Ilya Lyakhov

Discovery of Fur binding site clusters in Escherichia coli by information theory models

Fur is a DNA binding protein that represses bacterial iron uptake systems. Eleven footprinted Escherichia coli Fur binding sites were used to create an initial information theory model of Fur binding, which was then refined by adding 13 experimentally confirmed sites. When the refined model was scanned across all available footprinted sequences, sequence walkers, which are visual depictions of predicted binding sites, frequently appeared in clusters that fit the footprints (∼83% coverage). This indicated that the model can accurately predict Fur binding. Within the clusters, individual walkers were separated from their neighbors by exactly 3 or 6 bases, consistent with models in which Fur dimers bind on different faces of the DNA helix. When the E. coli genome was scanned, we found 363 unique clusters, which includes all known Fur-repressed genes that are involved in iron metabolism. In contrast, only a few of the known Fur-activated genes have predicted Fur binding sites at their promoters. These observations suggest that Fur is either a direct repressor or an indirect activator. The Pseudomonas aeruginosa and Bacillus subtilis Fur models are highly similar to the E. coli Fur model, suggesting that the Fur–DNA recognition mechanism may be conserved for even distantly related bacteria.

Hyperthermia-triggered intracellular delivery of anticancer agent to HER2+ cells by HER2-specific Affibody (ZHER2-GS-Cys)-Conjugated Thermosensitive Liposomes (HER2+ Affisomes)

We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2(+)affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2(+)affisomes). HER2(+)affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2(+) affisomes showed at least 10-fold increase in binding to HER2(+) cells (SK-BR-3) when compared to HER2(-) cells (MDA-MB-468) at 37°C. A competition experiment using free ZHER2-GS-Cys blocked HER2(+) affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2(+) affisomes accumulated in the cytosol of SK-BR-3 cells at 37°C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2(+) affisome/SK-BR-3 cell complexes with a 45°C (±1°C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20min at 45°C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2(+)affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2(+)affisomes. Brief exposure of liposome-cell complexes at 45°C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2(+)affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.

Affitoxin—A Novel Recombinant, HER2-specific, Anticancer Agent for Targeted Therapy of HER2-positive Tumors

Expression of the human epidermal growth factor receptor 2 (HER2) is amplified in 25% to 30% of breast cancers and has been associated with an unfavorable prognosis. Here we report the construction, purification, and characterization of Affitoxin—a novel class of HER2-specific cytotoxic molecules combining HER2-specific Affibody molecule as a targeting moiety and PE38KDEL, which is a truncated version of Pseudomonas exotoxin A, as a cell killing agent. It is highly soluble and does not require additional refolding, oxidation, or reduction steps during its purification. Using surface plasmon resonance technology and competitive binding assays, we have shown that Affitoxin binds specifically to HER2 with nanomolar affinity. We have also observed a high correlation between HER2 expression and retention of Affitoxin bound to the cell surface. Affitoxin binding and internalization is followed by Pseudomonas exotoxin A activity domain-mediated ADP-ribosylation of translation elongation factor 2 and, consequently, inhibition of protein synthesis as shown by protein expression analysis of HER2-positive cells treated with Affitoxin. Measured IC50 value for HER2-negative cells MDA-MB468 (65±2.63 pM) was more than 20 times higher than the value for low HER2 level-expressing MCF7 cells (2.56±0.1 pM), and almost 3 orders of magnitude higher for its HER2-overexpressing derivative MCF7/HER2 (62.7±5.9 fM). These studies suggest that Affitoxin is an attractive PE38-based candidate for treatment of HER2-positive tumors.

HER2-Affitoxin: A Potent Therapeutic Agent for the Treatment of HER2-Overexpressing Tumors

Purpose: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. Experimental Design: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. Results:In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T1/2 < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. Conclusions: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies. Clin Cancer Res; 17(15); 5071–81. ©2011 AACR.

