Immunol Res DOI 10.1007/s12026-014-8537-9

IMMUNOLOGY AT THE UNIVERSITY OF IOWA

Applying biodegradable particles to enhance cancer efficacy

Kawther K. Ahmed • Sean M. Geary • Aliasger K. Salem

Aliasger Salem

Ó Springer Science+Business Media New York 2014

Abstract One of the primary goals of our group and our collaborators here at the University of Iowa is to develop therapeutic cancer using biodegradable and biocompatible polymer-based vectors. A major advantage of using discretely packaged immunogenic cargo over non-encapsulated vaccines is that they promote enhanced cellular immunity, a key requirement in achieving antitumor activity. We discuss the importance of co-encapsulation of tumor and adjuvant, with specific focus on the synthetic oligonucleotide adjuvant, cytosine–phosphate–guanine oligodeoxynucleo- tides. We also discuss our research using a variety of polymers including poly(a-hydroxy acids) and polyanhydrides, with the aim of determining the effect that parameters, such as size and polymer type, can have on prophylactic and therapeutic tumor vaccine formulation efficacy. Aside from their role as vaccine vectors per se, we also address the research currently underway in our group that utilizes more novel applications of biodegradable polymer-based particles in facilitating other types of immune-based therapies.

Keywords Cancer vaccine Á Biodegradable polymer Á Poly(a-hydroxy acids) Á CpG Á PLGA

