Rama S. Verma

Humanized immunotoxins: A new generation of immunotoxins for targeted cancer therapy

Chemotherapy, radiation, and surgery are the conventional treatment modalities for cancer. The success achieved with these approaches has been limited due to several factors like chemoresistance to drugs, non‐specificity leading to peripheral toxicity, and non‐resectable tumors. To combat these problems, the concept of targeted therapy using immunotoxins was developed. Immunotoxins are chimeric proteins with a cell‐selective ligand chemically linked or genetically fused to a toxin moiety and can target cancer cells overexpressing tumor‐associated antigens, membrane receptors, or carbohydrate antigens. Ligands for these receptors or monoclonal antibodies or single chain variable fragments directed against these antigens are fused with bacterial or plant toxins and are made use of as immunotoxins. Pseudomonas exotoxin, anthrax toxin, and diphtheria toxin are the commonly used bacterial toxins. Ricin, saporin, gelonin, and poke weed antiviral protein are the plant toxins utilized in immunotoxin constructs. Several such fusion proteins are in clinical trials, and denileukin difitox is a FDA‐approved fusion protein. In spite of the promise shown by bacterial‐ and plant toxin‐based chimeric proteins, their clinical application is hampered by several factors like immunogenicity of the toxin moiety and non‐specific toxicity leading to vascular leak syndrome. In order to overcome these problems, a novel generation of immunotoxins in which the cytotoxic moiety is an endogenous protein of human origin like proapoptotic protein or RNase has been developed. This review summarizes the advances in this new class of fusion protein and the future directions to be explored. (Cancer Sci 2009)

An improved protocol for primary culture of cardiomyocyte from neonatal mice

The primary culture of neonatal mice cardiomyocyte model enables researchers to study and understand the morphological, biochemical, and electrophysiological characteristics of the heart, besides being a valuable tool for pharmacological and toxicological studies. Because cardiomyocytes do not proliferate after birth, primary myocardial culture is recalcitrant. The present study describes an improved method for rapid isolation of cardiomyocytes from neonatal mice, as well as the maintenance and propagation of such cultures for the long term. Immunocytochemical and gene expression data also confirmed the presence of several cardiac markers in the beating cells during the long-term culture condition used in this protocol. The whole culture process can be effectively shortened by reducing the enzyme digestion period and the cardiomyocyte enrichment step.

Therapeutic potential of anticancer immunotoxins.

Immunotoxins are chimeric proteins consisting of a tumor-specific ligand (antibody, growth factor or peptide) linked to a modified toxin. These molecules bind to cell surface receptors and are subsequently internalized by endocytosis, resulting in cell death. Advances in protein engineering and phage display have enabled the selection of high-affinity targeting moieties. Denileukin diftitox is the only FDA-approved immunotoxin, although others such as BL22 are currently in different phases of development. This review elaborates the key findings of the important clinical studies relating to various chimeric toxins.

Nanomaterials for early detection of cancer biomarker with special emphasis on gold nanoparticles in immunoassays/sensors

At the onset of cancer a selective protein or gene based biomarker gets elevated or modified in body fluids or tissues. Early diagnosis of these markers can greatly improve the survival rate or facilitate effective treatment with different modalities. Though the sophisticated imaging technologies like Magnetic Resonance Imaging, Positron Emission Tomography and Computed Tomography have the impact of nanotechnology on their improved performance, they are however unsuitable for early detection of cancer biomarkers or their quantification. Other approaches for cancer diagnosis based on cell morphology and microscopy (biopsies) are too not conclusive for early diagnosis of cancer. The only hope for early diagnosis of cancer in near future is by the detection of cancer biomarkers using immunoassays/sensors that are reformed by Nanotechnology. Attractive properties of nanoparticles have miraculously lifted up the design, fabrication, sensitivity and multiplexing of these immunoassays/sensors in biomarker detection. With this aspect we have explored the recent advancements in immunosensing techniques that were developed exploiting the unique properties of gold nanoparticles. We have also discussed the possible future trends with respect to gold nanoparticle-coupled microfluidic sensors; paper based analytical devices and the single-molecule biosensing.

Therapeutic targets and recent advances in protein immunotoxins

Targeted therapy has replaced the conventional methods of disease management with the advances in recombinant technology and increased understanding of molecular mechanisms of diseases. The immunotoxin strategy for diseases like cancer and a variety of autoimmune disorders has been used successfully in the past since its discovery. Since bacterial, fungal and plant toxins have various limitations like toxicity and immunogenicity, studies on fully humanized immunotoxins have gained attraction recently, which reduced toxicity significantly. Improved methods of antibody engineering have led to the emergence of various new formats of immunotoxins. This review summarizes the target moieties used in immunotoxin constructs in different diseases and describes the recent advances in immunotoxin targeting.

