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Focal ablation technologies are routinely used in the clinical management of inoperable solid tumors but they often result in incomplete ablations leading to high recurrence rates. Adjuvant therapies, capable of safely eliminating residual tumor cells, are therefore of great clinical interest. Interleukin-12 (IL-12) is a potent antitumor cytokine that can be localized intratumorally through coformulation with viscous biopolymers, including chitosan (CS) solutions. The objective of this research was to determine if localized immunotherapy with a CS/IL-12 formulation could prevent tumor recurrence after cryoablation (CA). Tumor recurrence and overall survival rates were assessed. Systemic immunity was evaluated in spontaneously metastatic and bilateral tumor models. Temporal bulk RNA sequencing was performed on tumor and draining lymph node (dLN) samples. In multiple murine tumor models, the addition of CS/IL-12 to CA reduced recurrence rates by 30–55%. Altogether, this cryo-immunotherapy induced complete durable regression of large tumors in 80–100% of treated animals. Additionally, CS/IL-12 prevented lung metastases when delivered as a neoadjuvant to CA. However, CA plus CS/IL-12 had minimal antitumor activity against established, untreated abscopal tumors. Adjuvant anti-PD-1 therapy delayed the growth of abscopal tumors. Transcriptome analyses revealed early immunological changes in the dLN, followed by a significant increase in gene expression associated with immune suppression and regulation. Cryo-immunotherapy with localized CS/IL-12 reduces recurrences and enhances the elimination of large primary tumors. This focal combination therapy also induces significant but limited systemic antitumor immunity.

The accurate analytical characterization of metastatic phenotype at primary tumor diagnosis and its evolution with time are critical for controlling metastatic progression of cancer. Here, we report a label-free optical strategy using Raman spectroscopy and machine learning to identify distinct metastatic phenotypes observed in tumors formed by isogenic murine breast cancer cell lines of progressively increasing metastatic propensities. Our Raman spectra-based random forest analysis provided evidence that machine learning models built on spectral data can allow the accurate identification of metastatic phenotype of independent test tumors. By silencing genes critical for metastasis in highly metastatic cell lines, we showed that the random forest classifiers provided predictions consistent with the observed phenotypic switch of the resultant tumors towards lower metastatic potential. Furthermore, the spectral assessment of lipid and collagen content of these tumors was consistent with the observed phenotypic switch. Overall, our findings indicate that Raman spectroscopy may offer a novel strategy to evaluate metastatic risk during primary tumor biopsies in clinical patients.

Purpose: IFN-α is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this study was to evaluate the effectiveness and compatibility of combining recombinant IFN-α with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen.Experimental Design: The phenotypic and functional effects of IFN-α were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-α administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-α and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice.Results: We identified a dose and schedule of IFN-α that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8+) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-α distal to the site of vaccination. The combination of IFN-α and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA+ adenocarcinomas. In mice with pancreatic tumors, IFN-α slowed tumor growth, induced CTL activity, and increased CD8+ tumor-infiltrating lymphocytes.Conclusions: These data suggest that IFN-α can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.

Supplementary Figures S1-S7 from The Antitumor and Immunoadjuvant Effects of IFN-α in Combination with Recombinant Poxvirus Vaccines

Supplementary Figure 2 from Intravesical Immunotherapy of Superficial Bladder Cancer with Chitosan/Interleukin-12

Supplementary Figure 1 from Intravesical Immunotherapy of Superficial Bladder Cancer with Chitosan/Interleukin-12

Supplementary Figure Legends 1-2 from Intravesical Immunotherapy of Superficial Bladder Cancer with Chitosan/Interleukin-12

Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan, a biocompatible, mucoadhesive polysaccharide, could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88% to 100% of mice bearing orthotopic bladder tumors were cured after four intravesical treatments with chitosan/IL-12. In contrast, only 38% to 60% of mice treated with IL-12 alone and 0% treated with BCG were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. [Cancer Res 2009;69(15):6192–9]

