Treatment of Hepatocellular Carcinoma by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant.

PURPOSE: To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Mice with bilateral, subcutaneous RIL-175 cell-derived HCCs were randomized to 4 groups: (a) phosphate-buffered saline (control), (b) cryoablation only (Cryo), (c) CPMV-treated only (CPMV), and (d) cryoablation plus CPMV-treated (Cryo + CPMV) (N = 11-14 per group). Intratumoral CPMV was administered every 3 days for 4 doses, with cryoablation performed on the third day. Contralateral tumors were monitored. Tumor growth and systemic chemokine/cytokine levels were measured. A subset of tumors and spleens were harvested for immunohistochemistry (IHC) and flow cytometry. One- or 2-way analysis of variance was performed for statistical comparisons. A P value of <.05 was used as the threshold for statistical significance. RESULTS: At 2 weeks after treatment, the Cryo and CPMV groups, alone or combined, outperformed the control group in the treated tumor; however, the Cryo + CPMV group showed the strongest reduction and lowest variance (1.6-fold ± 0.9 vs 6.3-fold ± 0.5, P < .0001). For the untreated tumor, only Cryo + CPMV significantly reduced tumor growth compared with control (9.2-fold ± 0.9 vs 17.8-fold ± 2.1, P = .01). The Cryo + CPMV group exhibited a transient increase in interleukin-10 and persistently decreased CXCL1. Flow cytometry revealed natural killer cell enrichment in the untreated tumor and increased PD-1 expression in the spleen. Tumor-infiltrating lymphocytes increased in Cryo + CPMV-treated tumors by IHC. CONCLUSIONS: Cryoablation and intratumoral CPMV, alone or combined, demonstrated potent efficacy against treated HCC tumors; however, only cryoablation combined with CPMV slowed the growth of untreated tumors, consistent with an abscopal effect.

The Antitumor Efficacy of CpG Oligonucleotides is Improved by Encapsulation in Plant Virus-Like Particles.

Oligodeoxynucleotides (ODNs) with CpG motifs have potent immunostimulatory effects on many subsets of immune cells. For example, Class B CpG-ODNs, such as ODN1826 induce the phagocytic activity of macrophages by activating the Toll-like receptor 9 signaling pathway. Systemic ODN delivery results in unfavorable pharmacokinetic profiles and can trigger adverse effects. To address this issue, plant virus-like particles (VLPs) are developed for the targeted delivery of ODN1826 to tumor-associated macrophages (TAMs). ODN1826 is encapsulated by the in vitro disassembly and reassembly of Cowpea chlorotic mottle virus (CCMV), producing VLPs that are structurally analogous to the native virus. The encapsulation of ODN1826 in CCMV-derived VLPs promotes ODN uptake by TAMs ex vivo and significantly enhance their phagocytic activity. The antitumor activity of the VLPs in vivo is also evaluated, revealing that the direct injection of ODN1826 VLPs into established tumors induces a robust antitumor response by increasing the phagocytic activity of TAMs in the tumor microenvironment. CCMV encapsulation significantly enhances the efficacy of ODN1826 compared to the free drug, slowing tumor growth and prolonging survival in mouse models of colon cancer and melanoma.

Active Delivery of VLPs Promotes Anti-Tumor Activity in a Mouse Ovarian Tumor Model.

Virus-like nanoparticles (VLPs) have been used as an attractive means in cancer immunotherapy because of their unique intrinsic immunostimulatory properties. However, for treating metastatic tumors in the peritoneal cavity, such as ovarian cancer, multiple injections of therapy are needed due to the large peritoneal space and fast excretion of therapy. Here, it is reported on the development of active VLP delivery vehicles for the treatment of peritoneal ovarian tumors using biocompatible Qß VLPs-loaded Mg-based micromotors. The autonomous propulsion of such Qß VLPs-loaded Mg-micromotors in the peritoneal fluid enables active delivery of intact immunostimulatory Qß VLPs to the peritoneal space of ovarian tumor bearing mice, greatly enhancing the local distribution and retention of Qß VLPs. Such improved distribution and longer retention time of Qß in the peritoneal cavity leads to enhanced immunostimulation and therefore increased survival rate of tumor-bearing mice compared to a passive Qß treatment. For clinical translation, the active delivery of VLPs holds great promise for tumor immunotherapy toward the treatment of different types of primary and metastatic tumors in the peritoneal cavity.

