Intralesional Vitamin D3 Injection in the Treatment of Recalcitrant Warts: A Novel Proposition.

BACKGROUND: Verruca vulgaris (viral warts) is a fairly common condition with a plethora of treatment options having variable success rates. Recalcitrant warts are refractory to treatment with often disappointing response and high recurrence rates. Lately, treatment with intralesional injections has gained momentum due to its effectiveness in clearing warts by stimulating the cell-mediated immunity. Vitamin D, when applied topically, regulates epidermal cell proliferation and is involved in the formation of antimicrobial peptides. We have attempted to use vitamin D3 to exploit its reported action as an immunotherapeutic molecule in addition to its topical effects. To our knowledge, there are no reports of intralesional vitamin D3 injections used in the treatment of extragenital recalcitrant warts. METHODS: Sixty-four patients with recalcitrant warts of varying sizes and duration were included in the study. About 0.2- to 0.5-mL vitamin D3 solution (600,000 IU, 15 mg/mL) was injected to the base of the wart. A maximum of 5 warts were injected per session at 3-week intervals until resolution or for a maximum of 4 treatments. Patients were followed up for 6 months after the last injection to detect any recurrence. RESULTS: Sixty patients completed the study. Complete response was seen in 54 of 60 (90%), partial response in 4 of 60 (6.66%), and no response in 2 of 60 (3.33%). The average number of injections required to achieve a complete resolution was 3.66. Complete resolution of distant warts was noticed in all patients. CONCLUSIONS: Intralesional vitamin D3 is a safe, effective, and an inexpensive treatment option for recalcitrant warts.

Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y.

The goal of this study was to develop a quantitative detection system for severe acute respiratory syndrome-associated coronavirus (SARS-CoV), targeting the nucleocapsid protein (NP), to determine the presence and degree of infection in suspected individuals. Because the NP is the viral protein shed during infection and its template mRNA is the most abundant subgenomic RNA, it is a suitable candidate for developing antibodies for diagnostic applications. In this study, we have prepared full-length SARS-CoV NP expressed in Escherichia coli and purified. Full-length NP was used for the preparation of mouse monoclonal antibody and chicken polyclonal IgY antibodies for the development of heterosandwich ELISA for early diagnostics of SARS-suspected individuals. The sensitivity of the developed heterosandwich ELISA can detect the viral antigen at 18.5 pg/mL of recombinant NP. This study describes ultrasensitive ELISA using 19B6 monoclonal antibody as the capture antibody and IgY as the detecting antibody against the most abundant SARS-CoV NP antigens. One of the most important findings was the use of inexpensive polyclonal IgY antibody to increase the sensitivity of the detection system for SARS-CoV at the picogram level. Furthermore, the immunoassay of SARS-CoV NP antigen developed could be an effective and sensitive method of diagnosing SARS-suspected individuals during a future SARS-CoV outbreak.

Screening of 14C-polyamines in the AT3B-1 rat prostate tumor model: the search for a new PET prostate imaging agent.

BACKGROUND: We have previously reported the polyamine uptake kinetics in various prostate and non-prostate cancer cell lines, concluding that the prostate cancer cell lines took up and accumulated polyamines at higher levels than non-prostate cell lines, with a view to their use as PET agents. The objective of the present study was to assess their in vivo accumulation in a rat prostate tumor model. MATERIALS AND METHODS: A comparative biodistribution study of the polyamines was conducted in AT3B-1 prostate tumors in male Copenhagen rats to determine which of the polyamines show preferential accumulation in the tumor. Tissue samples were collected one hour post administration of the polyamines (i.v.), and the radioactivity of the samples was measured by first combusting the tissue samples in a biological oxidizer and then assaying the trapped 14CO2 in a liquid scintillation counter. RESULTS: Putrescine exhibited the highest tumor accumulation followed by ornithine (4.1% and 1.8% of injected dose/g of the tumor respectively). The tumor-to-blood ratio was highest with putrescine followed by spermidine (18.7 and 12.9 respectively) and the order of tumor-to-normal prostate accumulation ratio was putrescine>ornithine>spermine>spermidine. CONCLUSION: The results indicated preferential accumulation of putrescine and ornithine in the prostate tumor.