BACKGROUND: Intravesical administration of interleukin 12 (IL-12) co-formulated with the biopolymer, chitosan (CS/IL-12), has demonstrated remarkable antitumor activity against preclinical models of bladder cancer. However, given historical concerns regarding severe toxicities associated with systemic IL-12 administration in clinical trials, it is important to evaluate the safety of intravesical CS/IL-12 prior to clinical translation. OBJECTIVE: To evaluate the pharmacokinetics as well as the local and systemic toxicities of intravesical CS/IL-12 immunotherapy in laboratory mice. METHODS: Local inflammatory responses in mouse bladders treated with intravesical IL-12 or CS/IL-12 were assessed via histopathology. Serum cytokine levels following intravesical and subcutaneous (s.c.) administrations of IL-12 or CS/IL-12 in laboratory mice were compared. Systemic toxicities were evaluated via body weight and liver enzyme levels. RESULTS: Intravesical IL-12 and CS/IL-12 treatments did not induce significant local or systemic toxicity. IL-12 dissemination and exposure from intravesical administration was significantly lower compared to s.c. injections. Weekly intravesical CS/IL-12 treatments were well-tolerated and did not result in blunted immune responses. CONCLUSIONS: Intravesical CS/IL-12 is safe and well-tolerated in mice. In particular, the lack of cystitis and acute inflammation justifies continued investigation of intravesical CS/IL-12 immunotherapy in larger animals and patients with bladder cancer.

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.

BackgroundUnresectable solid malignancies are responsible for a major proportion of total cancer-related mortalities, making focal ablation an attractive alternative. Nevertheless, there are high rates of recurrence after ablation [1,2]. The addition of an immune agonist to ablation has the potential to prevent this recurrence and improve treatment outcomes. The goal of this study is to determine if localized interleukin-12 (IL-12) can prevent primary tumor recurrence after cryoablation in both minimal ablation and metastasis models.MethodsLLC (LL/2) (ATCC) and MC38 (NCI) were implanted in 6–9 week old C57BL/6 mice. All tumors were treated at volumes of 200–500 mm3. LLC tumors with treated with three cycles of freeze/thaw and then monitored for tumor recurrence and lung metastasis 22–25 days after implantation. MC38 tumors were treated with a minimal cryoablation protocol where the tumor undergoes one cycle of freezing at 100% intensity up to the tumor margin by visual inspection, followed by one cycle of active thaw until the cryo probe can be removed. Ablation was performed using the Argon-Helium Visual-ICETM Cryoablation System (Boston Scientific). Interleukin-12 (IL-12) in 1.5% (w/v) chitosan acetate (CS) dissolved in dPBS was injected intratumorally within an hour after cryoablation, or as indicated. The dose of IL-12 was 1 ug unless otherwise indicated.ResultsWe established a model of 100% recurrence using the minimal cryoablation protocol. Using this protocol, we demonstrated that a single intratumoral injection of CS/IL-12 within an hour after cryoablation prevents recurrence in 7/8 mice while only 3/8 mice remained tumor-free without CS/IL-12 (Figure 1). Studies evaluating the impact of CS/IL-12 on tumor-specific T cell responses following cryoablation are ongoing. In the spontaneously metastatic LLC model, delivering CS/IL-12 two days before treatment, either resection or cryoablation, reduced the number of metastatic lung nodules and furthermore prevented the recurrence of the primary tumor after resection (Figure 2).Abstract 620 Figure 1CS/IL-12 prevents tumor recurrence after minimal cryoAbstract 620 Figure 2CS/IL-12 before cryo or surgery prevents metastasisConclusionsDue to the high percentage of solid malignancies that are unresectable at diagnosis, focal ablation is an attractive alternative, yet has a high rate of recurrence. We demonstrated that intratumoral neoadjuvant CS/IL-12 protects not only against primary recurrence after cryoablation, but also protects against lung metastasis and recurrence after resection. Further studies are necessary to explore the immune populations responsible for this therapeutic effect.AcknowledgementsThis work is supported by Boston Scientific, the NC State University Provost’s Fellowship, the NSF Graduate Research Fellowship and startup funds provided by the College of Engineering at NC State University.ReferencesWeld KJ, Landman J. Comparison of cryoablation, radiofrequency ablation and high-intensity focused ultrasound for treating small renal tumours. BJU Int. 2005;96(9):1224–1229. doi:10.1111/j.1464–410X.2005.05848.x.Guenther E, Klein N, Zapf S, et al. Prostate cancer treatment with Irreversible Electroporation (IRE): Safety, efficacy and clinical experience in 471 treatments. PloS One. 2019;14(4):e0215093. doi:10.1371/journal.pone.0215093Ethics ApprovalThe Institutional Animal Care and Use Committee at North Carolina State University approved of all animal protocols (#19–795) in compliance with The Guide for Care and Use of Laboratory Animals (National Research Council).

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC(50) value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.

