung capillary bed. Unbound immunoliposomes are taken up by liver and spleen within 3-5 min after injection. The level of lung accumulation increases significantly as the protein:lipid ratio of the immunoliposome increases. Approximately 50% of injected dose is accumulated in lung for 34A-liposomes, with an average of 935 antibody molecules per liposome. Immunoliposomes of larger size accumulate in lung more significantly than those of smaller size. Injection with higher doses also enhances the level of lung accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Rat monoclonal antibody distribution in mice: an epitope inside the lung vascular space mediates very efficient localization.

MoAb to an epitope on lung endothelial cells accumulates rapidly in the lung resulting in localization ratios of over 100. MoAb to a macrophage antigen found in spleen and lung has maximum localization ratios of 24 and 5, respectively, while MoAb specific for a human tumor grown in nude mice has a maximum ratio of about 4. Epitope concentrations in target organs (300-600 ng/mg protein) are comparable in all three systems, indicating that the MoAb to endothelium is efficient in localization to the lung.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed1990

Lipid composition is important for highly efficient target binding and retention of immunoliposomes.

By taking advantage of a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, we have prepared liposomes containing various amounts of antibody molecules (immunoliposomes). These immunoliposomes accumulate specifically in the lung when injected i.v. Two lipid compositions were used: phosphatidylcholine/cholesterol/phosphatidylserine (PS), 10:5:1 (mol/mol), a composition that allows liposomes to be readily taken up by the reticuloendothelial system (RES) (liver and spleen), and phosphatidylcholine/cholesterol/ganglioside GM1, 10:5:1 (mol/mol), a composition that allows liposomes to avoid or delay the RES uptake (the so-called stealth liposomes). Although an increase in the number of antibody molecules per liposome was accompanied by an increased level of lung binding of the immunoliposomes, differences due to the lipid composition were more profound. For example, stealth immunoliposomes containing an antibody/lipid ratio = 1:37 (wt/wt) accumulated in lung to a level of 60% of the injected dose, whereas PS-containing immunoliposomes with a higher antibody/lipid ratio (1:8) only accumulated 50% of the injected dose in the lung. Conjugation of antibody to the stealth liposome did not increase the rate of liposome uptake by liver; this rate was approximately 10-fold lower than that of the PS-containing liposomes without antibody. Stealth immunoliposomes with high antibody content also showed long retention in the lung. The t1/2 of lung residence for the stealth immunoliposomes with an antibody/lipid ratio = 1:11 (wt/wt) was approximately 24 hr. The fact that stealth immunoliposomes showed a longer retention time in the lung than the PS-containing immunoliposomes of similar antibody content suggests that macrophages may play a role in the removal of the bound immunoliposomes from the pulmonary endothelium. Alternatively, dissociated stealth immunoliposomes may reenter the circulation and rebind to the lung target, causing an apparent slow overall dissociation rate. These results can be understood on the basis of two competing kinetic processes: lung binding whose rate is directly proportional to the antibody content of the immunoliposomes and uptake by RES whose rate is significantly reduced in the case of the stealth liposomes. Even for a modest level of antibody content, the half-life for target binding of immunoliposomes was significantly shorter than the half-life of liver uptake of the liposomes, resulting in a favorable target binding. Significant immunoliposome binding to the lung is not due to the fact that tail vein-injected liposomes flow through the lung capillary bed before they encounter the liver, because portal vein-injected immunoliposomes showed the same rate and extent of target binding as the tail vein-injected ones.

Animals↗

PubMed1990

Second generation monoclonal antibodies to the human integrin alpha 6 beta 4.

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.

Animals↗

PubMed1990

The alpha 6 beta 1 (VLA-6) and alpha 6 beta 4 protein complexes: tissue distribution and biochemical properties.

A member of the integrin family, the alpha 6 beta 4 complex was previously identified on human and mouse carcinoma cell lines by using a rat monoclonal antibody to alpha 6. Here we describe two monoclonal antibodies that recognize epitopes on the beta 4 subunit of the human and mouse alpha 6 beta 4 complexes. The monoclonal antibodies against beta 4 were able to preclear alpha 6 beta 4, but not alpha 6 beta 1 from cell line extracts. A substantial fraction of the total beta 4 subunits present on the cell surface was not associated with alpha 6, as it could not be removed by anti-alpha 6 antibodies, but remained precipitable with anti-beta 4 antibodies. There was no evidence for novel alpha subunits associated with beta 4. The alpha 6 subunit consists of disulfide-linked heavy and light chains. The variability in size of these two chains from different cell types is largely due to differences in modifications of N-linked glycans. Additional heterogeneity may be caused by differential proteolytic cleavage of the alpha 6 precursor. Immunoperoxidase staining of tissue sections of neonatal and adult mice revealed that beta 4 expression is limited to epithelial tissues and peripheral nerves. The alpha 6 subunit has a wider distribution that includes all tissues and cells stained by antibodies against beta 4. Cells and tissue that are positive for alpha 6, but negative for beta 4, may express the alpha 6 beta 1 complex.

Animals↗

PubMed1989

Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions.

Monoclonal antibody was conjugated to N-glutaryl-phosphatidylethanolamine in the presence of octylglucoside by using N-hydroxysulfosuccinimide as a carboxyl-activation reagent. The conjugated antibody was then incorporated into liposomes by a simple dialysis method. The method is mild and is compatible with various lipid compositions of the liposomes. We have prepared immunoliposomes containing a lung endothelium-specific monoclonal antibody and showed excellent target binding (approximately 75% injected dose) of the immunoliposomes in mouse. Immunoliposomes can be prepared to contain other acidic lipids such as phosphatidylserine and various amounts of cholesterol. The presence of 20% or more cholesterol in liposomes resulted in high level of target binding. We have used in these experiments a new radioactive lipid-phase marker, 111In-DTPA-SA, which was very stable in vivo. The halflife of clearance in mouse exceeded 3 weeks.

Animals↗