Anti-tumor effects of the bacterium Caulobacter crescentus in murine tumor models.

Caulobacter crescentus is a gram negative, non-pathogenic bacterium, common in aquatic and soil environments. One feature of note is a protein surface layer (S-layer) composed of a single protein, organized as a self-assembled crystalline array that coats the bacterium. In the course of efforts to express cancer-associated peptides as genetic insertions into the S-layer, we noted a tumor suppressive effect of the unmodified bacterium. C. crescentus was examined for anti-tumor activity against three transplantable tumor mouse models: Lewis lung carcinoma cells transfected with the MUC1 gene in C57BL/6, murine mammary carcinoma (EMT-6) in BALB/c (both in prophylactic and therapeutic mode) and murine leukemia cells (L1210) in DBA2. Mice were immunized three times i.p. with C. crescentus (2 x 10(7) cells/mouse). In prophylactic mode, the mice were challenged with tumor cells two weeks after the last immunization. Immunization with live C. crescentus resulted in anti-tumor activity in all three transplantable tumor models, as measured by prolonged survival, reduced tumor mass or reduced number of lung nodules, compared to saline control groups. In the Lewis lung and the EMT-6 mammary carcinoma murine models the number of lung nodules as well as the tumor weight was lower in mice treated with C. crescentus, compared to the control group; for EMT-6, this was observed in prophylactic and therapeutic modes. In the murine leukemia and Lewis lung carcinoma models prolonged survival was observed in the groups of mice immunized with Caulobacters. In most cases the live C. crescentus cells were markedly more efficacious than heat killed or formalin fixed cells, despite the fact that they do not grow or persist in mice. The results suggest that C. crescentus may be a safe, bacterial immunomodulator for the treatment of tumors.

Monoclonal antibodies and synthetic tumor-associated glycoconjugates in the study of the expression of Thomsen-Friedenreich-like and Tn-like antigens on human cancers.

Synthetic carbohydrate haptens, which are conjugated to carrier human serum albumin molecules [synthetic tumor-associated glycoconjugates (S-TAGs)], were used to immunize mice for monoclonal antibody (MoAb) production. Two of the S-TAGs were composed of haptens related to the Thomsen-Friedenreich (TF) antigen, and their structures are beta Gal(1----3)-beta GalNAc (TF-beta) and beta Gal(1----3) alpha GalNAc (TF-alpha) (Gal = galactose; GaNAc = N-acetylgalactosamine). The third S-TAG was made up of Tn hapten groups of the structure alpha GalNAc-O-serine. MoAbs specific for TF-alpha and Tn were able to be generated. All MoAbs generated against TF-beta cross-reacted with TF-alpha but not with Tn. None of the TF-alpha-specific MoAbs reacted with human carcinomas, whereas several TF-beta and Tn MoAbs were found to react with most human lung, colon, and breast carcinomas. It is believed that this is the first report of the use of synthetic carbohydrate cancer antigens for the production of anticancer MoAbs.

Transport of folate compounds in L1210 cells: kinetic evidence that folate influx proceeds via the high-affinity transport system for 5-methyltetrahydrofolate and methotrexate.