Background Clinically, cryoablation is used to treat certain early stage prostate, liver, and kidney tumors in addition to bone and soft tissue sarcomas. However, for late-stage cancers, ablation is only an auxiliary step before complete resection. This leaves a gap of patients with advanced and inoperable tumors, where up to 90% of all pancreatic, and 80–85% of all prostate cancers are unresectable at diagnosis.1 2 Because cryoablation can release large amounts of antigen, it is uniquely capable of not only treating advanced, unresectable tumors, but also may induce an in situ vaccination response when combined with the appropriate immunotherapy. Previously, our results in single primary tumor models indicated that the addition of interleukin-12 (IL-12) to cryoablation (cryo) improved tumor burden and survival We hypothesized that intratumoral injection of IL-12 after cryo would activate a strong T cell response and induce systemic immunity in bilateral tumor models. Methods Panc02 cells were purchased from ATCC; MC38 and MB49 cells were acquired from the NIH. Female C57BL/6 mice were purchased from Jackson Laboratory. For primary tumor implantation, 1 × 106 Panc02 cells and 3 × 105 MC38 cells were injected subcutaneously (s.c.) in the right flank. For rechallenge, the same dose of cells was implanted on the left flank of cured mice. For bilateral models, in both the MB49 and MC38 models, 3 × 105 and 1.5 × 105 cells were injected s.c. in the right and left flanks respectively on the same day. For the Panc02 model, 1 × 106 cells were implanted s.c. on both the right and left flanks on the same day. Tumor volume was calculated as 0.5*a*b2 given the perpendicular long (a) and short (b) dimensions. Tumors measuring between 150–300 mm3 were cryoablated with three cycles of freeze/thaw using the Visual-ICETM Cryoablation System (Boston Scientific). The dose of IL-12 was 1 µg/mouse in 1.5% (w/v) chitosan acetate (CS) dissolved in DPBS, and then injected intratumorally within an hour after cryoablation. For the anti-PD-1 and isotype antibodies (BioXCell, clone: RMP1.14). 300µg was injected intraperitoneally every 3 days starting on the day of cryoablation for a total of 4 doses. Results In the bilateral MB49 mouse bladder cancer model, the median survival for the cryo alone group was 20 days post treatment (p.t.) compared to 23 days p.t. for cryo + CS/IL-12, which was not significant, and 12 days for the untreated control group. In the bilateral Panc02 model, the median survival for both the cryo alone and cryo + CS/IL-12 groups was the same at 20.5 days p.t., compared to 10 days p.t. for the untreated control. In the bilateral MC38 model, the addition of anti-PD-1 to cryo + CS/IL-12 did not significantly improve survival compared to isotype + cryo + CS/IL-12, with a median survival of 24 days p.t. and 16 days p.t. respectively (p=0.53, Log-rank test) (figure 1). However, addition of anti-PD-1 did significantly delay abscopal tumor growth up to 500 mm3 when compared to the isotype + cryo + CS/IL-12 (p=0.0398, Unpaired t test) (figure 2). Finally, the addition of IL-12 worsens memory in the MC38 model, where 100% of rechallenged cryo alone mice were protected (5/5) compared to only 43% protected of the cryo + CS/IL-12 group (3/7). Conclusions Conclusions: While cryoablation in combination with immunotherapy has the potential to treat advanced, unresectable primary tumors and distant untreated tumors, the addition of a single injection of IL-12 is not enough to induce a strong abscopal effect. Furthermore, it may actually worsen the establishment of effector memory cells. The addition of anti-PD-1 only slows abscopal tumor growth. Future work is needed to understand the mechanism of T cell priming in the context of the post-ablative tumor. Acknowledgements This work is supported by Boston Scientific, the NC State University Provost’s Fellowship, the NSF Graduate Research Fellowship and startup funds provided by the College of Engineering at NC State University. References van der Geest LGM. et al. Trends in treatment and survival of patients with nonresected, nonmetastatic pancreatic cancer: a population-based study. Cancer Med 2018; 7, 4943–4951. Takaki H. et al. Thermal ablation and immunomodulation: from preclinical experiments to clinical trials. Diagn. Interv. Imaging 2017; 98:651–659.

Interleukin-12 (IL-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. Unfortunately, IL-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. Early clinical trials in the mid-1990's showed that systemic delivery of IL-12 incurred dose-limiting toxicities. Nevertheless, IL-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. The development of strategies which maximize IL-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. Diverse IL-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. Several localized IL-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. Taken together, localized delivery systems are supporting an IL-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. This review begins with a brief historical account of cytokine monotherapies and describes how IL-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. The bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for IL-12-based cancer immunotherapies. Advantages and limitations of different delivery technologies are highlighted. Finally, perspectives on how IL-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered.

More than a century ago, the American surgeon William Coley noticed a correlation between cancer remissions and postoperative infections: some patients who had battled an infection also experienced a regression of their cancer. Because of these observations, Coley hypothesized that a patient's immune response to a bacterial infection could be leveraged to treat cancer. To test his hypothesis, Coley injected live bacteria into an inoperable tumor of one of his patients. The patient's tumor regressed, and Coley went on to experiment with direct injections of live, and later heat-killed, bacteria into more than a thousand patients over the next 40-plus years. Coley's toxins never achieved widespread clinical success due to concerns over reproducibility, although a strain of mycobacterium, bacillus Calmette-Guerin, is still routinely administered to treat early-stage bladder cancers.