Heterologous Prime-Boost Enhances the Antitumor Immune Response Elicited by Plant-Virus-Based Cancer Vaccine.

New cancer vaccine strategies are required to vanquish the self-tolerance and elicit robust immune responses against tumor-associated antigens and/or neoantigens. Contemporary approaches in nanomedicine center on the use of a single nanocarrier modified with multiple copies of multiple different functional domains, e.g., epitopes for vaccines. Therefore, we set out to develop a combinatorial approach toward the next-generation concept of epitope delivery: a prime-boost strategy in which the same epitope is delivered using different nanocarriers. We tested this concept in the setting of HER2+ breast cancer. We synthesized HER2-based cancer vaccines using three icosahedral plant viruses as carriers and evaluated the immune response as a result of repetitive, homologous immunization using BALB/c mice. Two of the vaccines induced a Th2-predominant response and the other a Th1-predominant response. To enhance the immunogenicity of the vaccines, we developed a heterologous prime-boost strategy with each of the vaccines administered only once, yielding higher titers of HER2-specific immunoglobulins and increasing the toxicity of the antisera toward cancer cells. The prime-boost also induced a Th1-predominant response. An in vivo tumor challenge showed that the prime-boost regimen reduced tumor growth and improved survival in mice. This novel strategy to elicit robust immune responses against weakly immunogenic antigens in principle could be broadly applicable to cancers and other diseases.

Combination of Plant Virus Nanoparticle-Based in Situ Vaccination with Chemotherapy Potentiates Antitumor Response.

Immunotherapeutics are gaining more traction in the armamentarium used to combat cancer. Specifically, in situ vaccination strategies have gained interest because of their ability to alter the tumor microenvironment to an antitumor state. Herein, we investigate whether flexuous plant virus-based nanoparticles formed by the potato virus X (PVX) can be used as an immunotherapeutic for in situ vaccine monotherapy. We further developed dual chemo-immunotherapeutics by incorporating doxorubicin (DOX) into PVX yielding a dual-functional nanoparticle (PVX-DOX) or by coadministration of the two therapeutic regimes, PVX immunotherapy and DOX chemotherapy (PVX+DOX). In the context of B16F10 melanoma, PVX was able to elicit delayed tumor progression when administered as an intratumoral in situ vaccine. Furthermore, the coadministration of DOX via PVX+DOX enhanced the response of the PVX monotherapy through increased survival, which was also represented in the enhanced antitumor cytokine/chemokine profile stimulated by PVX+DOX when compared to PVX or DOX alone. Importantly, coadministered PVX+DOX was better for in situ vaccination than PVX loaded with DOX (PVX-DOX). Whereas the nanomedicine field strives to design multifunctional nanoparticles that integrate several functions and therapeutic regimens into a single nanoparticle, our data suggest a paradigm shift; some therapeutics may need to be administered separately to synergize and achieve the most potent therapeutic outcome. Altogether, our studies show that development of plant viral nanoparticles for in situ vaccines for treatment is a possibility, and dual mechanistic therapeutics can increase efficacy. Nonetheless, combining immunotherapeutics with cytolytic chemotherapy requires detailed investigation to inform optimal integration of cytolytic and immunotherapies and maximize synergy and efficacy.

Polymer Structure and Conformation Alter the Antigenicity of Virus-like Particle-Polymer Conjugates.

Covalent conjugation of water-soluble polymers to proteins is critical for evading immune surveillance in the field of biopharmaceuticals. The most common and long-standing polymer modification is the attachment of methoxypoly(ethylene glycol) (mPEG), termed PEGylation, which has led to several clinically approved pharmaceuticals. Recent data indicate that brush-type polymers significantly enhance in vitro and in vivo properties. Herein, the polymer conformation of poly(ethylene glycol) is detailed and compared with those of water-soluble polyacrylate and polynorbornene (PNB) when attached to icosahedral virus-like particles. Small-angle neutron scattering reveals vastly different polymer conformations of the multivalent conjugates. Immune recognition of conjugated particles was evaluated versus PEGylated particles, and PNB conjugation demonstrated the most effective shielding from antibody recognition.

