In vivo Gold Nanoparticle Delivery of Peptide Vaccine Induces Anti-Tumor Immune Response in Prophylactic and Therapeutic Tumor Models

Gold nanoparticles (AuNPs) are promising vehicles for cancer immunotherapy, with demonstrated efficacy in immune delivery and innate cell stimulation. Nevertheless, their potential has yet to be assessed in the in vivo application of peptide cancer vaccines. In this study, it is hypothesized that the immune distribution and adjuvant qualities of AuNPs could be leveraged to facilitate delivery of the ovalbumin (OVA) peptide antigen and the CpG adjuvant and enhance their therapeutic effect in a B16-OVA tumor model. AuNP delivery of OVA (AuNP-OVA) and of CpG (AuNP-CpG) enhanced the efficacy of both agents and induced strong antigen-specific responses. In addition, it is found that AuNP-OVA delivery alone, without CpG, is sufficient to promote significant antigen-specific responses, leading to subsequent anti-tumor activity and prolonged survival in both prophylactic and therapeutic in vivo tumor models. This enhanced therapeutic efficacy is likely due to the adjuvant effect of peptide coated AuNPs, as they induce inflammatory cytokine release when cultured with bone marrow dendritic cells. Overall, AuNP-mediated OVA peptide delivery can produce significant therapeutic benefits without the need of adjuvant, indicating that AuNPs are effective peptide vaccine carriers with the potential to permit the use of lower and safer adjuvant doses during vaccination

Elimination of Metastatic Melanoma Using Gold Nanoshell-Enabled Photothermal Therapy and Adoptive T Cell Transfer

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b+Ly-6G/C+ myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control

Gold Nanoparticle Delivery of Modified CpG Stimulates Macrophages and Inhibits Tumor Growth for Enhanced Immunotherapy

Gold nanoparticle accumulation in immune cells has commonly been viewed as a side effect for cancer therapeutic delivery; however, this phenomenon can be utilized for developing gold nanoparticle mediated immunotherapy. Here, we conjugated a modified CpG oligodeoxynucleotide immune stimulant to gold nanoparticles using a simple and scalable selfassembled monolayer scheme that enhanced the functionality of CpG in vitro and in vivo. Nanoparticles can attenuate systemic side effects by enhancing CpG delivery passively to innate effector cells. The use of a triethylene glycol (TEG) spacer on top of the traditional poly-thymidine spacer increased CpG macrophage stimulatory effects without sacrificing DNA content on the nanoparticle, which directly correlates to particle uptake. In addition, the immune effects of modified CpGAuNPs were altered by the core particle size, with smaller 15 nm AuNPs generating maximum immune response. These TEG modified CpG-AuNP complexes induced macrophage and dendritic cell tumor infiltration, significantly inhibited tumor growth, and promoted survival in mice when compared to treatments with free CpG

Gold Nanoparticle Platforms for Antigen and Adjuvant Delivery in Cancer Immunotherapy

Cancer immunotherapy is a growing treatment modality with the promise to yield systemic and targeted treatments for cancer. Major modalities such as radiation, chemotherapy, and surgery are limited in that they are either too localized--as is the case with radiation and surgery—and cannot be effective in metastatic disease, or they are not targeted-- as with chemotherapy--thus leading to severe toxicities. Immunotherapy aims to stimulate the body’s immune system against disease, allowing one to circumvent such challenges because the immune system can act systemically and can have specific activity against cancer cells expressing antigens of interest. The development of an effective cancer vaccine and subsequent immune response requires the delivery of an antigen for immune recognition and of an adjuvant for an inflammatory response. Gold nanoparticles (AuNPs) are promising vaccine carriers because they are generally non-toxic, can be synthesized in the optimal sizes for lymphatic drainage and cell uptake, and can be readily conjugated with antigens and adjuvants for delivery. This thesis project characterizes AuNP distribution in the immune system and details the development of AuNP mediated delivery of antigens and adjuvants for cancer immunotherapy. In our work, we have detailed AuNP distribution within immune cells of the spleen and the tumor microenvironment, thereby identifying that AuNPs associate with a range of immune populations, including B cells, dendritic cells, and macrophages, all of which can be potentially targeted for immune modulation. Next we developed AuNP complexes capable of delivering the CpG oligonucleotide adjuvant, demonstrating that AuNP delivery promotes the therapeutic effect of CpG in vitro and in vivo. In addition, we studied AuNP mediated delivery of the ovalbumin (OVA) peptide antigen and showed that AuNP delivery enhances vaccination with the antigen in vivo, subsequently causing tumor inhibition and prolonged survival in both prophylactic and established tumor models. The thesis thus elucidates AuNP interactions with the immune system and demonstrates that the technology is an effective platform for delivery of immune modulatory agents