Ailments of the bladder are often treated via intravesical delivery-direct application of therapeutic into the bladder through a catheter. This technique is employed hundreds of thousands of times every year, but protocol development has largely been limited to empirical determination. Furthermore, the numerical analyses of intravesical delivery performed to date have been restricted to static geometries and have not accounted for bladder deformation. This study uses a finite element analysis approach with biphasic solute transport to investigate several parameters pertinent to intravesical delivery including solute concentration, solute transport properties and instillation volume. The volume of instillation was found to have a substantial impact on the exposure of solute to the deeper muscle layers of the bladder, which are typically more difficult to reach. Indeed, increasing the instillation volume from 50-100 ml raised the muscle solute exposure as a percentage of overall bladder exposure from 60-70% with higher levels achieved for larger instillation volumes. Similar increases were not seen for changes in solute concentration or solute transport properties. These results indicate the role that instillation volume may play in targeting particular layers of the bladder during an intravesical delivery.

Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.

Acidic fibroblast growth factors (FGF1s) are heparin binding proteins that regulate a wide array of key cellular processes and are also candidates for promising biomedical applications. FGF1-based therapeutic applications are currently limited due to their inherent thermal instability and susceptibility to proteases. Using a wide range of biophysical and biochemical techniques, we demonstrate that reversal of charge on a well-conserved positively charged amino acid, R136, in the heparin binding pocket drastically increases the resistance to proteases, thermal stability, and cell proliferation activity of the human acidic fibroblast growth factor (hFGF1). Two-dimensional NMR data suggest that the single point mutations at position-136 (R136G, R136L, R136Q, R136K, and R136E) did not perturb the backbone folding of hFGF1. Results of the differential scanning calorimetry experiments show that of all the designed R136 mutations only the charge reversal mutation, R136E, significantly increases (ΔTm = 7 °C) the thermal stability of the protein. Limited trypsin and thrombin digestion results reveal that the R136E mutation drastically increases the resistance of hFGF1 to the action of the serine proteases. Isothermal titration calorimetry data show that the R136E mutation markedly decreases the heparin binding affinity of hFGF1. Interestingly, despite lower heparin binding affinity, the cell proliferation activity of the R136E variant is more than double of that exhibited by either the wild type or the other R136 variants. The R136E variant due to its increased thermal stability, resistance to proteases, and enhanced cell proliferation activity are expected to provide valuable clues for the development of hFGF1- based therapeutics for the management of chronic diabetic wounds.

Background Although metastasis is ultimately responsible for about 90% of breast cancer mortality, the vast majority of breast-cancer-related deaths are due to progressive recurrences from non-metastatic disease. Current adjuvant therapies are unable to prevent progressive recurrences for a significant fraction of patients with breast cancer. Autologous tumor cell vaccines (ATCVs) are a safe and potentially useful strategy to prevent breast cancer recurrence, in a personalized and patient-specific manner, following standard-of-care tumor resection. Given the high intra-patient and inter-patient heterogeneity in breast cancer, it is important to understand which factors influence the immunogenicity of breast tumor cells in order to maximize ATCV effectiveness. Methods The relative immunogenicity of two murine breast carcinomas, 4T1 and EMT6, were compared in a prophylactic vaccination-tumor challenge model. Differences in cell surface expression of antigen-presentation-related and costimulatory molecules were compared along with immunosuppressive cytokine production. CRISPR/Cas9 technology was used to modulate tumor-derived cytokine secretion. The impacts of cytokine deletion on splenomegaly, myeloid-derived suppressor cell (MDSC) accumulation and ATCV immunogenicity were assessed. Results Mice vaccinated with an EMT6 vaccine exhibited significantly greater protective immunity than mice vaccinated with a 4T1 vaccine. Hybrid vaccination studies revealed that the 4T1 vaccination induced both local and systemic immune impairments. Although there were significant differences between EMT6 and 4T1 in the expression of costimulatory molecules, major disparities in the secretion of immunosuppressive cytokines likely accounts for differences in immunogenicity between the cell lines. Ablation of one cytokine in particular, granulocyte-colony stimulating factor (G-CSF), reversed MDSC accumulation and splenomegaly in the 4T1 model. Furthermore, G-CSF inhibition enhanced the immunogenicity of a 4T1-based vaccine to the extent that all vaccinated mice developed complete protective immunity. Conclusions Breast cancer cells that express high levels of G-CSF have the potential to diminish or abrogate the efficacy of breast cancer ATCVs. Fortunately, this study demonstrates that genetic ablation of immunosuppressive cytokines, such as G-CSF, can enhance the immunogenicity of breast cancer cell-based vaccines. Strategies that combine inhibition of immunosuppressive factors with immune stimulatory co-formulations already under development may help ATCVs reach their full potential. Electronic supplementary material The online version of this article (10.1186/s13058-018-1054-3) contains supplementary material, which is available to authorized users.

