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L J Foote

Publications and source records attributed to L J Foote.

At least 19 recordsLinked to original sources
PubMed2002

Monoclonal antibodies to CD44 epitopes on mouse endothelium.

CD44 is a widely expressed, plasma membrane protein. Many molecular forms of CD44 are possible as it is encoded by a gene with multiple exons that can be alternatively spliced and its deduced protein sequence contains numerous glycosylation sites. Through its role as an adhesion molecule, CD44 is involved in many and diverse biological processes, including angiogenesis, lymphogenesis, wound healing, inflammation, and cancer metastasis. We have developed a new panel of rat monoclonal antibodies (MAbs) to murine CD44 by immunization with mouse lung endothelial cells (LEII cells). The antibodies were characterized using immunoprecipitation, mass spectrometry, competition binding, and cross Western blot experiments with MAb 133-13A, which recognizes CD44 expressed on tumor cells. The new MAbs recognize three distinct epitope groups. MAbs 531-2A and 531-32A compete for binding with the MAb 133-13A that was described previously. MAb 531-30A identifies a CD44 epitope found on low molecular weight forms expressed in vivo, while MAb 531-22A appears to recognize an epitope specific for endothelial cells. This novel panel of anti-CD44 antibodies has potential for investigating the role of CD44 in disease pathogenesis models in the mouse. They may be particularly useful for examining the role of endothelial cells in these models.

Animals↗
PubMed2000

Labeling and distribution of linear peptides identified using in vivo phage display selection for tumors.

To develop targeting molecules to be used for vascular targeting of short half-lived alpha-emitters for radioimmunotherapy, linear peptide phage display libraries were selected in vivo for binding to IC-12 rat tracheal tumors growing in severe combined immune deficient mice. After three rounds of selection, 15 phage clones were analyzed for DNA sequence, and the deduced translation products of cDNA inserts were compared. Three consensus sequences were chosen from three separate experimental selection series and peptides of these sequences with added -gly-gly-tyr were obtained. Peptides were radiolabeled on tyrosine with (125)I and the biodistribution in tumor-bearing mice was determined. The radioiodinated peptides were stable in vitro and when injected in tumor-bearing mice approximately 3.0 %ID/g accumulated in the tumor; however, much of the (125)I was found in the gastrointestinal tract and thyroid, indicative of dehalogenation of the labeled peptide. Radiolabeling peptide 2 with N-succinimidyl-3-(125)I-iodobenzoate resulted in faster excretion, which in turn resulted in lower levels in tumor and other organs, especially thyroid and gastrointestinal tract. Peptide 2 was derivatized with the bifunctional isothiocyanates of cyclohexyl-B diethylenetriaminepentaacetic acid (DTPA) or CHX-A" DTPA by direct conjugation or with a hydroxylamine derivative of 1B4M-DTPA (2-(p-[O-(carboxamylmethyl)hydroxylamine]benzyl)-6-methyl-diethylenetriamine-N,N,N',N",N"-pentaacetic acid ) coupled at the N-terminus. The primary molecular species in the conjugated products were shown by mass spectrometry to have one DTPA per peptide. Peptide chelate conjugates were radiolabeled with (213)Bi and the products tested for biodistribution in tumor-bearing mice. The data show that chelation of (213)Bi to peptides was accomplished by both the direct method of DTPA attachment and by the method using the linker at the N-terminus. Only small amounts of peptide accumulated at tumor sites. We conclude that phage display is a powerful tool to select peptides with restricted binding specificity; however, the peptides isolated to date do not bind with high retention to tumor sites in vivo.

Amino Acid Sequence↗
PubMed1999

Analysis for TNF-alpha using solid-phase affinity capture with radiolabel and MALDI-MS detection.

Screening of mutant mice for subtle phenotypes requires sensitive, high-throughput analyses of sentinel proteins in functional pathways. The cytokine TNF-alpha is upregulated during inflammatory reactions associated with autoimmune diseases. We have developed a method to monitor the concentration of TNF-alpha under physiological conditions. TNF-alpha is captured, purified, and concentrated using monoclonal antibody-coated microbeads. The capture is efficient (> 80%) and can be used in the concentration range < 100 pg/mL to > 50 ng/mL, as determined by detection of 125I-labeled TNF-alpha. The bead capture of TNF-alpha can be combined with direct detection by MALDI-MS for sample concentrations of > 10 ng/mL. TNF-alpha can be captured and detected from diluted mouse serum, with minimal interferences observed in the MALDI spectrum. This method is adaptable to high-throughput sample handling with microfluidic devices and automated mass spectrometric analysis.

