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.

One potential cancer treatment selectively deposits heat to the tumor through activation of magnetic nanoparticles inside the tumor. This can damage or kill the cancer cells without harming the surrounding healthy tissue. The properties assumed to be most important for this heat generation (saturation magnetization, amplitude and frequency of external magnetic field) originate from theoretical models that assume non-interacting nanoparticles. Although these factors certainly contribute, the fundamental assumption of ‘no interaction’ is flawed and consequently fails to anticipate their interactions with biological systems and the resulting heat deposition. Experimental evidence demonstrates that for interacting magnetite nanoparticles, determined by their spacing and anisotropy, the resulting collective behavior in the kilohertz frequency regime generates significant heat, leading to nearly complete regression of aggressive mammary tumors in mice.

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.

Magnetic nanoparticles are being developed for a wide range of biomedical applications. In particular, hyperthermia involves heating the magnetic nanoparticles through exposure to an alternating magnetic field. These materials offer the potential to selectively treat cancer by heating cancer tissue locally and at the cellular level. This may be a successful method if there are enough particles in a tumor possessing a sufficiently high specific absorption rate (SAR) to deposit heat quickly while minimizing thermal damage to surrounding tissue. High SAR magnetic nanoparticles have been developed and used in mouse models of cancer. The magnetic nanoparticles comprise iron oxide magnetic cores (mean core diameter of 50 nm) surrounded by a dextran layer shell for colloidal stability. In comparing two similar systems, the saturation magnetization is found to play a crucial role in determining the SAR, but is not the only factor of importance. (A difference in saturation magnetization of a factor of 1.5 yields a difference in SAR of a factor of 2.5 at 1080 Oe and 150 kHz.) Variations in the interactions due to differences in the dextran layer, as determined through neutron scattering, also play a role in the SAR. Once these nanoparticles are introduced into the tumor, their efficacy, with respect to tumor growth, is determined by the location of the nanoparticles within or near the tumor cells and the association of the nanoparticles with the delivered alternating magnetic field (AMF). This association (nanoparticle SAR and AMF) determines the amount of heat generated. In our setting, the heat generated and the time of heating (thermal dose) provides a tumor gross treatment response which correlates closely with that of conventional (non-nanoparticle) hyperthermia. This being said, it appears specific aspects of the nanoparticle hyperthermia cytopathology mechanism may be very different from that observed in conventional cancer treatment hyperthermia.