Human acidic fibroblast growth factor 1 (hFGF1) is a protein intricately involved in cell growth and tissue repair. In this study, we investigate the effect(s) of understanding the role of a conserved proline (P135), located in the heparin binding pocket, on the structure, stability, heparin binding affinity, and cell proliferation activity of hFGF1. Substitution of proline-135 with a positively charged lysine (P135K) resulted in partial destabilization of the protein; however, the overall structural integrity of the protein was maintained upon substitution of proline-135 with either a negative charge (P135E) or a polar amino acid (P135Q). Interestingly, upon heparin binding, an increase in thermal stability equivalent to that of wt-hFGF1 was observed when P135 was replaced with a positive (P135K) or a negative charge (P135E), or with a polar amino acid (P135Q). Surprisingly, introduction of negative charge in the heparin-binding pocket at position 135 (P135E) increased hFGF1's affinity for heparin by 3-fold, while the P135K mutation, did not alter the heparin-binding affinity. However, the enhanced heparin-binding affinity of mutant P135E did not translate to an increase in cell proliferation activity. Interestingly, the P135K and P135E double mutations, P135K/R136E and P135/R136E, reduced the heparin binding affinity by ∼3-fold. Furthermore, the cell proliferation activity was increased when the charge reversal mutation R136E was paired with both P135E (P135E/R136E) and P135K (P135K/R136E). Overall, the results of this study suggest that while heparin is useful for stabilizing hFGF1 on the cell surface, this interaction is not mandatory for activation of the FGF receptor.

Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immu- nity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be � 98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 ± 0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (K d(app) =6 9 ±1 lM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.

Many life-threatening diseases are disseminated through biological fluids, such as blood, lymph, and cerebrospinal fluid. The migration of tumor cells through the vascular circulation is a mandatory step in metastasis, which is responsible for ∼90% of cancer-associated mortality. Circulating pathogenic bacteria, viruses, or blood clots lead to other serious conditions including bacteremia, sepsis, viremia, infarction, and stroke. Therefore, technologies capable of detecting circulating tumor cells (CTCs), circulating bacterial cells (CBCs), circulating endothelial cells (CECs), circulating blood clots, cancer biomarkers such as microparticles and exosomes, which contain important microRNA signatures, and other abnormal features such as malaria parasites in biological fluids may facilitate early diagnosis and treatment of metastatic cancers, infections, and adverse cardiovascular events. Unfortunately, even in a disease setting, circulating abnormal cells are rare events that are easily obscured by the overwhelming background material in whole blood. Existing detection methods mostly rely on ex vivo analyses of limited volumes (a few milliliters) of blood samples. These small volumes limit the probability of detecting CTCs, CECs, CBCs and other rare phenomena. In vivo detection platforms capable of continuously monitoring the entire blood volume may substantially increase the probability of detecting circulating abnormal cells and, in particular, increase the opportunity to identify exceedingly rare and potentially dangerous subsets of these cells, such as circulating cancer stem cells (CCSCs). In addition, in vivo detection technologies capable of destroying and/or capturing circulating abnormal cells may inhibit disease progression. This review focuses on novel therapeutic and diagnostic (theranostic) platforms integrating in vivo real-time early diagnosis and nano-bubble based targeted therapy of CTCs, CECs, CBCs and other abnormal objects in circulation. This critical review particularly focuses on nanotechnology-based theranostic (nanotheranostic) approaches, especially in vivo photoacoustic (PA) and photothermal (PT) nanotheranostic platforms. We emphasize an urgent need for in vivo platforms composed of multifunctional contrast nanoagents, which utilize diverse modalities to realize a breakthrough for early detection and treatment of harmful diseases disseminated through the circulation.

The clinical management of bladder cancer has not changed significantly in several decades. In particular, intravesical bacillus Calmette–Guérin (BCG) immunotherapy has been a mainstay for high-risk nonmuscle invasive bladder cancer since the late 1970s/early 1980s. This is despite the fact that bladder cancer has the highest recurrence rates of any cancer and BCG immunotherapy has not been shown to induce a tumor-specific immune response. We and others have hypothesized that immunotherapies capable of inducing tumor-specific adaptive immunity are needed to impact bladder cancer morbidity and mortality. This article summarizes the preclinical and clinical development of bladder cancer immunotherapies with an emphasis on the last 5 years. Expected progress in the near future is also discussed.