Animals↗
PubMed1999

Combination vascular targeted and tumor targeted radioimmunotherapy.

Rat MAb 201B, which binds to murine thrombomodulin, can deliver up to 50% of the injected dose of attached radioisotopes to the lung vascular endothelium. We have shown previously that intravenous injection of about 30 microCi of 213Bi-MAb 201B, which delivers about 15 Gy of alpha irradiation to the lung, is capable of eradicating small lung colonies (500-1000 cells) of the mammary tumor line, EMT-6. Larger tumors (> 5000 cells) were not completely cured by this vascular targeted radioimmunotherapy (VT-RAIT) approach. We reasoned that VT-RAIT might make the lung vessels serving the tumor cells more permeable, allowing MAb targeted to the tumor cells to extravasate more readily and mediate more efficient standard radioimmunotherapy (RAIT). Distribution experiments with the tumor targeted MAb 13A (RAIT MAb), following VT-RAIT, did not demonstrate a large increase in tumor uptake; however, microautoradiography did indicate that MAb 13A was distributed more evenly throughout the tumor when administered after VT-RAIT. Therapy experiments on lung tumors of approximately 5000 cells each, combining 213Bi-MAb 201B (VT-RAIT) with 213Bi-MAb 13A (RAIT) 24 hours later, resulted in a better outcome (3 cured/10 at risk) than for control groups: RAIT only (0/10), VT-RAIT only (1/10), or no therapy (0/10). RAIT therapy delivered 48 hours after VT-RAIT had no apparent benefit. 213Bi-MAb 201B VT-RAIT followed by 90Y-MAb 13A Fab' RAIT showed only a slight improvement in tumor cures (2/10) over that in control groups: (0/9), (0/10), (0/10), respectively. These results suggest that optimal timing, dosage, and choice of MAb for RAIT should enhance the double MAb therapy approach significantly.

Animals↗
PubMed1999

Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells.

Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.

Alpha Particles↗
PubMed1998

Monoclonal antibody to rat CD63 detects different molecular forms in rat tissue.

From mice immunized with rat endothelial cell membranes, we isolated several hybridomas secreting monoclonal antibodies (MAbs) to a 45-kDa glycoprotein expressed on the surface of cultured cells. One of these antibodies, 523-14A, was purified and used for immunoaffinity chromatography, Western blotting, and immunohistochemistry. The glycoprotein containing the antigen for MAb 523-14A, gp45, was isolated from rat lung endothelial cell membranes using wheat germ agglutinin and antibody affinity chromatography sequentially. Mass spectrometry of tryptic peptides from gel purified bands identified gp45 as rat CD63, a member of the transmembrane-4 superfamily. Western blot analyses of tissues from F344 rats showed that kidney, spleen, uterus, and ovaries expressed CD63 at high levels. Thymus, salivary gland, testicles, intestines, pancreas, and adrenals expressed lower amounts. Tissue cell types expressing CD63 were also examined and the results showed that, in addition to the expected expression on lymphoid cells, CD63 was expressed on many epithelial and muscle cells as well. The mobility of CD63 on SDS-PAGE varied widely, indicative of molecular masses ranging from 45 kDa in some tissues to nearly 60 kDa in others.

Animals↗
PubMed1993

Sequence of a cDNA encoding the beta 4 subunit of murine integrin.

A cDNA coding for the beta 4 subunit of murine integrin (m beta 4) has been cloned and sequenced using mRNA from a murine lung carcinoma as the template. The 5' sequence contains two AUG codons, the second of which initiates synthesis of the mature protein. The cDNA sequence has an open reading frame coding for 1748 amino acids (aa), including a signal peptide, cysteine-rich region, serine- and threonine-rich region, transmembrane domain, and a cytoplasmic domain of over 1000 aa. Overall, the deduced m beta 4 aa sequence has 88% identity with the human beta 4 subunit (h beta 4) sequence deduced from the sequence of placental mRNA. Reverse transcriptase-polymerase chain reaction using primers flanking splice sites for two variant forms of h beta 4 transcripts provided evidence for alternate splicing of RNA in the murine spleen and to a lesser extent in the skin, uterus, and thymus but was found at only one of the two alternative sites. Five potential glycosylation sites present in the extracellular domain of h beta 4 are conserved in m beta 4. One tyrosine in the terminal region of the cytoplasmic domain (position 1600) is conserved between m beta 4 and h beta 4 and has the consensus sequence for tyrosine phosphorylation. Finally, a genomic restriction map of m beta 4 shows that the gene is about 40 kb in length. No restriction-fragment length polymorphisms were detected between BALB/c liver and BALB/c lung carcinoma DNA.

