Vijaya B. Joshi

Biodegradable particles as vaccine antigen delivery systems for stimulating cellular immune responses.

There is a need for both new and improved vaccination formulations for a range of diseases for which current vaccines are either inadequate or non-existent. Biodegradable polymer-based vaccines fulfill many of the desired properties in achieving effective long-term protection in a manner that is safe, economical, and potentially more practicable on a global scale. Here we discuss some of the work performed with micro/nanoparticles made from either synthetic (poly[lactic-co-glycolic acid] [PLGA] and polyanhydrides) or natural (chitosan) biodegradable polymers. Our attention is focused on, but not limited to, the generation of antitumor immunity where we stress the importance of particle size and co-delivery of antigen and adjuvant.

Characterizing the antitumor response in mice treated with antigen-loaded polyanhydride microparticles.

Delivery of vaccine antigens with an appropriate adjuvant can trigger potential immune responses against cancer leading to reduced tumor growth and improved survival. In this study, various formulations of a bioerodible amphiphilic polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) with inherent adjuvant properties were evaluated for antigen-loading properties, immunogenicity and antitumor activity. Mice were vaccinated with 50:50 CPTEG:CPH microparticles encapsulating a model tumor antigen, ovalbumin (OVA), in combination with the Toll-like receptor-9 agonist, CpG oligonucleotide 1826 (CpG ODN). Mice treated with OVA-encapsulated CPTEG:CPH particles elicited the highest CD8(+) T cell responses on days 14 and 20 when compared to other treatment groups. This treatment group also displayed the most delayed tumor progression and the most extended survival times. Particles encapsulating OVA and CpG ODN generated the highest anti-OVA IgG(1) antibody responses in mice but these mice did not show significant tumor protection. These results suggest that antigen-loaded CPTEG:CPH microparticles can stimulate antigen-specific cellular responses and could therefore potentially be used to promote antitumor responses in cancer patients.

Biodegradable Microparticles Loaded with Doxorubicin and CpG ODN for In Situ Immunization Against Cancer

ABSTRACTIn situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen-presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. We designed a comprehensive approach to in situ immunization using poly(lactic-co-glycolic acid) (PLGA)-biodegradable microparticles (MPs) loaded with doxorubicin (Dox) and CpG oligodeoxynucleotides (CpG) that deliver Dox (chemotherapy) and CpG (immunotherapy) in a sustained-release fashion when injected intratumorally. Dox induces immunogenic tumor cell death while CpG enhances tumor antigen presentation by DCs. PLGA MPs allow their safe co-delivery while evading the vesicant action of Dox. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs. In vivo, Dox/CpG MPs combined with antibody therapy to enhance and maintain the T cell response generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent an efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.

Tumor lysate-loaded biodegradable microparticles as cancer vaccines

Cancer vaccines that use tumor lysate (TL) as a source of tumor-associated antigens (TAAs) have significant potential for generating therapeutic anti-tumor immune responses. Vaccines encompassing TL bypass the limitations of single antigen vaccines by simultaneously stimulating immunity against multiple TAAs, thereby broadening the repertoire of TAA-specific T-cell clones available for activation. Administration of TL in particulate form, such as when encapsulated in biodegradable microparticles, increases its immunostimulatory capacity and produces more robust immune responses than when TL is given in soluble form. These effects can be further enhanced by co-administering TL with adjuvants. A number of recent studies using polymeric microparticle delivery of TL, with or without adjuvants, have produced promising results in preclinical studies. In this review, we will discuss current experimental approaches involving TL being pursued in the oncoimmunology field, and comment on strategies such as combining specific chemotherapeutic agents with TL microparticle delivery that may eventually lead to improved survival outcomes for cancer patients.

Antigen-coated poly α-hydroxy acid based microparticles for heterologous prime-boost adenovirus based vaccinations

Adenoviruses show promising potential as vectors for cancer vaccines, however, their high immunogenicity can be problematic when it comes to homologous prime-boost strategies. In the studies presented here we show that heterologous prime-boost vaccinations involving ovalbumin (OVA)-antigen-coated microparticles as a prime, and adenovirus encoding OVA (AdOVA) as a boost, were equally as effective as homologous AdOVA prime-boosts at generating OVA-specific CD8(+) T-cell responses, which translated into effective tumor protection. OVA-coated biodegradable poly α-hydroxy acid-based microparticles of varying chemistries, when used as primes in heterologous prime-boost vaccinations, were comparable in terms of promoting OVA-specific CD8(+) T cells as well as providing protection against subsequent tumor challenge. These findings auger well for using poly α-hydroxy acid-based microparticles in prime-boost viral vaccination strategies geared toward the safer, and potentially more efficient, generation of anti-tumor immunity.

Three steps to breaking immune tolerance to lymphoma: a microparticle approach.