In Vivo Immune Cell Distribution of Gold Nanoparticles in Naïve and Tumor Bearing Mice

Gold nanoparticles (AuNP) have been widely used for drug delivery and have recently been explored for applications in cancer immunotherapy. Although AuNPs are known to accumulate heavily in the spleen, the particle distribution within immune cells has not been thoroughly studied. Here, cellular distribution of Cy5 labeled 50 nm AuNPs is characterized within the immune populations of the spleen from naïve and tumor bearing mice using flow cytometry. Surprisingly, approximately 30% of the detected AuNPs are taken up by B cells at 24 h, with about 10% in granulocytes, 18% in dendritic cells, and 8% in T cells. In addition, 3% of the particles are detected within myeloid derived suppressor cells, an immune suppressive population that could be targeted for cancer immunotherapy. Furthermore, it is observed that, over time, the particles traveled from the red pulp and marginal zone to the follicles of the spleen. Taking into consideration that the particle cellular distribution does not change at 1, 6 and 24 h, it is highly suggestive that the immune populations carry the particles and migrate through the spleen instead of the particles migrating through the tissue by cell-cell transfer. Finally, no difference is observed in particle distribution between naïve and tumor bearing mice in the spleen, and nanoparticles are detected within 0.7% of dendritic cells of the tumor microenvironment. Overall, these results can help inform and influence future AuNP delivery design criteria including future applications for nanoparticle-mediated immunotherapy

Ovarian Grafts 10 Days after Xenotransplantation: Folliculogenesis and Recovery of Viable Oocytes.

Ovarian xenotransplantation is a promising alternative to preserve fertility of oncologic patients. However, several functional aspects of this procedure remained to be addressed. The aim of this study was evaluate the feasibility of xenotransplantation as a strategy to maintain bovine ovarian grafts and produce oocytes. Adult ovarian cortical pieces were xenotransplanted to the dorsal subcutaneous of female NOD-SCID mice (n = 62). Grafts were recovered ten days after xenotransplantation. Host and graft weights; folliculogenesis progression; blood perfusion, relative gene expression and number of macrophage and neutrophil of xenografts; in vitro developmental competence of graft-derived oocytes were evaluated. Folliculogenesis was supported in the grafts, as indicated by the presence of primordial, primary, secondary, antral, and atretic follicles. The xenografts showed a greater volumetric density of atretic follicles and higher hyperemia and number of host-derived macrophage and neutrophil (P<0.05), when compared to non-grafted fragments. There was a higher blood perfusion under the back skin in the transplantation sites of host animals than in control and non-grafted (P<0.01). BAX and PRDX1 genes were up-regulated, while BCL2, FSHR, IGF1R and IGF2R were down-regulated, when compared to the control (P<0.01). Twenty seven oocytes were successfully harvested from grafts, and some of these oocytes were able to give rise to blastocysts after in vitro fertilization. However, cleavage and blastocyst rates of xenograft derived oocytes were lower than in control (P<0.01). Despite showing some functional modifications, the ovarian xenografts were able to support folliculogenesis and produce functional oocytes

PTT promotes the expansion of adoptively transferred pmel T cells.

<p>Primary B16-F10 tumors were established on day 0, and contralateral tumors were established on day 6. Primary tumors were ablated by PTT on day 10 followed by pmel ATCT on day 11. On day 20, mice were euthanized and tissues were harvested for analysis. IFN-γ secretion in response to hgp100 by cells isolated from the (<b>A</b>) spleen and (<b>B</b>) TDLN. Treatment groups consisted of untreated tumor bearing mice (n = 5), PTT alone (n = 4), ATCT alone (n = 5), and dual PTT/ATCT (n = 5). The data is representative of two or more experiments. *p<0.05, **p<0.01; ANOVA followed by Student's t-test with multiple comparison adjustment.</p

Progression of folliculogenesis, morphometry and inflammatory cell numbers in xenografted and in control ovarian fragments.

<p>(a) Primordial, (b) primary, (c) secondary, (d) antral and (e) atretic follicles were noted 10 days after xenotransplantation. (g) Volumetric density (%) of ovarian pieces before and after xenotransplantation showing a higher incidence of follicular atresia and hyperemia (f; <i>P<</i>0.01) after this procedure. (h, i) As demonstrated using (h) NAG and (i) MPO assays in xenografted ovarian wet tissue, higher concentration of host-derived macrophages and neutrophils were observed (<i>P<</i>0.01). Black arrows = healthy follicles; red arrows = atretic follicles; white arrows = blood vessels; black arrowhead = hyperemia; asterisks = oocytes. Bars in a, b, c and e = 80 μm and d and f = 150 μm.</p