Alternative Splicing↗
PubMed1993

CD44 expression on murine tissues.

CD44 is a cell surface glycoprotein found on lymphoid and epithelial cells. Its primary function on lymphocytes and macrophages is to mediate interaction with endothelium, while its function on epithelial cells is not known. The protein has many different forms, generated by alternative mRNA splicing and by post-translational modification, which may mediate different functions. During previous work on murine lung tumor cells, mAb 133-13A was isolated and shown to recognize a surface glycoprotein, P100, of 90-100 x 10(3) M(r). Amino acid sequence analysis of purified P100 indicates that it is CD44. Since few data exist to indicate which forms of CD44 are present in different normal tissues, mAb 133-13A was used to analyze CD44 expression in mouse tissue. Quantitative data on the distribution of CD44(P100) in mice show that spleen, thymus, liver, intestine, uterus and choroid of the eye are major sites of expression. In addition, epithelia of adrenals, esophagus and trachea are CD44(P100) positive. Previous work on human cell lines has implicated a high molecular mass (130-160 x 10(3) M(r)) form of the glycoprotein as the form expressed in epithelial cells and carcinomas. Isolation of CD44 proteins from lymphoid tissues in the mouse indicate that, as in human lymphoid tissue, the low molecular mass form (80-90 x 10(3) M(r)) is predominately expressed. These data show that both small (approximately 81 x 10(3) M(r)) and large forms of the glycoprotein are expressed in basal epithelia of esophagus and trachea and in salivary gland, while only the small form is expressed in epithelium of the adrenal cortex and in the murine lung and mammary carcinomas studied. While these data cannot distinguish between specific splice variants, they show that the large forms of CD44 are minor components in normal tissue and seem to be found only in basal epithelium. The CD44 of low M(r) found in epithelial tissues is probably associated with lymphoid cell types in the tissues.

Amino Acid Sequence↗
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↗
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↗
PubMed1989

Analysis of the tumor-associated antigen TSP-180. Identity with alpha 6-beta 4 in the integrin superfamily.

The tumor-associated antigen complex, TSP-180, was previously defined in carcinoma cell lines and found to be expressed in higher amounts in tumor than in normal tissue. Here, the mouse TSP-180 complex is shown to consist of three related proteins (bands 1, 2, and 3) associated with a distinct protein (band 5) that is probably derived from a precursor protein (band 4). All of these proteins are cell surface glycoproteins, and the largest protein (band 1) can be readily labeled with 32PO4. The mouse TSP-180 complex described here strongly resembles the recently described human integrin alpha 6-beta 4 complex. This homology was confirmed using two distinct rat anti-alpha 6 monoclonal antibodies, each of which recognized both human alpha 6-beta 4 and mouse TSP-180 complexes. Furthermore, the TSP-180 band 5 protein (mouse alpha 6) had an N-terminal sequence identical to that of human alpha 6. Finally, two different monoclonal antibodies are described, 346-11A and 439-9B, which directly recognize the multiple forms of mouse and human beta 4 proteins, respectively.

Animals↗
PubMed1987

Rat monoclonal antibodies to mouse lung components for analysis of fibrosis.

