Rendall R. Strawbridge

Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.

Optimization of humanized IgGs in glycoengineered Pichia pastoris

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.

Feasibility studies of electrical impedance spectroscopy for early tumor detection in rats

Electrical impedance spectroscopy (EIS) has been previously reported as a technique for non invasive assessment of tissue change. Our previous in vivo studies demonstrated the ability of EIS to non-invasively detect and longitudinally follow tumor growth. This study was designed to determine the ability of EIS to detect tumors at a very early stage post-implantation. Complex impedance measurements were collected from eight rats with one control and one tumor implanted leg six or seven days after tumor cell inoculation. Legs were also imaged with computed tomography (CT) and ultrasound (US) in an effort to determine EIS resolution and sensitivity. Six of the animals were sacrificed immediately after imaging, and tissue was collected for histology and later co-registration of the pathology with the imaging techniques. Results show that EIS is able to repeatedly detect small tumors (<3 mm) and tumor-associated changes, whereas CT and US were not routinely capable of detecting pathological developments on this scale.

Real-time in vivo Cherenkoscopy imaging during external beam radiation therapy

Abstract. Cherenkov radiation is induced when charged particles travel through dielectric media (such as biological tissue) faster than the speed of light through that medium. Detection of this radiation or excited luminescence during megavoltage external beam radiotherapy (EBRT) can allow emergence of a new approach to superficial dose estimation, functional imaging, and quality assurance for radiation therapy dosimetry. In this letter, the first in vivo Cherenkov images of a real-time Cherenkoscopy during EBRT are presented. The imaging system consisted of a time-gated intensified charge coupled device (ICCD) coupled with a commercial lens. The ICCD was synchronized to the linear accelerator to detect Cherenkov photons only during the 3.25-μs radiation bursts. Images of a tissue phantom under irradiation show that the intensity of Cherenkov emission is directly proportional to radiation dose, and images can be acquired at 4.7  frames/s with SNR>30. Cherenkoscopy was obtained from the superficial regions of a canine oral tumor during planned, Institutional Animal Care and Use Committee approved, conventional (therapeutically appropriate) EBRT irradiation. Coregistration between photography and Cherenkoscopy validated that Cherenkov photons were detected from the planned treatment region. Real-time images correctly monitored the beam field changes corresponding to the planned dynamic wedge movement, with accurate extent of overall beam field, and expected cold and hot regions.

Recognition of endogenous ecotropic murine leukaemia viruses by anti-AKR/Gross virus cytotoxic T lymphocytes (CTL): epitope variation in a CTL-resistant virus.

AKR/Gross virus-specific cytotoxic T lymphocytes (CTL) from C57BL/6 (B6) mice are H-2Kb-restricted and recognize epitopes encoded by the prototype endogenous ecotropic murine leukaemia virus (Emv) AKR623. Four CTL epitopes have been identified by the use of synthetic peptides corresponding to AKR623-encoded amino acid sequences. Here we present both functional and nucleotide sequence data indicating that three closely related Emv share all of these CTL epitopes. We also found that one other murine leukaemia virus (MuLV) was not susceptible to lysis by these CTL. This is the ecotropic component of the LP-BM5 virus complex that causes murine AIDS. Nucleotide sequencing revealed that three of the four epitopes, including the immunodominant peptide, are altered in this virus. The other epitope was unchanged. These data implied that the inability of anti-AKR/Gross virus CTL to lyse cells infected with the LP-BM5 ecotropic (BM5eco) MuLV was due to the functional loss of three of the four CTL epitopes. Using recombinant vaccinia and Sindbis virus vectors, we have shown that the BM5eco-encoded form of the immunodominant epitope, which differs only by an arginine for lysine substitution at the N-terminal residue, fails to induce a CTL response in B6 mice. Immunization with BM5eco-infected cells also failed to induce MuLV-specific CTL. In light of the long in vivo passage history of the LP-BM5 complex in B6 mice, our results are consistent with a contribution of CTL-mediated immune selection to the evolution of the BM5eco MuLV.

Imaging and modification of the tumor vascular barrier for improvement in magnetic nanoparticle uptake and hyperthermia treatment efficacy.