Folate is transported into L1210 mouse leukemia cells by the same system that mediates the uptake of methotrexate and reduced folate compounds. This conclusion is supported by the following observations: (a) methotrexate competitively inhibits the influx of folate and the Ki is comparable to the Kt for methotrexate influx; (b) the profile for inhibition of folate influx by methotrexate is monophasic and complete inhibition is achieved at high concentrations of the competitor; (c) folate inhibits the influx of methotrexate and the Ki is comparable to the Kt for folate influx; (d) the N-hydroxysuccinimide ester of methotrexate, a potent and specific irreversible inhibitor of the reduced folate system, also blocks the influx of folate; (e) folate and methotrexate influx are both inhibited by low concentrations of p-chloromercuriphenylsulfonate; and (f) folate influx fluctuates with the anionic composition of the medium in the same fashion as the influx of methotrexate. Measurements of folate influx can be complicated by the fact that the 3H-labeled substrate is susceptible to decomposition and that labeled breakdown products at concentrations as low as 1% contribute appreciably to the observed uptake. Of these products, 6-hydroxymethylpterin appears to account for most of the extraneous uptake. Impurities can be eliminated by subjecting the [3H]folate to preparative thin-layer chromatography immediately prior to use.

Analysis of human tumor associated Thomsen-Friedenreich antigen.

The Thomsen-Friedenrich (TF) antigen is a precursor structure of MN blood group antigens and is also expressed by about 90% of human carcinomas. The immunodominant group of TF antigen [beta-galactosyl(1-3)-alpha-N-acetylglactosamine] is present in cryptic form in normal RBC and is revealed by neuraminidase treatment. A murine monoclonal antibody (Mab 49H.8) developed against neuraminidase treated human RBC was reactive against a variety of human tumors. We have characterized the human tumor associated TF antigen detected by this antibody from a human transitional bladder carcinoma cell line (647V), a human colon adenocarcinoma cell line (LS174T), and a pleural effusion fluid of a breast adenocarcinoma patient (PE 89). A heterologous sandwich radioimmunoassay for TF antigen was developed using Mab 49H.8 as the catcher and 125I-peanut agglutinin as the probe. Detergent extracts of 647V and LS174T cells, media conditioned by culturing these cells, and PE 89 were shown to contain the antigen by this assay. The specificity of the antigen capture by Mab 49H.8 in this assay was demonstrated by its selective inhibition by nitrophenyl-beta-D-galactoside, phenyl-beta-D-galactoside, and a TF hapten. Preliminary studies on TF antigen in serum samples using this assay showed that about 53.7% of the carcinoma samples contained an antigen concentration greater than 200 units/ml whereas for 90.9% of the normal samples the antigen concentration was below 200 units/ml. These studies demonstrated that the TF antigen is shed by the tumor cells both in vitro and in vivo. The TF antigen was sensitive to treatment with alkali (0.1 M NaOH for 5 h at 37 degrees C) and periodate (10 mM sodium periodate for 1 h at room temperature), was resistant to acidic pH (50 mM acetate buffer, pH 4.5, for 5 h at 37 degrees C), and could be extracted with perchloric acid (0.6 M for 1 h at 4 degrees C). The antigen was shown to be a high molecular weight glycoprotein (Mr greater than 1,000,000) by gel filtration chromatography. The density of the antigen was estimated to be about 1.35 g/ml by cesium chloride density gradient centrifugation. The antigen could be isolated from conditioned media by a combination of affinity chromatography and gel filtration with an overall purification of about 61,432-fold and a final recovery of 53.2%.(ABSTRACT TRUNCATED AT 400 WORDS)

A general affinity method to purify peroxidase-tagged antibodies.

Antibodies tagged with enzymes, e.g. horseradish peroxidase (HRPO) are used extensively in a broad range of immunoassay, immunohistochemical, and prodrug-based immunotherapeutic applications. These antibodies may be polyclonal, monoclonal, bispecific or genetically engineered in origin. Often, purification of the antibody is the single greatest obstacle to obtaining immunoprobes with high specific activity [Milstein and Cuello, Nature 305 (1983) 537]. We have circumvented this problem by utilising benzhydroxamic acid-agarose to purify the antibodies tagged with HRPO as a preformed immune complex. Benzhydroxamic acid has been shown to have affinity for the active site of HRPO [de Ropp et al., Biochemistry 38 (1999) 1077]. A preliminary ammonium sulfate precipitation of 250 ml of bispecific antibody supernatant was performed and the pellet resuspended and dialysed against phosphate buffer (pH 7.0). This fraction was incubated with HRPO, then loaded on the affinity column which was washed, and the labelled bispecfic monoclonal antibodies were eluted under mild conditions (borate buffer pH 9.0). The effective yield of this bispecific antibody-HRPO complex was 30 assay plates or 3000 wells. We have also successfully co-purified covalent polyclonal-HRPO conjugates and HRPO-labelled streptavidin using a similar strategy to obtain enzyme-labelled probes with high specific activities for a multitude of applications.