Introduction metastases, and chemotherapy is often limited by its highly toxic side effects and the capacity of tumor cells to develop Cancer remains a challenging health problem and is the multidrug resistance [4]. Therefore, improved therapies are second leading cause of death in the USA [1]. Although required that reduce morbidity for cancer patients during incidence and mortality rates have marginally declined for treatment while ultimately extending their survival. Cancer many cancer types over the past decade, this is speculated vaccines are a promising therapy in this regard in that they to be primarily due to a combination of preventative have been shown to be well tolerated by patients, as they measures (i.e. reducing risk factors) and enhanced early rarely cause serious side effects, along with being capable detection, rather than a significant increase in the effec- of promoting tumor-specific immune responses entailing tiveness of treatment modalities per se [2, 3]. Conventional the activation of CD8? T cells [also referred to as cyto- treatments involve surgery and radiotherapy, to remove the toxic T lymphocytes (CTLs)] [5–7]. These CTLs are not main tumor bulk (usually the primary tumor), along with only potentially capable of eradicating existing tumor cells chemotherapy to ensure complete elimination of tumor but can also provide the host with immune memory such cells in order to prevent or combat metastases. However, that tumor recurrence can be prevented. surgery and radiotherapy cannot address the problem of The fact that the can be instrumental in the fight against cancer was recognized more than a century ago through the work of William Coley [8, 9]. At the time K. K. Ahmed Á S. M. Geary Á A. K. Salem (&) there was little understanding of the immune system and its Division of Pharmaceutics and Translational Therapeutics, components, however, many advances in our understand- College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA ing have been made since that have resulted in significant e-mail: [email protected] developments in the field of oncoimmunology. These 123 University of Iowa Immunology 2014 include the findings that tumors express tumor ; addition, the use of biodegradable over non-biodegradable that the host possesses -specific T cells particles offers another advantage of eliminating the need capable of being activated; that immunosuppression, par- for carrier removal [30]. It is also generally the case that ticularly within the tumor microenvironment, plays a key biodegradable particles are less toxic than non-biodegrad- role in abrogating antitumor immunity; and that the innate able particles and can be readily studied for their effects on immune system and various adjuvants play an important dendritic cells (e.g. maturation and particle uptake) in vitro role in determining the nature and efficacy of immune prior to progression to in vivo studies [31, 32]. Biode- responses [6, 10–14]. The primary aim of any cancer gradable polymeric particles are versatile delivery systems vaccine is to alert the patient’s immune system to the that can be fabricated using different polymers such as presence of the cancer and to activate an effective - poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) mediated tumor-specific attack. For a cancer vaccine to be (PLA), polyanhydrides, chitosan, or other types of polymer effective, it is important that the antigenic material [tumor (Fig. 1)[33, 34]. Different methods can be employed for antigen(s)] be delivered in a particulated rather than soluble their synthesis, and the choice of the method would pri- form as this promotes uptake and cross-presentation of marily be governed by the property of the cargo (hydro- antigen (i.e. presentation of antigen such that CTL philic/hydrophobic) and desired particle size [35, 36]. responses are favored) by professional antigen presenting Particle size is a flexible parameter, and depending on the cells known as dendritic cells [15, 16]. It is also important intended use can vary from less than a 100 nm to more than for the particulated antigen to be co-delivered with an 10 lm. adjuvant capable of promoting maturation The literature is rich with preclinical studies using a and TH1-type immune responses, as this will result in more wide diversity of biodegradable particle-based formula- potent T cell activation and IFN-c production [17–19]. tions as cancer vaccines. The type of polymers used to Many different approaches and formulations have been fabricate particles for cancer vaccines have included well- investigated as potential cancer vaccines and include whole established poly(a-hydroxy acid)-based polymers, such as tumor cells, tumor cell lysates, dendritic cells pulsed PLA and PLGA, as well as more novel polymers such as ex vivo with tumor antigen, as well as the use of biode- polyanhydrides. Microparticles based on the polyanhy- gradable particles as vaccine delivery vehicles co-encap- drides 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane sulating antigen and adjuvant [20–22]. Biodegradable (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) particles, aside from their potential as vaccine carriers, may have been reported to have inherent immune adjuvant also have the potential to improve upon other vaccine properties [37, 38]. Polyanhydride particles (1–3 lmin strategies. For instance, they could be used to deliver tumor diameter) co-loaded with ovalbumin (OVA) and the syn- lysates in particulated rather than soluble form, to form thetic adjuvant, CpG, were fabricated using a double- cell–particle hybrid structures where the particle may carry emulsion solvent evaporation method [35] and tested as a a range of immune stimulatory agents and thereby enhance vaccine in a murine study [39]. Vaccination with these the antitumor potential of whole-cell tumor vaccines [23], microparticles resulted in higher levels of OVA-specific or to improve delivery of tumor antigen to dendritic cells CD8? T cells and provided superior prophylactic protec- pulsed ex vivo. Many of these possibilities are being tion against an OVA-expressing tumor challenge, as dem- investigated by various groups within the University of onstrated by slower tumor progression and enhanced Iowa and are mentioned below. survival of mice, compared to mice vaccinated with soluble OVA and CpG [39]. In a separate study, various poly(a-hydroxy acid)-based Biodegradable polymeric particles as cancer particles were investigated as priming agents in a heterol- vaccine vectors ogous prime/boost cancer vaccine strategy where the ade- novirus (encoding OVA) was used as the boost [40]. This Biodegradable particles, such as polymeric particles, lipo- study involved the comparison of particles (1–3 lm somes, and virus-like particles have shown potential as diameter) fabricated from PLA, PLGA, or PLGA-COOH vectors for cancer vaccines in both clinical and preclinical polymers and coated with OVA by passive adsorption. All settings [24]. The use of particles as delivery vehicles three particle types demonstrated the ability to stimulate offers many potential advantages over non-particulated similar levels of murine dendritic cell maturation in vitro as vaccine systems that include protection of the payload from defined by CD86 upregulation. Using mice, it was estab- premature degradation, enhancing intracellular delivery of lished that all three particle types were also capable of cargo [25, 26], providing sustained release of contents [27– generating long-term immune memory responses in vivo as 29], and enabling co-delivery (to dendritic cells) of mul- determined by the sustained presence of OVA-specific tiple components [e.g. immunogen(s) and adjuvant(s)]. In CD8? T cells in the blood and the ability to resist 123 University of Iowa Immunology 2014