The predicted success of nanoparticle based cancer therapy is due in part to the presence of the inherent leakiness of the tumor vascular barrier, the so called enhanced permeability and retention (EPR) effect. Although the EPR effect is present in varying degrees in many tumors, it has not resulted in the consistent level of nanoparticle-tumor uptake enhancement that was initially predicted. Magnetic/iron oxide nanoparticles (mNPs) have many positive qualities, including their inert/nontoxic nature, the ability to be produced in various sizes, the ability to be activated by a deeply penetrating and nontoxic magnetic field resulting in cell-specific cytotoxic heating, and the ability to be successfully coated with a wide variety of functional coatings. However, at this time, the delivery of adequate numbers of nanoparticles to the tumor site via systemic administration remains challenging. Ionizing radiation, cisplatinum chemotherapy, external static magnetic fields and vascular disrupting agents are being used to modify the tumor environment/vasculature barrier to improve mNP uptake in tumors and subsequently tumor treatment. Preliminary studies suggest use of these modalities, individually, can result in mNP uptake improvements in the 3-10 fold range. Ongoing studies show promise of even greater tumor uptake enhancement when these methods are combined. The level and location of mNP/Fe in blood and normal/tumor tissue is assessed via histopathological methods (confocal, light and electron microscopy, histochemical iron staining, fluorescent labeling, TEM) and ICP-MS. In order to accurately plan and assess mNP-based therapies in clinical patients, a noninvasive and quantitative imaging technique for the assessment of mNP uptake and biodistribution will be necessary. To address this issue, we examined the use of computed tomography (CT), magnetic resonance imaging (MRI), and Sweep Imaging With Fourier Transformation (SWIFT), an MRI technique which provides a positive iron contrast enhancement and a reduced signal to noise ratio, for effective observation and quantification of Fe/mNP concentrations in the clinical setting.

Assessment of intratumor non-antibody directed iron oxide nanoparticle hyperthermia cancer therapy and antibody directed IONP uptake in murine and human cells.

Hyperthermia, as an independent modality or in combination with standard cancer treatments such as chemotherapy and radiation, has been established in vitro and in vivo as an effective cancer treatment. However, despite efforts over the past 25 years, such therapies have never been optimized or widelyaccepted clinically. Although methods continue to improve, conventionally-delivered heat (RF, ultrasound, microwave etc) can not be delivered in a tumor selective manner. The development of antibody-targeted, or even nontargeted, biocompatible iron oxide nanoparticles (IONP) now allows delivery of cytotoxic heat to individual cancer cells. Using a murine mouse mammary adenocarcinoma (MTGB) and human colon carcinoma (HT29) cells, we studied the biology and treatment of IONP hyperthermia tumor treatment. Methods: Cancer cells (1 x 106) with or without iron oxide nanoparticles (IONP) were studied in culture or in vivo via implanted subcutaneously in female C3H mice, Tumors were grown to a treatment size of 150 mm3 and tumors volumes were measured using standard 3-D caliper measurement techniques. Mouse tumors were heated via delivery of an alternating magnetic field, which activated the nanoparticles, using a cooled 36 mm diameter square copper tube induction coil which provided optimal heating in 1.5 cm wide region of the coil. The IONPs were dextran coated and had a hydrodynamic radius of approximately 100 nm. For the in vivo studies, intra-tumor, peritumor and rectal (core body) temperatures were continually measured throughout the treatment period. Results: Although some eddy current heating was generated in non-target tissues at the higher field strengths, our preliminary IONP hyperthermia studies show that whole mouse AMF exposure @160 KHz and 400 or 550 Oe, for a 20 minutes (heat-up and protocol heating), provides a safe and efficacious tumor treatment. Initial electron and light microscopic studies (in vitro and in vivo) showed the 100 nm used in our studies are rapidly taken up and retained by the tumor cells. Additional in vitro studies suggest antibodies can significantly enhance the cellular uptake of IONPs.