Physalis Mottle Virus-Like Particles as Nanocarriers for Imaging Reagents and Drugs.

Platform technologies based on plant virus nanoparticles (VNPs) and virus-like particles (VLPs) are attracting the attention of researchers and clinicians because the particles are biocompatible, biodegradable, noninfectious in mammals, and can readily be chemically and genetically engineered to carry imaging agents and drugs. When the Physalis mottle virus (PhMV) coat protein is expressed in Escherichia coli, the resulting VLPs are nearly identical to the viruses formed in vivo. Here, we isolated PhMV-derived VLPs from ClearColi cells and carried out external and internal surface modification with fluorophores using reactive lysine-N-hydroxysuccinimide ester and cysteine-maleimide chemistries, respectively. The uptake of dye-labeled particles was tested in a range of cancer cells and monitored by confocal microscopy and flow cytometry. VLPs labeled internally on cysteine residues were taken up with high efficiency by several cancer cell lines and were colocalized with the endolysosomal marker LAMP-1 within 6 h, whereas VLPs labeled externally on lysine residues were taken up with lower efficiency, probably reflecting differences in surface charge and the propensity to bind to the cell surface. The infusion of dye and drug molecules into the cavity of the VLPs revealed that the photosensitizer (PS), Zn-EpPor, and the drugs crystal violet, mitoxantrone (MTX), and doxorubicin (DOX) associated stably with the carrier via noncovalent interactions. We confirmed the cytotoxicity of the PS-PhMV and DOX-PhMV particles against prostate cancer, ovarian and breast cancer cell lines, respectively. Our results show that PhMV-derived VLPs provide a new platform technology for the delivery of imaging agents and drugs, with preferential uptake into cancer cells. These particles could therefore be developed as multifunctional tools for cancer diagnosis and therapy.

POxylation as an alternative stealth coating for biomedical applications.

Polyethylene glycol (PEG) polymers are currently used in a variety of medical formulations to reduce toxicity, minimize immune interactions and improve pharmacokinetics. Despite its widespread use however, the presence of anti-PEG antibodies indicates that this polymer has the potential to be immunogenic and antigenic. Here we present an alternative polymer, poly(2-oxazoline) (POx) for stealth applications, specifically shielding of a proteinaceous nanoparticle from recognition by the immune system. Tobacco mosaic virus (TMV) was used as our testbed due to its potential for use as a nanocarrier for drug delivery and molecular imaging applications.

The Protein Corona of Plant Virus Nanoparticles Influences their Dispersion Properties, Cellular Interactions, and In Vivo Fates.

Biomolecules in bodily fluids such as plasma can adsorb to the surface of nanoparticles and influence their biological properties. This phenomenon, known as the protein corona, is well established in the field of synthetic nanotechnology but has not been described in the context of plant virus nanoparticles (VNPs). The interaction between VNPs derived from Tobacco mosaic virus (TMV) and plasma proteins is investigated, and it is found that the VNP protein corona is significantly less abundant compared to the corona of synthetic particles. The formed corona is dominated by complement proteins and immunoglobulins, the binding of which can be reduced by PEGylating the VNP surface. The impact of the VNP protein corona on molecular recognition and cell targeting in the context of cancer and thrombosis is investigated. A library of functionalized TMV rods with polyethylene glycol (PEG) and peptide ligands targeting integrins or fibrin(ogen) show different dispersion properties, cellular interactions, and in vivo fates depending on the properties of the protein corona, influencing target specificity, and non-specific scavenging by macrophages. Our results provide insight into the in vivo properties of VNPs and suggest that the protein corona effect should be considered during the development of efficacious, targeted VNP formulations.

3D bioprinting cowpea mosaic virus as an immunotherapy depot for ovarian cancer prevention in a preclinical mouse model.

Implantable polymeric hydrogels loaded with immunostimulatory cowpea mosaic virus (CPMV) were fabricated using digital light processing (DLP) printing technology. The CPMV-laden hydrogels were surgically implanted into the peritoneal cavity to serve as depots for cancer slow-release immunotherapy. Sustained release of CPMV within the intraperitoneal space alleviates the need for repeated dosing and we demonstrated efficacy against ovarian cancer in a metastatic mouse model.