The prevalence of obesity, an established risk factor for several chronic diseases, including cancer, has risen dramatically over the past 4 decades. Dietary change and/or increased physical activity are the most commonly recommended lifestyle-based strategies for preventing or reversing obesity. One of several physiological systems that may be enhanced by dietary change and exercise is the immune system. In this study, we examined the effects of energy restriction (ER; 30% reduction relative to control energy intake) and/or exercise (EX; voluntary wheel running) on systemic and mucosal immune function. Female C57BL/6 mice were randomized into 4 treatment conditions: 1) controls consumed ad libitum (AL); 2) AL with access to running wheels (AL + EX); 3) 30% ER; and 4) 30% ER with access to running wheels (ER + EX). Both ER and EX reduced spleen weight and the number of splenic T and B lymphocytes (P < 0.05). ER enhanced natural killer (NK) cell function, but reduced concanavalin A (Con A)-induced T-cell proliferation (P < 0.05). In contrast, EX enhanced Con A-induced proliferation and cytokine production from Peyer's patch cells (P < 0.05). These data suggest that ER and EX enhance some, but not all, components of the immune system and are likely working via different biological mechanisms to regulate NK and T-cell function.

Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses both clinically and preclinically. This study evaluates the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 12-24h. Furthermore, a single s.c. injection of 20 microg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20 microg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20 microg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20 microg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4(+) proliferation, peptide-specific CD8(+) pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF.

The development of safe, novel adjuvants is necessary to maximize the efficacy of new and/or available vaccines. Chitosan is a non-toxic, biocompatible, biodegradable, natural polysaccharide derived from the exoskeletons of crustaceans and insects. Chitosan's biodegradability, immunological activity and high viscosity make it an excellent candidate as a depot/adjuvant for parenteral vaccination. To this end, we explored chitosan solution as an adjuvant for subcutaneous vaccination of mice with a model protein antigen. We found that chitosan enhanced antigen-specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold. Strong increases in antibody titers together with robust delayed-type hypersensitivity (DTH) responses revealed that chitosan induced both humoral and cell-mediated immune responses. When compared with traditional vaccine adjuvants, chitosan was equipotent to incomplete Freund's adjuvant (IFA) and superior to aluminum hydroxide. Mechanistic studies revealed that chitosan exhibited at least two characteristics that may allow it to function as an immune adjuvant. First, the viscous chitosan solution created an antigen depot. More specifically, less than 9% of a protein antigen, when delivered in saline, remained at the injection site after 8 h. However, more than 60% of a protein antigen delivered in chitosan remained at the injection site for 7 days. Second, chitosan induced a transient 67% cellular expansion in draining lymph nodes. The expansion peaked between 14 and 21 days after chitosan injection and diminished as the polysaccharide was degraded. These mechanistic studies, taken together with the enhancement of a vaccine response, demonstrate that chitosan is a promising and safe platform for parenteral vaccine delivery.

The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. In order to characterize and improve the interstitial transport, we developed a reproducible single molecule detection method to observe the electromobility of DNA in a range of pulsed, high field strength electric fields typically used during electric field-mediated gene delivery. Using agarose gel as an interstitium phantom, we investigated the dependence of DNA electromobility on field magnitude, pulse duration, pulse interval, and pore size in the interstitial space. We observed that the characteristic electromobility behavior, exhibited under most pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, and pulse interval of the applied pulse sequences, as well as the pore size of the fibrous matrix through which the DNA migrated. Our data also suggest the existence of a minimum pulse amplitude required to initiate electrophoretic transport. These results are useful for understanding the mechanisms of DNA electromobility and improving interstitial transport of genes during electric field-mediated gene delivery.

Interstitial transport of DNA is a rate-limiting step in electric field-mediated gene delivery in vivo. Interstitial transport of macromolecules, such as plasmid DNA, over a distance of several cell layers, is inefficient due to small diffusion coefficient and inadequate convection. Therefore, we explored electric field as a novel driving force for interstitial transport of plasmid DNA. In this study, agarose gels were used to mimic the interstitium in tissues as they had been well characterized and could be prepared reproducibly. We measured the electrophoretic movements of fluorescently labeled plasmid DNA in agarose gels with three different concentrations (1.0%, 2.0% and 3.0%) subjected to electric pulses at three different field strengths (100, 200 and 400 V/cm) and four different pulse durations (10, 50, 75, 99 ms). We observed that: (1) shorter pulses (10 ms) were not as efficient as longer pulses in facilitating plasmid transport through agarose gels; (2) plasmid electromobility reached a plateau at longer pulse durations; and (3) plasmid electromobility increased with applied electric energy, up to a threshold, in all three gels. These data suggested that both pulse strength and duration needed to be adequately high for efficient plasmid transport through extracellular matrix. We also found that electric field was better than concentration gradient of DNA as a driving force for interstitial transport of plasmid DNA.

