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.

Autonomic nervous system regulates secretion of anti-inflammatory prohormone SMR1 from rat salivary glands.

The autonomic nervous system regulates the secretion of bioactive proteins and peptides from salivary glands that can be important in systemic physiological responses. The prohormone submandibular rat-1, which is highly expressed in rat submandibular glands, can be cleaved to produce polypeptides with analgesic and anti-inflammatory activities. Human genes related to submandibular rat-1 have conserved biological functions and are potentially important in pain suppression, erectile function, and inflammation. In this study we describe the differential expression and posttranslational modification of submandibular rat-1 protein in salivary glands, the urogenital tract, lung, blood, and saliva in male Sprague-Dawley and Brown Norway rats. Submandibular rat-1 protein is secreted into saliva after the administration of beta-adrenergic or cholinergic agonists. Removal of the sympathetic ganglion that innervates the salivary glands results in increased levels of submandibular rat-1 protein in salivary glands. The secretion of submandibular rat-1 in response to physiological stress may provide a large pool of submandibular rat-1-derived peptide products that can promote analgesia and decrease inflammation locally and systemically. This pathway may be conserved among mammals and may constitute an important anti-inflammatory and analgesic response to stress.

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.

Use of bispecific antibodies in molecular velcro assays whose specificity approaches the theoretical limit of immunodetection for Bordetella pertussis.

A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular "velcro" enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.

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.

Effect of cyclooxygenase-2 (COX-2) inhibitors on prostate cancer cell proliferation.

BACKGROUND: The tricyclic cyclooxygenase-2 (COX-2) inhibitors celecoxib and rofecoxib exhibit different magnitudes of antiproliferative activity against the human prostate cancer cell lines LNCaP and PC-3. We investigated the correlation between the COX-2 inhibitory potencies, antiproliferative activities and the nature of the central ring system of novel tricyclic COX-2 inhibitors belonging to the diarylcycloalkyl and diarylheterocyclic classes possessing a central 3-membered cyclopropyl (1 and 2), a 5-membered isoxazoline (3), an isoxazole (4) or 2-(5H) furanone (5), and a 6-membered pyran-2-one (6a-c) ring system. MATERIALS AND METHODS: Novel tricyclics were synthesized and evaluated in vitro for their COX-1/COX-2 inhibitory activities and their abilities to inhibit cell proliferation in prostate (AT3B-1, PC-3 and LNCaP) and breast (MCF-7) cancer cell lines. A molecular modeling study was carried out to characterize the electronic nature of the central ring systems of the novel tricyclic COX-2 inhibitors. RESULTS: The isoxazoline (3), which exhibited excellent COX-2 inhibitory potency and selectivity, showed growth inhibition in all the cell lines tested with IC50 values of 349 microM (AT3B-1), 378 microM (PC-3), 100 microM (LNCaP) and 200 microM (MCF-7), respectively. The rofecoxib analog (5) and 6-membered pyran-2-ones (6a-c) showed weak inhibition (MCF-7, AT3-B-1 and PC-3) inspite of possessing good COX-2 inhibitory potencies and selectivities. CONCLUSION: This study demonstrates that the antiproliferative activity profiles exhibited by the novel tricyclic COX-2 inhibitors is dependent on the electronic nature of the central ring system and is independent of their COX-2 inhibitory potencies and selectivities. Accordingly, the 2,3-dimethyl-5-(4-methylsulfonylphenyl)-4-phenyl-4-isoxazoline (3), which possesses an electron rich central 5-membered isoxazole ring, could serve as a lead compound to develop novel drugs to treat prostate cancer.

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 second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis.

We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.

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.

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.

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.

Modern trends in radioimmunotherapy of cancer: pretargeting strategies for the treatment of ovarian cancer.

A review of published data on some of the problems associated in treating cancer using radioimmunotherapy is presented. Potential improvements for this type of therapy using pretargeting strategies are discussed and preliminary results on a novel multistep regimen to treat human ovarian cancer are presented. A pretargeting strategy using a biotinylated, anti-CA125 monoclonal antibody (MAb) to attract biotinylated long-circulating liposomes to the surface of CA125-expressing ovarian cancer cells, was employed. Confocal laser scanning microscopy and fluorescent labels were used to establish the biodistribution patterns in NIH:OVCAR-3 (CA-125 positive) and SK-OV-3 (CA-125 negative) human ovarian cancer cells. Shedding kinetics of the pretargeted stage were measured using 125I labeled MAbs. No significant internalization of the MAb used in the pretargeting step was observed by 4 hrs. The antibody was gradually internalized starting at 6 hrs, and most of the labelled MAb was detected in cytoplasm by 24 hrs. Shedding and exocytosis of the antigen-MAb complex was not significant for up to 6-hours following administration of the iodinated MAb. Biotinylated liposomes were shown to specifically target the biotinylated MAb/streptavidin complex on the cell surface. We have demonstrated that by a three-step pretargeting approach, biotinylated liposomes can be specifically delivered to cells pretargeted with biotinylated MAb/SAv complex. The slow internalization and shedding properties of the two MAbs are ideal for multistep pretargeting methods. A successful multistep linkage was established with the biotinylated MAb B27.1, streptavidin and biotinylated liposomes to OVCAR-3 cells, but not to SK-OV-3 cells.