Intratumoral iron oxide nanoparticle hyperthermia and radiation cancer treatment

The potential synergism and benefit of combined hyperthermia and radiation for cancer treatment is well established, but has yet to be optimized clinically. Specifically, the delivery of heat via external arrays /applicators or interstitial antennas has not demonstrated the spatial precision or specificity necessary to achieve appropriate a highly positive therapeutic ratio. Recently, antibody directed and possibly even non-antibody directed iron oxide nanoparticle hyperthermia has shown significant promise as a tumor treatment modality. Our studies are designed to determine the effects (safety and efficacy) of iron oxide nanoparticle hyperthermia and external beam radiation in a murine breast cancer model. Methods: MTG-B murine breast cancer cells (1 x 106) were implanted subcutaneous in 7 week-old female C3H/HeJ mice and grown to a treatment size of 150 mm3 +/- 50 mm3. Tumors were then injected locally with iron oxide nanoparticles and heated via an alternating magnetic field (AMF) generator operated at approximately 160 kHz and 400 - 550 Oe. Tumor growth was monitored daily using standard 3-D caliper measurement technique and formula. specific Mouse tumors were heated using a cooled, 36 mm diameter square copper tube induction coil which provided optimal heating in a 1 cm wide region in the center of the coil. Double dextran coated 80 nm iron oxide nanoparticles (Triton Biosystems) were used in all studies. Intra-tumor, peri-tumor and rectal (core body) temperatures were continually measured throughout the treatment period. Results: Preliminary in vivo nanoparticle-AMF hyperthermia (167 KHz and 400 or 550 Oe) studies demonstrated dose responsive cytotoxicity which enhanced the effects of external beam radiation. AMF associated eddy currents resulted in nonspecific temperature increases in exposed tissues which did not contain nanoparticles, however these effects were minor and not injurious to the mice. These studies also suggest that iron oxide nanoparticle hyperthermia is more effective than non-nanoparticle tumor heating techniques when similar thermal doses are applied. Initial electron and light microscopy studies of iron oxide nanoparticle and AMF exposed tumor cells show a rapid uptake of particles and acute cytotoxicity following AMF exposure.

Modulation of hypoxia by magnetic nanoparticle hyperthermia to augment therapeutic index.

A hypoxic microenvironment in solid tumors has been known to cause resistance to standard therapies and to increase the malignant potential of tumors. The utilization of magnetic nanoparticle hyperthermia (mNPH) has shown promise in improving therapeutic outcome by (1) killing of hypoxic tumor cells directly and (2) increasing tumor oxygenation and therefore susceptibility to therapies. In this study, the interaction of a hypoxic microenvironment with mNPH efficacy was investigated in a human breast cancer orthotopic xenograft model. Using electron paramagnetic resonance (EPR) to assess in vivo oxygen concentration in tumors repeatedly and non-invasively, we found that mNPH increased tumor pO₂ from 3.5 to 68.8 mmHg on average for up to 10 days. Tumors treated once with mNPH showed growth delay. On Transmission Electron Microscopy, magnetic nanoparticles (mNPs) were localized intracellularly in multiple vesicles in the cytoplasm of cells within tumors 48 h after incubation of mNP. In conclusion, mNPH increased tumor oxygenation in vivo and resulted in decreased growth of hypoxic tumors. Future studies will establish tumor pO₂-guided multimodal therapies, such as mNPH and radiation, to improve therapeutic efficacy.

Comparison of Iron Oxide Nanoparticle and Waterbath Hyperthermia Cytotoxicity

The development of medical grade iron oxide nanoparticles (IONP) has renewed interest in hyperthermia cancer therapy. Because of their modifiable size and heating capabilities under an AC magnetic field (alternating magnetic field, AMF), IONPs have the potential to damage or kill cells in a manner more therapeutically efficient than previous hyperthermia techniques. The use of IONPs in hyperthermia cancer therapy has prompted numerous questions regarding the cytotoxic mechanism associated with IONP heat therapy and if such mechanism is different (more or less effective) with respect to conventional hyperthermia techniques. In this in vitro study, we determine the immediate and long-term (24 hours) cytotoxic effects of isothermal IONP hyperthermia treatment versus a conventional global heating technique (water bath). Using the same heating time and temperature we showed significantly greater cytotoxicity in IONP-heated cells as opposed to water bath-treated cells. We postulate that the difference in treatment efficacy is due to the spatial relationship of particle-induced thermal damage within cells. Although the exact mechanism is still unclear, it appears likely that intracellular IONPs have to achieve a very high temperature in order to heat the surrounding environment; therefore it is reasonable to assume that particles localized to specific areas of the cell such as the membrane can deliver exacerbated injury to those areas. In this experiment, although detectable global temperature for the particle-heated cells stands comparable to the conventional heat treatment, particle-induced cell death is higher. From the results of this study, we propose that the mechanism of IONP hyperthermia renders enhanced cytotoxicity compared to conventional waterbath hyperthermia at the same measured thermal dose.