Fig. 1 Scanning electron microscope image showing a, b blank PLGA particles of different sizes. Particles are spherical with smooth surfaces. Scale bar = 5 lm, c blank polyanhydride particles prepared using 50:50 CPTEG/ CPH, d OVA-loaded polyanhydride particles prepared using 50:50 CPTEG/ CPH. e blank polysulfonamide particles, f OVA-loaded polysulfonamide particles. All particles were prepared using the double-emulsion solvent evaporation. Scale bar is indicated on each image

subsequent tumor challenge [with the OVA-expressing particle diameters of 0.3, 1, 7, and 17 lm. It was found that thymoma cell line (E.G7)]. These studies suggest that the smallest sized particles (0.3 lm) were capable of being heterologous prime–boost vaccinations may be an effective more readily taken up by in vitro cultured murine bone alternative to homologous prime–boost vaccinations marrow-derived dendritic cells than the larger sized parti- involving adenoviruses, thereby avoiding the issues of cles. These 0.3 lm particles also resulted in enhanced toxicity and neutralizing adenovirus-specific antibodies. maturation of dendritic cells in vitro and enhanced OVA- Besides the type of polymer used, there have been a specific CD8? T cell responses in vivo subsequent to number of studies documenting the effect of particle size prime/boost (Day 0/Day 7) intraperitoneal vaccinations on generating immune responses, resulting in what appears (Fig. 2). to be conflicting data [41–43]. These differences may stem from a variety of variable parameters including differences in materials used to fabricate the particles, the type of The use of CpG as an adjuvant in cancer vaccines antigen loaded, the method of antigen loading, and the route of administration of these particles [44]. Recently, a Unmethylated cytosine–phosphate–guanine oligodeoxynu- study looking at the effect of particle size on murine CTL cleotides (CpG) are synthetic single-stranded DNA motifs responses was performed using PLGA particles (encapsu- that act as pathogen-associated molecular patterns and are lating OVA and CpG) [45]. The study involved using the rarely found in the mammalian genome and more abun- double-emulsion solvent evaporation method to fabricate dantly present in bacterial DNA. The adjuvant properties of PLGA particles of narrow size distributions and mean CpG were recognized well before the identification of toll-

