Biomedical subjects

M R Suresh

Publications and source records attributed to M R Suresh.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

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.

Animals↗

PubMed2003

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.

Alkaline Phosphatase↗

PubMed2002

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.

Antibodies, Monoclonal↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Antibodies, Monoclonal↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

A single chain Fv specific against Western equine encephalitis virus.

A recombinant single chain Fv (scFv) specific against Western equine encephalitis virus (WEE) was developed and characterized. The scFv was generated from 11D2 hybridoma producing anti-WEE antibody reactive to E1 component of viral envelope glycoprotein. V(L) and V(H) gene segments of 11D2 scFv were generated and joined together with a (gly4ser)3 linker by polymerase chain reaction (PCR). The resulting scFv was successfully expressed in P. pastoris expression system. Fifteen individual plasmids were tested and six of them were shown to drive scFv expression. DNA sequence analysis from three productive plasmids showed that they all carried the same VL and V(H) gene segments with a few base differences. Comparison of 11D2 scFv DNA sequence to the Kabat database showed that VH of 11D2 antibody belonged to subgroup IIID and subfamily XIV, while VL domain did not belong to any known subgroup or subfamily. Western blot analysis of 11D2 scFv using anti-c-myc antibody for detection showed different band pattern among clones derived from different plasmids. This was thought to be due to the different glycosylation where amino acid substitution occurred. Successful purification of 11D2 scFv could be done by immobilized metal affinity chromatography with an unoptimized yield of 700 microg/L. Functional studies showed that 11D2 scFv could bind to its respective WEE antigen as demonstrated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The binding affinity of 11D2 scFv is reasonably good compared to the parental 11D2 bivalent monoclonal antibody (MAb). Thus, 11D2 scFv and its derivatives have a potential use as immunotherapeutic and immunodiagnostic agents of WEE infections.

Amino Acid Sequence↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Relative affinity and specificity of monoclonal antibodies against prostate-specific antigen.

The relative affinities of a panel of antibodies submitted to the ISOBM TD-3 PSA Workshop were determined by direct-binding ELISA. The Workshop antibodies were also tested for reactivity with the prostate-specific antigen alpha1-antichymotrypsin complex (PSA-ACT) and cross-reactivity with porcine pancreatic kallikrein. There was a wide range of affinities observed for the panel of antibodies. Twelve antibodies failed to react with the PSA-ACT complex, and 1 antibody was found to recognize an epitope on ACT but not on PSA. Only 2 antibodies were found to react with porcine pancreatic kallikrein, a protein with 64% sequence homology with human glandular kallikrein-2 (hK2).

Antibodies, Monoclonal↗

PubMed1998

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.

Animals↗

PubMed1998

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography.

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid-hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid-hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.

Amino Acid Sequence↗