Approximately 9 of 10 breast cancer-related deaths are attributable to metastasis. Yet, less than 4% of breast cancer patients are initially diagnosed with metastatic cancer. Therefore, the majority of breast cancer-related deaths are due to recurrence and progression of nonmetastatic disease. There is tremendous clinical opportunity for novel adjuvant strategies, such as immunotherapies, that have the potential to prevent progressive recurrences. In particular, autologous tumor cell-based vaccines can train a patient's immune system to recognize and eliminate occult disease. Autologous tumor cell-based vaccines have several advantages including safety, multivalency and patient specificity. Furthermore, because lumpectomy or mastectomy is indicated for the vast majority of breast cancer patients, resected tumors offer a readily available, patient-specific source of tumor antigen. Disadvantages of autologous tumor cell-based vaccines include poor immunogenicity and production inconsistencies. This review summarizes recent progress in the development of autologous breast tumor vaccines and offers insight for overcoming existing limitations.

Bladder cancer is a highly recurrent disease in need of novel, durable treatment strategies. This study assessed the ability of an intravesical immunotherapy composed of a coformulation of the biopolymer chitosan with interleukin-12 (CS/IL-12) to induce systemic adaptive tumor-specific immunity. Intravesical CS/IL-12 immunotherapy was used to treat established orthotopic MB49 and MBT-2 bladder tumors. All mice receiving intravesical CS/IL-12 immunotherapy experienced high cure rates of orthotopic disease. To investigate the durability and extent of the resultant adaptive immune response, cured mice were rechallenged both locally (intravesically) and distally. Cured mice rejected 100 % of intravesical tumor rechallenges and 50–100 % of distant subcutaneous rechallenges in a tumor-specific manner. The ability of splenocytes from cured mice to lyse targets in a tumor-specific manner was assessed in vitro, revealing that lytic activity of splenocytes from cured mice was robust and tumor specific. Protective immunity was durable, lasting for at least 18 months after immunotherapy. In an advanced bladder cancer model, intravesical CS/IL-12 immunotherapy controlled simultaneous orthotopic and subcutaneous tumors in 70 % of treated mice. Intravesical CS/IL-12 immunotherapy creates a robust and durable tumor-specific adaptive immune response against bladder cancer. The specificity, durability, and potential of this therapy to treat both superficial and advanced disease are deserving of consideration for clinical translation.

Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation–coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated. Chitosan particle sizes, which ranged from 300 nm to 3 μm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, were inversely related to the hydration strength of precipitant salts, protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. This study demonstrates that chitosan nano/microparticles with high protein loading efficiencies can be engineered with well-defined sizes and controllable release kinetics through manipulation of specific formulation parameters.

There is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8+ T cells are required for initial tumor rejection, but CD4+ T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4+ and CD8+ effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer.

The vast majority of breast cancer-related deaths are due to progressive recurrences of non-metastatic disease. Current adjuvant therapies aimed at preventing breast cancer recurrence are ineffective for tens of thousands of women each year. In addition, current adjuvant therapies carry significant co-morbidities. Active, specific immune-based strategies have the potential to train a patient's immune system to recognize and eliminate occult metastases. Here, we explore autologous tumor cell vaccines (ATCVs) as a strategy to prevent tumor recurrence and improve survival. Because >90% of breast cancer patients undergo resection, autologous tumor cells are readily available for the production of personalized vaccine. Patient-specific vaccination is particularly attractive for breast cancer which is highly heterogeneous. The major disadvantage of ATCVs, is poor immunogenicity. Thus, in order to develop an effective, immunogenic ATCV against breast cancer, we wanted to understand the features of immunogenicity. In this study, we evaluated the immunogenicities of two murine breast cancer cell lines, EMT6 and 4T1. For tumor protection studies, BALB/c female mice were given priming and booster vaccinations, ten days apart, with 1,000,000 irradiated (100Gy) cells. Ten days after the booster vaccination, mice were challenged with live tumor cells. Mice vaccinated with EMT6 cells were completely protected against a live EMT6 challenge in 80% of mice. However, mice vaccinated with irradiated 4T1 cells failed to provide any protection against a live 4T1 challenge. Most interestingly, when mice were vaccinated with a mixture of irradiated EMT6 and 4T1 cells, the protective response against EMT6 challenge was significantly diminished as 60% of mice developed tumors. This finding implied that non-immunogenic 4T1 cells released one or more immunosuppressive factors that inhibited anti-EMT6 immunity. Thus, we investigated the levels of different immunosuppressive cytokines, GM-CSF, IL-6, MCP-1, TGF-β and VEGF released by both 4T1 and EMT6 cells before and after irradiation. Of the different cytokines measured, we found that only GM-CSF is produced at significantly higher levels by 4T1 cells than EMT6 cells. Since at higher levels (>200pg/ml) GM-CSF can induce the accumulation of large amounts of myeloid derived suppressor cells (MDSCs) in the tumor site and lymphoid organs, we believe it to be the reason for the failure of the hybrid vaccine. Future studies will determine if blocking GM-CSF production by 4T1 cells will decrease the accumulation of MDSCs and subsequently increase anti-tumor immunity. Citation Format: Ravindranathan S, Kurtz SL, Smith SG, Koppolu B, Nguyen KG, Zaharoff DA. Characterization of an autologous tumor cell vaccine against breast cancer in mice. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-16.

