Cindy Zer

siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells.

BackgroundDoxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant administration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, we examined the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase on breast cancer cells. We found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, we observed an enhanced and sustained apoptosis induction in breast cancer cells.MethodssiRNA targeting the mRNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT mRNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay. The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice.The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examined in vitro by MTT assay, FACS and SA-β-galactosidase staining.ResultsThe hTERT siRNA effectively knocked down the mRNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control. In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells. The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5 μM doxorubicin treatment had a comparable effect but with a slower kinetics. The combination of hTERT siRNA and 0.5 μM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments.ConclusionThe study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer. When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.

Protein microarray: sensitive and effective immunodetection for drug residues

BackgroundVeterinary drugs such as clenbuterol (CL) and sulfamethazine (SM2) are low molecular weight (<1000 Da) compounds, or haptens, that are difficult to develop immunoassays due to their low immunogenicity. In this study, we conjugated the drugs to ovalbumin to increase their immunogenicity for antiserum production in rabbits and developed a protein microarray immunoassay for detection of clenbuterol and sulfamethazine. The sensitivity of this approach was then compared to traditional ELISA technique.ResultsThe artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA) showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g) than the ci-ELISA (0.1 ng/g) for detection of CL residues.ConclusionsThe protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.

Development of a High Affinity, Non-covalent Biologic to Add Functionality to Fabs

Functionalization of monoclonal antibodies (mAbs) requires chemical derivatization and/or genetic manipulation. Inherent in these methods are challenges with protein heterogeneity, stability and solubility. Such perturbations could potentially be avoided by using a high affinity, non-covalent intermediate to bridge the desired functionality to a stable mAb. Recently, we engineered a binding site for a peptide named “meditope” within the Fab of trastuzumab. Proximity of the meditope site to that of protein L suggested an opportunity to enhance the meditopes moderate affinity. Joined by a peptide linker, the meditope-protein L construct has a KD ~ 180 pM - a 7000-fold increase in affinity. The construct is highly specific to the engineered trastuzumab, as demonstrated by flow cytometry. Moreover, the fusion of a bulky GFP to this construct did not affect the association with cell surface antigens. Collectively, these data indicate this specific, high affinity construct can be developed to rapidly add new functionality to mAbs.

Mechanically interlocked functionalization of monoclonal antibodies

Because monoclonal antibodies (mAbs) have exceptional specificity and favorable pharmacology, substantial efforts have been made to functionalize them, either with potent cytotoxins, biologics, radionuclides, or fluorescent groups for therapeutic benefit and/or use as theranostic agents. To exploit our recently discovered meditope–Fab interaction as an alternative means to efficiently functionalize mAbs, we used insights from the structure to enhance the affinity and lifetime of the interaction by four orders of magnitude. To further extend the lifetime of the complex, we created a mechanical bond by incorporating an azide on the meditope, threading the azide through the Fab, and using click chemistry to add a steric group. The mechanically interlocked, meditope–Fab complex retains antigen specificity and is capable of imaging tumors in mice. These studies indicate it is possible to “snap” functionality onto mAbs, opening the possibility of rapidly creating unique combinations of mAbs with an array of cytotoxins, biologics, and imaging agents.Meditope-Fab is a peptide-antibody complex potentially useful for drug delivery and diagnostic, but a short half-life prevents its use in vivo. Here the authors engineer the complex to improve its stability, create functionalized antibodies by click chemistry and use them for in vivo tumor imaging.

Abstract 598: Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Therapeutic antibodies can bind to cell-surface receptors, prompting receptor internalization and effectively blocking further stimulation by the cognate receptor ligands. Receptor internalization is also exploited by antibody-drug conjugates (ADC) to promote effective delivery of cytotoxic payload to the inside of the cell. Meditope Biosciences has developed a way to use its SnAP (Site-specfi novel Antibody Platform) technology to promote enhanced internalization of antibody receptor complexes. The Meditopes SnAP technology functionally enables antibodies to bind to specific peptides, termed “meditopes’, and this property can be used to directly facilitate receptor crosslinking when meditope enabled antibodies are bound to cell surface receptors. Here we show that the affinity of the meditope enabled antibodies (trastuzumab anti-HER2 and gemtuzumab anti-CD33) for cell surface receptors results in the formation of an antibody receptor complex that can be detected by native gel electrophoresis and gel filtration. Binding of the meditope peptide to the enabled antibody does not interfere with normal antigen-antibody interaction and the formation of the peptide bound antibody:receptor complexes results in accelerated internalization of the complex, as shown by decreased surface fluorescence of Alexa 488-labeled SnAP peptides as well as by increased pHrodo-Red increased fluorescence of internalized antibodies when they reach the endosomal compartment. Finally, as a result this increased internalization, the downregulation of specific antibody mediated signaling events are much more effectively downregulated in the presence of the meditope peptide complex as compared to the antibody alone. Citation Format: Calin D. Dumitru, Elisabeth Gardiner, John Williams, Cindy Zer. Utilization of Meditope Biosciences SnAP technology to enhance antibody internalization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 598.