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.

Colonization and immune responses in mice to Helicobacter pylori expressing different Lewis antigens.

BACKGROUND AND AIMS: A mouse model was established to compare colonization by the H. pylori Sydney strain SS1 with several clinical isolates expressing different Lewis antigens on their surface. In addition, both humoral and cell mediated immune responses were determined for different H. pylori strains. METHODS: Mice were inoculated intragastrically separately with the Sydney strain as well as with five clinical isolates of H. pylori expressing different Lewis (Le) antigen phenotypes. Colonization of the mouse stomach by the bacteria was monitored from two to fourteen weeks post inoculation by four independent methods namely, urease, PCR (using CagA primers), bacterial culture and histology. Antibody titers and cellular immune responses were monitored by ELISA and antigen stimulation test respectively. RESULTS: Different degrees of colonization were observed in C57, CD1 and Balb/c mice inoculated with H. pylori strain SS1 (Le(x), Le(y)) and clinical isolates UA948 (Le(a), Le(x)), UA861 (alpha-glucosyl polyLacNAc), UA1258 (Le(y)), UA802 (Le(y)) and UA1264 (no Le antigen) starting from week two post inoculation. All three mice strains mounted high immune responses against different H. pylori antigens. Treatment of mice with vancomycin prior to inoculation has no effect either on colonization of the stomach or the immune response of the mice. Histological evaluation established colonization after 10 weeks post inoculation but not gastritis. CONCLUSIONS: Stomach of mice can be colonized consistently, with H. pylori strain SS1, and colonization was also achieved with all clinical isolates that were not mouse adapted. These strains could be detected more consistently by PCR in the early stages, then by culture only after 8 - 10 weeks. In our study, Lewis(x) expressing bacterial strain (UA948) failed to colonize Balb/c mice, whereas the Le(y) expressing strain (UA1258) did not colonize C57/BL6 mice.

Construction and characterization of monoclonal antibodies against western equine encephalitis virus.

A repertoire of mouse monoclonal antibodies (MAbs) against western equine encephalitis virus (WEE) was constructed and characterized. Anti-WEE antibodies were expressed from hybridomas and purified by protein G chromatography. Each of the antibodies was functionally assessed by indirect enzyme-linked immunosorbent assays (ELISAs), Western blotting, and immunoprecipitations. All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis (VEE), and eastern equine encephalitis (EEE). Because many of the antibodies were highly reactive to WEE antigen in one or more of the assays, these antibodies are excellent candidates for immunodetection and immunotherapy studies.

Development and characterization of a bispecific single-chain antibody directed against T cells and ovarian carcinoma.

Bispecific antibodies with specificity for tumor antigen and CD3 have been shown to redirect the cytotoxicity of T cells against relevant tumor. Our objective was to generate single-chain bispecific antibodies (bsSCA) that could retarget mouse cytotoxic T lymphocytes (CTL) to destroy human ovarian carcinoma in a xenogeneic setting. A bsSCA, 2C11 x B43.13, was constructed by genetic engineering and expressed in mammalian cells. Molecular characteristics, binding properties, and ability to retarget CTL were studied. Western blot analysis showed that the product is a 65-kDa protein. Purification of antibodies could be done by single-step affinity chromatography using protein L-agarose with an unoptimized yield of 200 microg/L. BsSCA 2C11 x B43.13 was capable of binding to mouse CD3 and human CA125 as detected by FACS analysis of EL4 and OVCAR Nu3H2 cells, respectively. It could also bridge activated splenic T cells and human ovarian carcinoma as demonstrated by a bridge FACS assay. Redirected mouse CTL could mediate human target cell lysis in a 20-h 51Cr release assay despite that they are xenogeneic. Prolonged incubation of redirected CTL and tumor targets resulted in a dramatic reduction in tumor cell number. CD28 co-stimulation enhanced redirected CTL function in both types of assays. BsSCA 2C11 x B43.13 thus can be used as a preclinical immunotherapeutic model for human ovarian cancer in a xenogeneic setting.

Bispecific MAb aided liposomal drug delivery.

We have developed a new method for specifically delivering liposomal model drugs to tumor cells. Bispecific monoclonal antibodies (bsMAb) (174H.64 x anti-biotin) which can bind tumor-specific antigen and biotin were developed and characterized. Biotinylated stealth liposome loaded with model drug 99mTc-DTPA can bind to the biotin-binding arm of bsMAb. This targeted liposomal delivery strategy was tested in mouse KLN-205 squamous carcinoma model. bsMAbs were administered 24h in advance into tumor allograft bearing mice, which allow them to bind to tumor cells through the anti-tumor binding arm. After clearance of circulating bsMAb, biotinylated stealth liposomes were introduced to specifically bind to the tumor sites where bsMAb localized earlier. The results show that pretargeted bsMAb can enhance liposomal drug targeting by four times, 3.61% dose/g vs. 0.89% dose/g. This bsMAb/liposome strategy show the broad possibilities of selective delivery of cytotoxic drugs or genes to the specific targets.

Development of a functional monoclonal single-chain variable fragment antibody against Venezuelan equine encephalitis virus.

We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.