Bispecific monoclonal antibodies against a viral and an enzyme: utilities in ultrasensitive virus ELISA and phage display technology.

A quadroma (hybrid-hybridoma) secreting bispecific antibodies with one paratope specific for M13 bacteriophage coat protein and another paratope specific for alkaline phosphatase (AP) was developed by electro-fusion of the two parental hybridomas and selected by a fluorescence activated cell sorter (FACS). The anti-phage M13/anti-AP bsMAbs were purified from anti-phage M13 monospecific MAb by a novel affinity method using Mimetic Blue A6XL as immune complexes with AP. The purified bsMAbs with potentially every molecule uniformly bound with AP generated an immuno-probe with the theoretical highest specificity. An ultrasensitive sandwich ELISA for detecting viruses was developed by using this bsMAb coupled with an amplified ELISA procedure. The sensitivity of the assay was increased 1000 times compared with conventional ELISA to achieve detection of 100 phage particles which is approximately 2.3 fg of phage coat protein. This type of bsMAb probe and ELISA format can be used to design new body fluid assays for viral load of HIV, hepatitis and other human pathogens as rapid and inexpensive alternatives to the PCR based method. This unique bispecific probe also allowed rapid and sensitive detection of bound M13/fd phage clones while panning for specific phages displaying peptide mimics against an antigen from a phage display peptide library. Furthermore, we demonstrate the principle virus purification using bsMAb as affinity ligand with a mild phosphate buffer elution. The results indicate that bsMAb could be used to develop affinity chromatography for purifying highly contagious and pathogenic viruses avoiding procedures employing prolonged high-speed centrifugation.

Development of a bispecific monoclonal antibody as a universal immunoprobe for detecting biotinylated macromolecules.

Bispecific monoclonal antibody (BsMab) combining two different antigen binding sites, anti-biotin and anti-HRPO paratopes, could be used as a universal immunoprobe for detecting all biotinylated macromolecules. First, a mouse hybridoma cell line secreting monospecific anti-biotin Mab was generated and characterized. Second, a quadroma cell line which could continuously secrete bsMab (anti-biotin x anti-HRPO) was developed by a nonselective microelectrofusion method. The supernatant containing bsMab was collected from tissue culture medium and purified with two affinity columns. This bsMab has comparable avidity to commercial streptavidin-HRPO when tested against biotinylated macromolecules. Compared to streptavidin, this bsMab can bind the enzyme and thus eliminate the need for chemical conjugation. This bsMab can be used as a promising immunoprobe for detecting many macromolecules bearing biotin markers, such as protein, phage, liposome and DNA in different bioassay systems.

A rapid non-selective method to generate quadromas by microelectrofusion.

A simple non-selective methodology was developed and standardized to generate desired hybrid-hybridoma or quadroma secreting bifunctional antibodies. This novel protocol is based on microelectrofusion on a meander chamber using a few hundred cells of each of the two parental hybridomas with no laborious drug selection procedures. Seeding approximately 10 cells per well in a 96-well microtitre plate after fusion in 200 microliters standard medium containing 20% FBS and 10% Origen growth factor generated positive quadromas secreting bispecific antibodies with good stability after the second reclone. Compared to the conventional PEG fusion and other methods this simple protocol is both rapid and economical. Generally, conventional methods to make quadromas and triomas require the introduction of drug selection markers into one or both of the parental cells, a procedure that could take 3-6 months. Utilizing the non-selective microelectrofusion method described here, we have generated several quadromas in a very short time. Further, such a protocol could also be potentially adopted to generate human hybridomas with few B cells isolated from peripheral blood lymphocytes enriched by antigen specific panning or affinity microelectrofusions.