Targeted therapy of cancer using diphtheria toxin-derived immunotoxins

The mortality rate in cancer patients demands novel therapy. One of the novel approaches developed in recent decades includes immunotoxins. Cancer cells frequently have specific growth factor receptors/antigens overexpressed on their surface; this is the principle of selective targeting of immunotoxins. Ligands recognizing these receptors and antigens can be conjugated to modified toxins. Continuous efforts are being made (i) to investigate molecules exclusively expressed on cancer cells, (ii) to improve the specificity and efficacy of these immunotoxins, (iii) to eliminate side effects (iv) to decrease immunogenicity and (v) to improve pharmacokinetics and ensure better drug delivery.

Development of a targeted siRNA delivery system using FOL-PEG-PEI conjugate

Receptor mediated delivery of siRNA enables silencing of target genes in specific tissues. Folate receptor (FR) is an attractive target for tumor-selective gene delivery. The focus of this study was to deliver the dihydrofolate reductase (DHFR) siRNA expressing plasmid and to silence the DHFR gene in FR positive KB cells, by complexing the plasmid with a folate-polyethylene glycol-polyethylenimine (FOL-PEG-PEI) conjugate, as a gene carrier. A DHFR siRNA sequence was cloned into a pSUPER-RNAi vector and complexed with the FOL-PEG-PEI conjugate. The complex was characterized by particle size analyzer, gel retardation and DNase protection assay. The FOL-PEG-PEI/pSUPER-siDHFR complex was transfected to FR overexpressing (KB) and FR negative (A549) cells. The transfection effiencies and gene inhibition were analyzed by fluorescence microscopy and RT-PCR. The pSUPER-siDHFR/PEI-PEG-FOL complex delivered the siRNA vector and inhibited DHFR gene in KB cells, while A549 cells were unaffected. Lipofectamine mediated transfection of pSUPER-siDHFR, delivered the vector and inhibited the DHFR gene in both KB and A549 cells. FR mediated delivery of siDHFR complexed with PEI-PEG-FOL conjugate inhibits the DHFR expression in FR positive cells alone. This strategy can be extended to deliver a wide range of drugs and post-transcriptional gene silencing therapeutics.

Effect of perturbation of specific folate receptors during in vitro erythropoiesis.

Although antisera to specific placental folate receptors inhibits the uptake of 5-methyltetrahydrofolate into cultured malignant human cells, little is known of the functional significance of folate receptors in normal human cells. Human bone marrow cells were therefore assayed for erythropoietic burst-forming units in the presence of an antihuman placental folate receptor serum and preimmune serum to determine the role of such a receptor in erythroid differentiation. When marrow cells were assayed in the presence of anti-receptor antiserum, there was (i) a threefold increase in erythropoietic burst formation and a twofold increase in the number of cells per erythroid burst; (ii) morphological evidence for nuclear/cytoplasmic dissociation of orthochromatic normoblasts composing erythroid bursts (megaloblastic erythropoiesis); (iii) intracellular folate deficiency with a 70% reduction of intracellular folate in antiserum treated cells as compared with control cells; and (iv) complete reversal of antiserum-induced changes on preincubation of antiserum with purified human placental folate receptor. These data support the conclusion that folate receptors on marrow cells provide an important function in the cellular uptake of folates during in vitro erythropoiesis. This process of folate uptake also appears to play a pivotal role in the differentiation and proliferation of erythroid progenitor cells.

Identification of high affinity folate binding proteins in human erythrocyte membranes.

Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging.

Antibacterial activity and chemical composition of essential oil of Pamburus missionis.

AIM OF THE STUDY The aerial parts of Pamburus missionis (Wight) Swingle (Rutaceae) are traditionally used in the treatment of swelling, chronic rheumatism, paralysis, and puerperal diseases.The aim of this work was to investigate the chemical composition and antibacterial activity of essential oil of Pamburus missionis against different bacterial strains. MATERIALS AND METHODS The essential oil was obtained from fresh leaves by hydro distillation method and its chemical composition was analyzed by GC and GC-MS. The antibacterial activity was determined by disc diffusion and micro broth dilution assay. RESULTS GC-MS analysis of the essential oil revealed the presence of 1-tridecanol (38.4%), n-hexadecanoic acid (16.1%), oxygenated monoterpenes (14.4%), monoterpene hydrocarbons (3.1%) and eugenol (1.9%) as the major components. The oil exhibited antibacterial activity at 100 and 450 microg against the test organisms with inhibition zones of 7-25 mm and minimal inhibitory concentrations values in the range of 10-100 and >100 mg/ml. CONCLUSION The present study reveals the antibacterial potency of essential oil of Pamburus missionis. The use of Pamburus missionis in the treatment of various ailments and puerperal diseases can be attributed to its antibacterial property.