Makkouk and colleagues show that intratumoral delivery of doxorubicin in polylactide-co-glycolide micro-particles combined with antibodies against OX40 and CTLA-4 induced T cell–dependent systemic responses that enhanced T-cell infiltration into and eradication of distant tumors and improved survival in mouse models of lymphoma. In situ immunization aims at generating antitumor immune responses through manipulating the tumor microenvironment. On the basis of recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (i) inducing immunogenic tumor cell death with the chemotherapeutic drug doxorubicin. Doxorubicin enhances the expression of “eat-me” signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DC). Because of the vesicant activity of doxorubicin, microparticles made of biodegradable polymer poly(lactide-co-glycolide) or PLGA can safely deliver doxorubicin intratumorally and are effective vaccine adjuvants, (ii) enhancing T-cell activation using anti-OX40 and (iii) sustaining T-cell responses by checkpoint blockade using anti–CTLA-4. In vitro, doxorubicin microparticles were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by tumor cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared with soluble doxorubicin. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T-cell infiltration into distant tumors, leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation as an immunotherapy for patients with lymphoma. Cancer Immunol Res; 3(4); 389–98. ©2015 AACR.

The absence of CpG in plasmid DNA-chitosan polyplexes enhances transfection efficiencies and reduces inflammatory responses in murine lungs.

Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200–400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(−)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(−)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(−)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.

Production of Antigen-Loaded Biodegradable Nanoparticles and Uptake by Dendritic Cells

Particle-based cancer vaccines prepared from biodegradable polymers are a potentially attractive way of delivering antigen alone, or in combination with adjuvant molecules, to dendritic cells (DC). Non-viral modes of vaccination must ensure the activation of cellular immune responses in addition to humoral responses if an effective antitumor immune response is to be initiated. Here we describe in detail a method for manufacturing poly(D,L-lactide-co-glycolide) (PLGA) particles loaded with purified protein/antigen. We also describe a method for generating bone marrow-derived DC in vitro. These DC can be subsequently used to measure the adjuvant properties of the particle formulations.

Poly(galactaramidoamine) is an efficient cationic polymeric non-viral vector with low cytotoxicity for transfecting human embryonic kidney (HEK293) and murine macrophage (RAW264.7) cells

Poly(galactaramidoamine) (PGAA) is a cationic co-polymer of dimethyl-meso-galactarate and pentaethylenehexamine. PGAA electrostatically complexes with plasmid DNA (pDNA) to form nano-sized particles. In this study, we show that PGAA-pDNA polyplexes generate high transfection efficiencies in human embryonic kidney (HEK293) and murine macrophage-like (RAW264.7) cell lines. PGAA-pDNA mediated transfection is a function of the amine:phosphate (N/P) ratio at which the polyplexes are prepared. The maximum expression of luciferase was obtained using polyplexes prepared at an N/P ratio of 40. Polyplexes prepared at increasing N/P ratios did not significantly increase in size but did result in decreasing luciferase expression. Cellular toxicity increased as the N/P ratios at which the polyplexes were prepared increased.

Abstract 2885: Therapeutic microparticle-based tumor lysate vaccination reduces spontaneous lung metastases in a murine breast cancer model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Although existing interventions are highly efficacious for the treatment of localized breast cancers, disseminated tumors remain incurable. Therapeutic induction of systemic anti-tumor immunity is a promising approach for treating metastatic disease. We have developed a novel heterologous prime/boost vaccination protocol that significantly reduces metastatic tumor burdens in a pre-clinical murine model of breast cancer. In our studies, mice were orthotopically challenged with Luciferase-expressing 4T1 adenocarinoma cells. Six days later, mice received a priming therapy that consisted of parental 4T1 tumor lysates encapsulated in poly(lactic-co-glycolic) acid microparticles administered contralateral to the established tumor. On day eleven, mice received a boost therapy that consisted of 4T1 lysates and a cocktail of Toll-like receptor agonists. At the conclusion of the assay (day twenty-five), tumor burdens from excised lungs were quantified using bioluminescent imaging. Therapy-receiving mice showed a significant reduction in lung tumors (Students T-test: p = 0.002, n = 37 control mice vs. 25 treated mice) with an average 46.66% decrease relative to no therapy control mice. Independent quantification of lung metastases was also performed using a standard 6-thioguanine assay. There was a strong correlation (R^2 = 0.8803) between bioluminescent intensity and 6-thioguanine colony number, demonstrating that the decrease in bioluminescence signal due to therapy is not merely explained by the in vivo loss of Luciferase expression by 4T1 cells. We are currently characterizing the vaccination-induced differences in the localized immune environment of metastatic lung tumors. Effector T cell activation/exhaustion kinetics and the influx of immunosuppressive cell populations (myeloid-derived suppressor cells and regulatory T cells) are now being explored. These studies will help elucidate the immunological mechanism(s) responsible for the diminished metastatic tumor outgrowth observed with our therapy. In conclusion, our data demonstrate that therapeutic vaccination of tumor lysates is efficacious in controlling spontaneous lung metastases in an aggressive model of murine breast cancer. Citation Format: Brett P. Gross, Amaraporn Wongrapanich, Meghan Francis, Vijaya B. Joshi, Aliasger K. Salem, Lyse A. Norian. Therapeutic microparticle-based tumor lysate vaccination reduces spontaneous lung metastases in a murine breast cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2885. doi:10.1158/1538-7445.AM2014-2885