Six rat monoclonal antibodies to mouse lung membrane fraction have been characterized. Each has unique binding properties and can be used to stain particular lung components in paraffin sections. One antibody, 133-13A, recognizes a 100-kDa glycoprotein on lung tumor cells, but stains only macrophage-like cells in normal or fibrotic lung sections as determined by immunogold electron microscopy. The monoclonal antibody 273-34A binds to a 112-kDa protein on the surface of normal mouse lung fibroblasts. Immunogold electron microscopy demonstrates antibody binding to capillary endothelial cells, but not to fibroblasts. Type I, or Type II cells. Staining of fibrotic sections with MoAb 273-34A is dramatically enhanced over staining of normal lung. A third antibody, 370-8A, gives a general staining pattern throughout normal lung that is intensified in fibrotic lung. Another MoAb 328-41A mediates intense nuclear fluorescence of lung tumor cells and cells in lung sections. It binds to 14- and 17-kDa proteins that may be high mobility group (HMG) nuclear proteins. Enzyme inhibition studies and immunofluorescence staining patterns on normal lung indicate that elastin may be the target of MoAb 328-35B. MoAb 327-5B binds to normal mouse lung fibroblasts and red blood cell membranes. These last three MoAbs stain macrophages in fibrotic lung, but give a general pattern of light epithelial cell stain in normal lung sections indicating macrophage engulfment of the normal cell antigen during fibrosis. These antibodies should be useful in identifying cell types and molecular mechanisms involved in early stages of fibrosis induced by different chemical insults.

Animals↗
PubMed1986

Tumor antigen on benign adenomas and on murine lung carcinomas quantitated by a two-site monoclonal antibody assay.

TSP-180 is a Mr 180,000 cell surface protein found on several murine lung carcinomas but not on fibroblast or sarcoma cell lines. Monoclonal antibody 135-13C binds to TSP-180 with high affinity but could not be used to quantitate the protein in tumors and normal tissue (Cancer Res., 41: 3465-3470, 1981). TSP-180 was purified from a transplantable BALB/c carcinoma (line 1 cells) by immunoaffinity chromatography and used as an immunogen to produce another monoclonal antibody (346-11A) to a different epitope on the molecule. A two-site solid-phase radioimmunoassay for TSP-180 was developed to quantitate TSP-180 in tissue extracts. The assay can detect as little as 3 ng of TSP-180 in samples up to 10 mg of protein. Analyses of several lung carcinoma cell lines confirmed that TSP-180 is present in all (five of five) cell lines ranging from 40 to 800 ng per mg of cell protein extract. Mouse tissues from normal and tumor-bearing mice were analyzed for TSP-180. Values for line 1 tumors growing i.m. were about 70 ng of TSP-180 per mg of protein. Normal tissue from normal and tumor-bearing mice contained low levels of TSP-180 from less than 0.3 ng/mg of protein for liver to a high of about 11 ng/mg of protein for leg muscle. Finally, small benign urethane-induced adenomas (1 to 2 mm) from BALB/c mice had moderate amounts of TSP-180 (11 ng/mg of protein), while two primary lung adenocarcinomas in the same animals had 20 and 47 ng/mg of protein, respectively, suggesting that TSP-180 expression may increase with increasing cancer of these tumors. Analyses of individual urethane-induced lung tumors from A/J mice showed that 11 of 13 carcinomas had high levels of TSP-180, while only 1 of 6 adenomas had a detectable amount of TSP-180, and that was at a moderate level. Specific quantitation of tumor markers in normal, benign, and malignant lung tissue may be helpful in identifying different levels of gene expression as tumors progress to more malignant states.

Adenoma↗
PubMed1983

Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging.

For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. We have studied the binding of 125I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind (formula; see text). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10(8) M-1 are not likely to be useful for drug targeting or tumor imaging.

Animals↗
PubMed1981

Analysis of surface proteins of mouse lung carcinomas using monoclonal antibodies.

Hybridoma cultures secreting monoclonal antibodies were derived from fusions of parent myeloma P3-X63-Ag8 with spleen cells of F344 rats which had been immunized with the BALB/c alveolar lung carcinoma line 1. Screening of several thousand hybridoma cultures identified 55 cell lines making antibody that bound to the line 1 cell surface. Of 15 of these antibodies tested, seven could be identified as binding to P100, a surface protein expressed on both normal and tumor cells, and five reacted with a tumor surface protein with a molecular weight of 180,000 (TSP-180) not found on control cell lines. Monoclonal antibodies to TSP-180 bound to lung tumor cells but not to normal cells or tumors of other organs. Five different hybridoma cultures secreting antibody to TSP-180 were cloned, and monoclonal antibodies were purified by affinity chromatography. Radioiodinated monoclonal antibodies were used to determine affinity constants for binding to TSP-180 as it is expressed on four different mouse lung tumors. Binding constants ranged from 1.2 X 10(7) to 1.5 X 10(8) liters/mol for the different antibodies.

Animals↗