Renal tubular receptor imaging with iodine-131-labeled peanut lectin: pharmacokinetics and renal clearance mechanism in animals.

Intravenously administered peanut lectin (PNA), iodinated with 131I ([131I]PNA), is rapidly cleared from the plasma by the kidneys in dogs (clearance [total body] = 17.52 +/- 8.74 ml/min). Dynamic gamma camera renal scintigraphy demonstrated renal accumulation and excretion phases of the [131I]PNA renogram in dogs and rabbits (% injection dose-at-peak = 21.8 +/- 3.3% and 19.6 +/- 4.3%, time-to-peak = 44.6 +/- 4.8 min and 37.2 +/- 6.9 min, respectively). Immunoperoxidase staining of kidney sections, following i.v. administered PNA, demonstrated predominant accumulation by the proximal tubules of mice, rabbits, and dogs. The basement membrane was intensely stained at early times p.i. while intracellular and luminal PNA was evident within 1 hr. Urine analysis confirmed the presence of intact [131I]PNA in the bladder contents, while protein degradation products, and a small percentage of the free iodide (less than 5%) were noted within 1 hr p.i. The relative proportion of free iodide increased at later times p.i. (greater than 6 hr). A receptor mediated excretion mechanism is proposed for the clearance of PNA and may be useful for the study of renal tubular function.

Importance of pH in defining the specificity and clinical utility of monoclonal antibodies--an evaluation of two anti-CA 19-9 monoclonal antibodies.

The effect of pH on the binding characteristics of monoclonal antibodies against tumor associated antigens has been studied, using anti-CA 19-9 monoclonal antibodies as an example. The 1116-NS-19-9 monoclonal antibody is used in several commercially available two-step sandwich IRMAs for the measurement of the CA 19-9 antigen in cancer patient sera (Centocor CA 19-9 RIA, Malvern, PA, U.S.A., or similar kits by Centocor licensees). The B25.10 monoclonal antibody is in use in the Truquant GITM RIA (Biomira Inc., Edmonton, Alberta, Canada), a competitive inhibition assay measuring the CA 19-9 antigen. We determined the Ka value of the B25.10 and the 1116-NS-19-9 antibodies at pH 4.5 and 7.3 on a solid phase coated with CA-19-9 bearing mucin. The 1116-NS-19-9 antibody had a Ka value of 4.1 x 10(8) at pH 4.5 and approximately 0.3 x 10(8) at pH 7.3. The Ka value of B25.10 was in the range of 0.7-4.1 x 10(9) at either pH, but somewhat lower at pH 4.5. At pH 4.5 (the pH optimum for binding of 1116-NS-19-9 to the CA 19-9 antigen Del Villano, B.C., 1984]) the binding of 125I-labeled B25.10 to the solid phase was comparably inhibited by 1116-NS-19-9 and B25.10. However, at neutral pH (pH 7.2) the apparent affinity of the 1116-NS-19-9 antibody for the CA 19-9 antigen is significantly diminished [Del Villano, B.C., 1984] while that of B25.10 is slightly higher. Consequently, the ability of the 1116-NS-19-9 to inhibit the binding of the B25.10 antibody to the CA 19-9 solid phase is greatly reduced in the physiological pH range. The consequences of these findings for in vivo applications are discussed.

Radiolabelled peanut lectin for the scintigraphic detection of cancer.

To investigate the role of intravenously administered, radioiodinated peanut lectin (131I-PNA) in the non-invasive detection of cancer, the pharmacokinetics and scintigraphic distribution of this novel tumor-seeking compound were studied in 8 patients with metastatic cancer of the colon, breast or lung. Scintigraphic localization of 131I-PNA was apparent at certain anatomical sites of known metastases in 2 patients and in a further 2 patients an adjacent malignant pleural effusion was visualized. The rapid clearance of radioactivity from the whole body and plasma with marked renal concentration and rapid urinary excretion of significant amounts of intact 131I-PNA (mol. wt. 107,000, pI 5.95) implied that this molecule was excreted selectively by the renal tubules. PNA or other lectins may find a role in the scintigraphic detection of selected types of cancer.