HER2-and EGFR-specific Affiprobes—Novel Recombinant Optical Probes for Cell Imaging

The human epidermal growth factor receptors, EGFR and HER2, are members of the EGFR family of cell‐surface receptors/tyrosine kinases. EGFR‐ and HER2‐positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate, compared to EGFR‐ or HER2‐negative cancers. The details of HER2 proto‐oncogenic functions are not deeply understood, partially because of a restricted availability of tools for EGFR and HER2 detection (A. Sorkin and L. K. Goh, Exp. Cell Res. 2009, 315, 683–696). We have created photostable and relatively simple‐to‐produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called affiprobes, consist of a targeting moiety, a HER2‐ or EGFR‐specific Affibody® molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of affiprobes. Affiprobes are able to stain both live cells and frozen tumor xenograph sections. This type of optical probe can easily be extended for targeting other cell‐surface antigens/ receptors.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy.

Affibody-DyLight Conjugates for In Vivo Assessment of HER2 Expression by Near-Infrared Optical Imaging

Purpose Amplification of the HER2/neu gene and/or overexpression of the corresponding protein have been identified in approximately 20% of invasive breast carcinomas. Assessment of HER2 expression in vivo would advance development of new HER2-targeted therapeutic agents and, potentially, facilitate choice of the proper treatment strategy offered to the individual patient. We present novel HER2-specific probes for in vivo evaluation of the receptor status by near-infrared (NIR) optical imaging. Experimental Design Affibody molecules were expressed, purified, and labeled with NIR-fluorescent dyes. The binding affinity and specificity of the obtained probe were tested in vitro. For in vivo validation, the relationship of the measured NIR signal and HER2 expression was characterized in four breast cancer xenograft models, expressing different levels of HER2. Accumulation of Affibody molecules in tumor tissue was further confirmed by ex vivo analysis. Results Affibody-DyLight conjugates showed high affinity to HER2 (KD = 3.66±0.26). No acute toxicity resulted from injection of the probes (up to 0.5 mg/kg) into mice. Pharmacokinetic studies revealed a relatively short (37.53±2.8 min) half-life of the tracer in blood. Fluorescence accumulation in HER2-positive BT-474 xenografts was evident as soon as a few minutes post injection and reached its maximum at 90 minutes. On the other hand, no signal retention was observed in HER2-negative MDA-MB-468 xenografts. Immunostaining of extracted tumor tissue confirmed penetration of the tracer into tumor tissue. Conclusions The results of our studies suggest that Affibody-DyLight-750 conjugate is a powerful tool to monitor HER2 status in a preclinical setting. Following clinical validation, it might provide complementary means for assessment of HER2 expression in breast cancer patients (assuming availability of proper NIR scanners) and/or be used to facilitate detection of HER2-positive metastatic lesions during NIR-assisted surgery.

Optical Imaging of Ovarian Cancer Using HER-2 Affibody Conjugated Nanoparticles

Computed Tomography (CT), Ultrasound (US), and Magnetic Resonance Imaging (MRI) have been the mainstay of clinical imaging regimens for the detection of ovarian cancer. However, without tumor specific contrast enhancement, these imaging modalities lack specificity and sensitivity in the detection of small primary and disseminated tumors in the peritoneal cavity. Herein, we illustrate a fairly new near infrared (NIR) optical imaging approach developed in our laboratory for the noninvasive detection of ovarian tumors using a HER-2 targeted nanoparticle-based imaging agent in an orthotopic mouse model of ovarian cancer. We used multimodal imaging approaches to detect the disease accurately and rapidly by utilizing a single imaging agent, NIR dye-labeled HER-2 affibody conjugated iron oxide nanoparticles. This agent targets HER-2 receptors, which are overexpressed in ovarian tumors. This chapter outlines materials and methods for the: (1) production of HER-2 targeted nanoparticles; (2) establishment of an orthotopic human ovarian cancer xenograft model; (3) monitoring of tumor growth by bioluminescence imaging; (4) administration of targeted nanoparticles followed by NIR optical imaging for the detection of orthotopic ovarian cancers with targeted accumulation of the nanoparticle imaging probes.