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Fig. 2 OVA-specific T cell frequency in mouse peripheral blood obtained from submandibular bleeds of mice. All groups were lymphocytes (PBLs) after prime/boost (Day 0/Day 7) vaccination statistically compared using ANOVA followed by Tukey’s post test with OVA (100 lg) and CpG ODN (50 lg) in different particulate (*p \ 0.05). Reproduced with kind permission from Springer Science and soluble formulations, as determined by tetramer staining and flow and Business Media: Joshi et al., Fig. 7. Ó 2012 American cytometric analysis on Day 14 (a) and Day 21 (b). PBLs were Association of Pharmaceutical Scientists [45] like receptors (TLR) and the full elucidation of its mech- encapsulation of CpG into submicron particles for systemic anism of action [46–49]. Subsequently, CpG was found to injection may be of benefit since this could result in pref- trigger innate immune responses and act as an adjuvant for erential delivery of CpG to metastases using the enhanced adaptive immune responses through the binding of TLR-9 permeation and retention effect. expressed by B cells, natural killer cells, and, most Dr. George Weiner’s laboratory in the Department of importantly, dendritic cells [50–53]. The use of CpG in Internal Medicine (University of Iowa) has reported on many cancer vaccines is being investigated in preclinical and studies investigating CpG in the context of a murine B cell clinical studies of different types of cancer such as mela- lymphoma model. In early pioneering work in mice, they noma [54], lymphoma [55], neuroblastoma [56], and compared the immunostimulatory potency of CpG with [57]. A variety of innovative approaches complete Freund’s adjuvant in a B cell tumor antigen vaccine have been investigated and have been reviewed in detail and found both adjuvants to be similar but that CpG gener- elsewhere [58, 59]. ated less toxicity [63]. This group went further to show the Here, at the University of Iowa, a number of researchers potential for the use of CpG as an adjuvant in humans by are investigating the effect of incorporating CpG into showing dendritic precursor cells derived from human cancer vaccines with the main goal being to develop a peripheral blood were stimulated by CpG to undergo matu- clinically translatable formulation that can be used as an ration, upregulate costimulatory proteins such as CD54 and alternative or adjunct therapy for cancer patients. For CD86, and promote TH1 cytokine production [64]. example, it was shown by our group, in a therapeutic murine tumor model, that combining intratumoral CpG treatment with subcutaneous adenovirus (encoding the Biodegradable particles carrying CpG as cancer model tumor antigen, OVA) vaccination resulted in sig- vaccines nificantly enhanced antitumor activity and increased sur- vival times compared to mice treated with adenovirus or Particles offer the advantage of reducing the effective CpG alone (Fig. 3)[60]. The results clearly support the delivery dose required to achieve desired biological or role of CpG in boosting adenovirus cancer vaccines, and it therapeutic responses and thereby reducing toxicity. In was noted that the presence of CpG resulted in significantly fact, it has been reported that CpG conjugated to particles less regulatory T cells (Tregs) within the tumor microen- was effective as an adjuvant for a prophylactic tumor vironment. It has been suggested by this group that prostate vaccine at doses as low as 4 lg, generating potent CTL cancer patients may be good candidates for this type of responses comparable to levels induced by 10–100 lgof treatment as they have previously shown in murine models soluble CpG [65]. This group showed that intradermal of prostate cancer that the subcutaneous administration of (footpad) prime/boost (Day 0/Day 14) vaccination with the combination of adenovirus (encoding human prostate- Pluronic-stabilized poly(propylene sulfide) particles specific antigen (PSA)) and CpG (delivered subcutane- (*25–30 nm diameter) conjugated to CpG and admixed ously) is capable of generating enhanced antitumor with similar particles conjugated to OVA could protect responses and superior anti-PSA-specific CD8? T cells against challenge with the aggressive B16-F10-OVA mel- compared to vaccination with the adenovirus alone [7, 61, anoma cells significantly better than control formulations. 62]. It would be of interest if in future studies, Based on supporting in vitro and in vivo data, this group 123 University of Iowa Immunology 2014

Fig. 3 Comparison of the therapeutic effect of adenovirus type 5 Ad5-OVA followed by IT injections of CpG (50 lg) on Day 7 (91); encoding OVA (Ad5-OVA) vaccination combined with no, single, or Days 7, 10, and 13 (93); Days 7, 10, 13, and 16 (94) post-tumor multiple intratumoral injections of CpG, on tumor volume. Individual challenge, or no CpG treatment (i.e. Ad5-OVA alone). Reproduced tumor progression (volume) over time for indicated treatments. with kind permission from Springer Science and Business Media: Tumor-challenged mice were vaccinated with either Ad5-LacZ or Geary et al., Fig. 1. Ó Springer-Verlag 2011 [60]

Fig. 4 Flow cytometry data of degradable polymer microparticles increased from 8.4 % at 30 min to 92 % by 16 h. Reproduced with (mean size of 1.4 ± 0.3 mm, polydispersity 0.24 ± 0.01) that have permission from Lippincott Williams and Wilkins/Wolters Kluwer been surface labeled with rhodamine and then incubated with Health: J Immunother, Zhang et al. Ó 2007 by Lippincott Williams & dendritic cells. At the highest concentration (1 mL of 1 % solids), Wilkins [66] the percentage of dendritic cells that had taken up microparticles proposed that the particles traveled to the draining lymph loaded with OVA and CpG were more immunostimulatory node where the CpG-conjugated particles stimulated cross- in terms of TH1-type responses than other formulations presentation (of OVA) by, and activation (as determined by such as OVA-loaded PLGA microparticles with soluble upregulation of CD80, CD86 and IL-12p70) of, CD11b-ve CpG. dendritic cells. Our group has compared the benefit of Co-delivery of Ag and adjuvant, in an associated form, using particulated CpG versus soluble CpG as an adjuvant to the same DC is important for optimal immunogenicity of for the model antigen, OVA [66]. In this study, PLGA a vaccine as was demonstrated by Blander and Medzhitov (75:25) microparticles were initially shown to be readily (2006) [67]. In a related study, Zhang et al. [66] compared taken up by a dendritic cell line (DC2.4) in vitro (Fig. 4). the immunogenicity of a fusion molecule, OVA-CpG, We went on further to show that PLGA microparticles co- to CpG and OVA co-loaded into PLGA (75:25)