Bispecific and bifunctional single chain recombinant antibodies.

Bispecific and bifunctional monoclonal antibodies as second generation monoclonals, produced by conventional chemical or somatic methods, have proved useful in the immunodiagnosis and immunotherapy of cancer and other diseases. Recombinant antibodies produced by genetic engineering techniques have also become available for use in preclinical and clinical studies. Furthermore, through genetic engineering, it is possible to remove or add on key protein domains in order to create designer antibody molecules with two or more desired functions. This review summarizes the strategies for development of single chain variable fragment (scFv) bifunctional and bispecific antibodies. The advantages and disadvantages as well as the problems of generating the various bispecific and bifunctional antibody constructs are reported and discussed. Since conventionally prepared bispecific and bifunctional monoclonal antibodies have already shown promise in clinical trials and results from preclinical studies of recombinant bispecific antibodies are encouraging, clinical trials in humans of recombinant bispecific and bifunctional antibodies, as a new generation of biologicals, are likely to be the thrust in the next decade and beyond.

Development of a biotin mimic tagged ScFv antibody against western equine encephalitis virus: bacterial expression and refolding.

Single chain antibodies (ScFvs) are heavy and light chain variable domains connected by an artificial linker. Because of their smaller size, ScFvs show improved tissue penetration in vivo and reduced immunogenicity, making them ideal for therapeutic applications. We have cloned a ScFv against western equine encephalitis (WEE) using rDNA technology. The ScFv was generated from a hybridoma cell line (11D2) specific to the WEE virus E1 glycoprotein and is arranged in the V(L)-V(H) orientation with a (gly(4)ser)(3) linker. This ScFv was engineered successfully with a biotin mimic tag (11 amino acid peptide) and cloned in the pET22b+ expression vector. The ScFv was expressed as a approximately 32kDa protein in Escherichia coli as inclusion bodies, with an estimated yield of 20-40 mg/l. Different refolding protocols were used to solubilise the inclusion bodies. Most of the functional ScFv was generated when the inclusion bodies were solubilized in a detergent, air oxidised in the presence of CuSO(4) and then denatured in urea buffer in comparison to other protocols. The product was renatured finally in Tris arginine buffer (pH 8.0). Refolded protein was dialysed against phosphate buffer saline (PBS) (pH 7.3) to remove the Tris and arginine. Our refolding protocol generated up to a 50% yield of soluble protein, which retained antigen-binding activity with whole inactivated WEE virus as demonstrated by ELISA and Western blot analysis. This 11D2-biotin mimic ScFv complexed with streptavidin horseradish peroxidase (St-HRPO) will be useful as a detector reagent in the ultrasensitive ELISA detection of WEE virus antigen.

Transport of folate compounds, pterins and adenine in L1210 mouse leukemia cells.

