Incorporation of CdSe/ZnS semiconductor quantum dots (QDs) into viral particles provides a new paradigm for the design of intracellular microscopic probes and vectors. Several strategies for the incorporation of QDs into viral capsids were explored; those functionalized with poly(ethylene glycol) (PEG) can be self-assembled into viral particles with minimal release of photoreaction products and enhanced stability against prolonged irradiation.

Alphaviruses are animal viruses holding great promise for biomedical applications as drug delivery vectors, functional imaging probes, and nanoparticle delivery vesicles because of their efficient in vitro self-assembly properties. However, due to their complex structure, with a protein capsid encapsulating the genome and an outer membrane composed of lipids and glycoproteins, the in-vitro self-assembly of viruslike particles, which have the functional virus coat but carry an artificial cargo, can be challenging. Fabrication of such alphavirus-like particles is likely to require a two-step process: first, the assembly of a capsid structure around an artificial core, second the addition of the membrane layer. Here we report progress made on the first step: the efficient self-assembly of the alphavirus capsid around a functionalized nanoparticle core.

Viral nanoparticles used for biomedical applications must be able to discriminate between tumor or virus-infected host cells and healthy host cells. In addition, viral nanoparticles must have the flexibility to incorporate a wide range of cargo, from inorganic metals to mRNAs to small molecules. Alphaviruses are a family of enveloped viruses for which some species are intrinsically capable of systemic tumor targeting. Alphavirus virus-like particles, or viral nanoparticles, can be generated from in vitro self-assembled core-like particles using nonviral nucleic acid. In this work, we expand on the types of cargo that can be incorporated into alphavirus core-like particles and the molecular requirements for packaging this cargo. We demonstrate that different core-like particle templates can be further enveloped to form viral nanoparticles that are capable of cell entry. We propose that alphaviruses can be selectively modified to create viral nanoparticles for biomedical applications and basic research.

Size polydispersity of immature human immunodeficiency virus type 1 particles represents a challenge for traditional methods of biological ultra-structural analysis. An in vitro model for immature HIV-1 particles constructed from recombinant Gag proteins lacking residues 16–99 and the p6 domain assembled around spherical nanoparticles functionalized with DNA. This template-directed assembly approach led to a significant reduction in size polydispersity and revealed previously unknown structural features of immature-like HIV-1 particles. Electron microscopy and image reconstruction of these particles suggest that the Gag shell formed from different protein regions which are connected by a “scar” – an extended defect connecting the edges of two continuous, regularly packed protein layers. Thus, instead of a holey protein array, the experimental model presented here appears to consist of a continuous array of ~5000 proteins enveloping the core, in which regular regions are separated by extended areas of disorder.

We encapsulated gadolinium oxide (Gd2O3) nanoparticles within phospholipid micelles as a novel low cytotoxic T1-weighted MRI imaging contrast agent (MGdNPs) that can also deliver small molecules such as DNA plasmids. MGdNPs show relatively good MRI relaxivity values, negligible cytotoxicity, excellent cellular uptake and expression of DNA plasmids in vivo. Biodistribution studies in mice show that intranasal and intraperitoneal administration of MGdNPs can effectively target specific organs.

Virus life stages often constitute a complex chain of events, difficult to track in-vivo and in real-time. Challenges are associated with spatial and time limitations of current probes: most viruses are smaller than the diffraction limit of optical microscopes while the entire time-scale of virus dynamics spans over 8 orders of magnitude. Thus, virus processes such as entry, disassembly, and egress have generally remained poorly understood. Here we discuss photothermal heterodyne imaging (PHI) as a possible alternative to fluorescence microscopy in the study of single virus-like nanoparticle (VNP) dynamics, with relevance in particular to virus uncoating. Being based on optical absorption rather than emission, PHI could potentially surpass some of the current limitations associated with fluorescent labels. As proof-of-principle, single VNPs self-assembled from 60 nm DNA-functionalized gold nanoparticles (DNA-Au NPs) encapsulated in a Gag protein shell of the Human immunodeficiency virus (HIV-1) were imaged and their photothermal response compared with DNA-Au NPs. For the first-time, the protein stoichiometry of a single virus-like particle was estimated by a method other than electron microscopy.

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy.

Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PCa). Current therapies target the ligand-binding domain (LBD) of AR by inhibiting production or binding of ligand. In castration-resistant PCa (CRPC), AR isoforms lacking the LBD are highly expressed, resulting in constitutive, ligand-independent AR signaling. Therapies to block ligand-independent AR signaling have not been introduced into clinical use. We have developed bispecific antibodies (bsAbs) that penetrate PCa cells and bind to the N-terminal domain of the AR, inhibiting AR signaling. The 3E10-AR441 bsAb was engineered by connecting two single-chain variable fragments (scFv) with a linker. One half of the bsAb molecule is scFv 3E10, derived from a lupus anti-DNA antibody. This module enters cells through the ENT2 nucleoside salvage receptor and locates in the nucleus. The other half is an scFv based on the anti-AR monoclonal antibody AR441. The 3E10-AR441 bsAb is expressed in yeast as a single recombinant protein. Treatment of LNCaP with bsAb resulted in nuclear accumulation of the antibody, visualized by confocal microscopy. The 3E10-AR441 bsAb also engaged its target under denaturing and non-denaturing conditions. The bsAb could detect AR when used to probe an AR immunoblot. It could also immunoprecipitate WT AR, as well as mutant/variant AR lacking the LBD (AR(Q640X), Arv7). The scFv 3E10 alone did not bind to AR. BsAb binding affinity to AR was assessed using a competitive sandwich-type ELISA. An anti-AR antibody-coated microtiter plate was used to capture WT AR or mutant\variant AR from cell lysates. AR441-HRP antibody conjugate was mixed with increasing concentrations of 3E10-AR441 or AR441 as competitors. Binding affinity of 3E10-AR441 to WT AR (180nM) was higher than that of the parental AR441 MoAb (7nM). Affinity of bsAb was 3 fold higher to WT AR than to an AR(Q640X) mutant. The bsAb blocked genomic AR signaling in LNCaP cells, as measured by dihydrotestosterone (DHT) activation of both artificial (ARE-luciferase) and endogenous (PSA) reporters. The magnitude of inhibition was similar to that seen with enzalutamide at 5 μM. The 3E10 scFv alone had minimal effect on reporter gene expression. Non-genomic AR signaling was measured by calcium release upon DHT treatment. Calcium5 dye was added to LNCap treated with variable doses of bsAb or 3E10. The fluorescence intensity is proportional to the amount of calcium released. 3E10-AR441 blocked DHT-induced release of calcium while 3E10 did not. Current work is focused on the design of 3E10-AR441 derivatives with enhanced binding affinity towards AR mutants lacking the LBD. 3E10-AR441 bsAb is an attractive therapeutic agent due to its ability to inhibit AR function in a ligand-independent manner. Citation Format: Nancy L. Goicochea, Maria Garnovskaya, Mary Blanton, Grace Chan, Richard Weisbart, Michael Lilly. Cell-penetrating bispecific antibodies for targeting androgen receptor signaling in advanced prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 642. doi:10.1158/1538-7445.AM2015-642