Abstract 5298: Affibody – NIR conjugate for optical imaging of HER2-expressing tumors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Amplification of the HER2/neu gene and/or overexpression of the protein have been identified in approximately 20% of invasive breast carcinoma, Particularly in breast cancer, elevated HER2 status is associated with increased proliferation and survival of cancer cells and thus contributes to poor therapy outcomes and unfavorable prognosis. Affibody molecules are unique class of artificial ligands. They are relatively small, (∼7-kDa), affinity proteins, structurally based on a 58-amino-acid scaffold derived from the Z domain of the Staphylococcus aureus- protein, and were obtained by combinatorial protein engineering. Optical imaging is a powerful tool allowing non-invasive in vivo analysis of macroscopic distribution of fluorescent labels. Introduction of Near-Infrared fluorescent beacons significantly improved the light penetration making the Over the past several years, there has been an explosion of reports describing successful in vivo NIR fluorescence imaging using antibodies or antibodies fragment or small molecules as contrast agents. Here we report the construction, in vitro and in vivo evaluation of a new Affibody based probe for Near Infrared imaging of HER2-positive tumors. Unique, C-terminal cysteine residue of HER2-Affibody molecules were modified with maleimide derivatives of optical beacons. The conjugates maintained high affinity to HER2 receptors with estimated KD value 3.66 ±0.26 nM using HER2 positive BT474 cells. Competition assay showed slight affinity reduction due to labeling (IC50=24.1 nM at 50nM labeled Affibody). No dye accumulation was observed for “non-specific” Affibody molecules (raised against Taq polymerase). HER2-Affibody binding was well correlated with receptor expression in five different cell lines as determined by FACS and HER2 Elisa respectively. Receptor specificity was also confirmed by immunofluorescence imaging: Cells with high HER2 level (SKBR3) showed almost exclusive membrane retention of the probe even after long period of incubation. Neither membrane retention nor intracellular uptake of the probe was observed for HER2-negative MDA MB-468 cells. In vivo characteristic of the probe was performed in mice subcutaneous tumor models using NIR fluorescence small-animal imager. The probe showed relatively fast clearance rate from the circulation after intravenous administration (T1/2=31.8±10.42 min). The highest accumulation was recorded for BT474 tumors 90 min post probe administration. No signal was detected in tumors injected with HER2-nonspecyfic probe or for HER2 negative tumors receiving HER2-Affibody. Interestingly, accumulation rate better correlated with HER2 expression level in the tumor than maximum uptake. HER2-dependend probe accumulation was confirmed also by IHC analysis. Our results suggest that optical imaging using Affibody-based probes, may become a noninvasive alternative tool for assessment of HER2 expression in breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5298. doi:10.1158/1538-7445.AM2011-5298

Abstract 1151: HER2- and EGFR-specific Affiprobes - Novel recombinant optical probes for cell imaging

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The human epidermal growth factor receptors EGFR (Her1, ErbB1) and HER2 (HER2/neu, ErbB2, or c-erb-b2) are members of EGFR (ErbB) family of cell-surface receptors/tyrosine kinases: EGFR, HER2, HER3, and HER4. There are several identified ligands for these receptors (except HER2), including EGF and TGF-alpha, that induce receptor dimerization followed by tyrosine autophosphorylation, which leads to cell proliferation. EGFR and HER2 are found overexpressed in a number of cancer types, such as breast, ovarian, salivary gland, stomach, kidney, colon, prostate, and non-small cell lung cancer. Research has shown that EGFR- or HER2-positive cancers represent a more aggressive disease with greater likelihood of recurrence, poorer prognosis, and decreased survival rate compared to EGFR- or HER2-negative cancers. The details of HER2 proto-oncogenic function, its internalization mechanism, and subcellular trafficking are not fully understood, partially because the availability of imaging reagents and tools for detection of EGFR and HER2 is still restricted to medium/low affinity antibody conjugated to small-molecule dyes. We have created photo-stable and relatively simple-to-prepare imaging probes for in vitro staining of EGFR and HER2. These new reagents, called Affiprobes, consist of the targeting moiety, a HER2- or EGFR-specific Affibody® molecule, and a fluorescent detection moiety mCherry (Red), or EGFP (Green). These fluorescent proteins were chosen due to their high stability and solubility and low toxicity for eukaryotic cells. Furthermore, HER2 and EGFR receptors can be detected simultaneously using corresponding optical filter sets. Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio for the Affiprobes. This type of optical probe can be easily extended to targeting other cell-surface antigens/receptors using different Affibody® molecules and fluorescent proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1151.