L1210 mouse leukemia cells provide a convenient model for examining the mechanisms and components involved in the active transport of various metabolites and drugs. One of these transport systems exhibits a broad specificity for folate compounds, including 4-amino antagonists such as methotrexate. The primary substrate for this system is 5-methyltetra-hydrofolate (Kt = 1 microM), the principal circulating form of the vitamin in mammals. 5-Formyltetrahydrofolate (Kt = 5 microM) and Methotrexate (Kt = 5 microM) are also taken up efficiently, but folate (Kt = 100 microM) is a relatively poor substrate. Vmax for this system is ca. 15 pmoles/min/mg protein. Energy for substrate internalization is provided by an anion-exchange mechanism, and regulation appears to be mediated by cyclic AMP. The system can be inhibited irreversibly by treatment of the cells with photo-activated azido AMP or carbodiimide-activated folate compounds. The latter method allows the membrane-associated binding protein to be labeled in situ, thereby providing a means for identifying it during subsequent solubilization and purification. Guidance for this latter project is provided by previous experience in the purification to homogeneity of a similar folate-binding protein from Lactobacillus casei. L1210 cells also contain an efficient system for the transport of adenine (Kt = 20 microM; Vmax = 200 pmoles/min/mg protein). Uptake of adenine is linked with its conversion to AMP via PRPP-dependent adenine phosphori-bosyltransferase. Pterins, which have a close structural similarity to adenine (as well as to a portion of the folate molecule), are also transported into L1210 cells. Transport of [3H] 6-hydroxymethylpterin (Kt = 20 microM) was inhibited by 6-formylpterin, 6-methylpterin and 6-carboxypterin with Ki values of 42, 100 and 350 microM, respectively. Adenine (Ki = 20 microM) and various other purines were also good inhibitors of pterin transport. Present evidence indicates that adenine and pterins use separate transport systems, but isolation of the components of these systems may further delineate their interrelationships.

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro.

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.

Comparative uptake of polyamines by prostate and non-prostate cancer cell lines.

The Km and Vmax of [14C]-radiolabeled polyamines were determined for PC-3 and AT3B-1 cell lines. With PC-3 Km values are in the following order: ornithine> spermidine> spermine> putrescine, while with AT3B-1 it was spermidine> ornithine> spermine> putrescine. To determine which of these polyamines exhibit higher accumulation, the relative uptake of all the four amines was studied with prostate (PC-3, AT3B-1, LNCaP) and non-prostate (MCF-7, KLN-205, OVCAR) cell lines at 10 and 20 microM after 1 hour. Spermine and spermidine accumulated at higher levels in prostate (AT3B-1 and LNCaP) over non-prostate cell lines (p < 0.01). Putrescine accumulated more in PC-3 and LNCaP than the non-prostate cancer cells.

Passive immunization with an anti-substance P antibody prevents substance P- and neurokinin A-induced bronchospasm in anesthetized guinea-pigs.

In a guinea-pig model of asthma, active immunization against substance P (SP) prevented the development of airways' hyperresponsiveness and reduced bronchospastic responses to SP (i.v.). The rat-mouse heterohybridoma NC1/34 secretes a specific, rat IgG1, anti-substance P antibody (alpha-SP Ab) which was isolated and purified by passing supernatant from cultures through thiophilic gel. Purity of antibody was about 50% (SDS-PAGE). The relative affinities of the alpha-SP Ab for SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were estimated by ELISA using a constant amount of SP coupled (glutaraldehyde) to bovine serum albumin (BSA) to capture the antibody, alone and in the presence of increasing concentrations of the neuropeptides. At alpha-SP Ab dilutions of 1 in 5,000 to 1 in 32,000, CGRP did not prevent antibody binding to SP-BSA conjugate bound to the plates, but both SP and NKA prevented binding. In this system, the relative affinity of the alpha-SP Ab, at dilutions of 1 in 5,000 and 1 in 10,000, was about 50 times greater for SP than NKA. Whether passive immunization with alpha-SP Ab prevented bronchospastic responses to SP and NKA (i.v.), in vivo, was determined in groups of anesthetized guinea-pigs by recording pulmonary flow resistance (RL) and dynamic pulmonary elastance (EL). Injection of alpha-SP Ab (i.v., 5:1 molar ratio: alpha-SP Ab:SP total dose) did not alter baseline values of RL and EL, but markedly inhibited increases in RL and EL induced by SP and NKA (i.v.) without affecting responses to methacholine (i.v.). A control, "irrelevant" rat IgG-type antibody at a similar concentration had no effect on responses to SP or NKA. These findings indicate that passive immunization with a monoclonal alpha-SP Ab can prevent the bronchospastic effects of exogenous SP and NKA in guinea-pigs.