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Fig. 5 T cell proliferation following vaccinations with immune Becton–Dickinson). (MP empty microparticle only, MP-CpG MP stimulatory antigen-loaded particles (ISAP). Splenocytes harvested containing CpG only, MP-Alum-TL MP containing alum and B16 from naı¨ve mice, mice vaccinated with microparticles only or mice tumor lysate, MP-GM-TL MP containing GM-CSF and B16 tumor vaccinated with various components of the immune stimulatory lysate, MP-CpG-TL (ISAP) MP containing CpG and B16 tumor antigen-loaded particles were restimulated with tumor lysate and lysate, and MP-GM-CpG-TL MP containing GM-CSF plus CpG plus incubated with carboxyfluorescein succinimidyl ester (CFSE). After tumor lysate). Reproduced with kind permission from Springer 7 days of incubation, cells were harvested and stained with anti-CD8 Science and Business Media: Goforth et al., Fig. 4. Ó Springer- Ab and then subjected to flow cytometric analysis (FACScan, Verlag 2008 [69] microparticles (*2.3 lm diameter). In this study, it was ) and CpG resulted in substantial CD4? and found that the particle formulation more efficiently stim- CD8? T cell proliferation in the spleen (Fig. 5). It was ulated TH1-type immune responses (as measured by OVA- established that microparticles were taken up by dendritic specific IgG2a) over the fusion molecule after intraperito- cells in the peritoneal cavity and from there transported to the neal prime/boost (Day 0/Day 14) vaccination of mice [68]. spleen and that subsequent immune responses were TH1as Indeed, both formulations were significantly better at indicated by increased IFN-c production by isolated inducing TH1-type responses over soluble OVA and CpG splenocytes [18, 69]. Despite substantial generation of T or Alum/OVA. It is possible that the microparticle for- lymphocyte responses in vivo, the microparticles containing mulation may have promoted enhanced immune responses tumor lysate and CpG had only a modest antitumor effect in a over the fusion molecule through a combination of pro- therapeutic model where mice were challenged with B16.F1 viding a depot for sustained release and providing protec- melanoma cells. It was discovered that a combination of the tion of its immunogenic cargo. vaccination and the presence of the tumor contributed to Another particle-based vaccine formulation where CpG Treg expansion and that co-delivery of anti-Treg therapy has shown advantageous effects is in the context of tumor (anti-CD25) with the microparticle vaccination resulted in lysates. Tumor lysates are being investigated as possible significantly increased antitumor activity in vivo. cancer vaccines where the tumor cells, autologous or allo- geneic, are lysed primarily by repeated freeze–thaw cycles and the resultant crude tumor antigen preparation can be used Future directions as a vaccine in soluble form or, alternatively, can be encapsulated in particles. Goforth et al. [69] showed that The use of biodegradable particles as vectors for cancer intraperitoneal vaccination of mice with PLGA (75:25) mi- vaccines is a growing field, and a number of groups within croparticles loaded with tumor lysate (from B16.F1 the University of Iowa and the Iowa Hospital are currently

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01A2/UI Mayo Clinic Lymphoma SPORE), and the Lyle and Sharon Bighley Professorship. The authors thank Vijaya Joshi and Dr. Jiaqiang Xu (College of Pharmacy, University of Iowa) for their contribution to preparing SEM images.

Conflict of interest The authors declare they have no conflict of interest.

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