Introduction: Intravesical immunotherapy with mycobacterium bovis baccillus Calmette-Guerin (BCG) has been the standard of care for high grade non-muscle invasive bladder tumors for nearly 40 years. While BCG has been successful at lowering recurrence rates and preventing progression, it does not engage adaptive memory and is associated with recurrence rates as high as 50%. Thus the search for treatment strategies that promote a durable anti-tumor memory response is warranted. One such strategy developed by our group is the intravesical administration of a simple coformulation of the TH1 polarizing cytokine interleukin-12 (IL-12) and the biopolymer chitosan (chitosan/IL-12). Chitosan is not bioactive but is cationic and viscous in solution and acts as a delivery vehicle for IL-12. It is presumed to enhance three aspects of delivery. 1) Chitosan's positive charge interacts with the urothelium's tight junctions to transiently increase its permeability. 2) Mucoadhesive forces between the urothelium's mucosa and chitosan prolong the contact of IL-12 with the urothelium. 3) The viscous nature of chitosan limits expulsion of IL-12 during voiding of the bladder. Our group has recently demonstrated that four weekly intravesical administrations of chitosan/IL-12 can 1) eliminate established orthotopic in >90% of mice, 2) provide protective local immunity, and 3) promote durable systemic immunity. The purpose of this study is to investigate the mechanisms of intravesical chitosan/IL-12 immunotherapy. Specifically, we look at the role of natural killer cells (NK), CD8+ cytotoxic T-cells, and CD4+ T-helper cells in chitosan/IL-12 therapy of orthotopic tumors as well as their importance for maintaining systemic immunity to those bladder tumors. Methods: Orthotopic bladder tumors were generated via intravesical implantation of 75,000 MB49 cells into female C57BL/6J mice. Briefly, anesthetized mice were catheterized with a 24G x 3/4” Teflon catheter and the cells injected after a 10 minute wash with Poly-L-Lysine. Upon onset of hematuria, usually one week post implantation, mice received four twice-weekly intravesical administrations of either saline or chitosan/IL-12 immunotherapy (1 µg IL-12 in 100µl of 10 mg/ml chitosan). For immune subset depletion studies, four daily administrations of 100 µg anti-CD4 (GK1.5), anti-CD8 (2.43), or anti-NK1.1 (PK136) antibodies were given prior to implantation. Depletion was maintained for the duration of the study with 100 µg antibody given twice per week. For systemic rechallenge studies, mice were injected with 300,000 MB49 in the right flank. Results: Mice were depleted of NK, CD8+, or CD4+ lymphocytes prior to tumor inoculation to investigate the importance of immune cell types on the efficacy of intravesical chitosan/IL-12. Mice depleted of CD8+ T-cells (n=9) failed to eliminate orthotopic tumors, but did extend survival over the saline treated control. Mice depleted of CD4+ or NK+ immune cells elicited a more mixed result with 4/8 and 3/8 mice respectively surviving long term, free of tumors. Mice that were not depleted, but were given chitosan/IL-12 therapy eliminated their tumors in 7/9 instances. In a separate study to investigate the importance of T-cell subtypes in maintaining systemic immunity, mice previously cured of orthotopic disease were depleted of CD8+ or CD4+ T-cells and then rechallenged subcutaneously. Eight out of 10 CD8-depleted mice rejected the tumors, while only 1/9 CD4-depleted mice rejected the rechallenge. All naïve mice grew subcutaneous tumors (n=9). All mice previously cured of orthotopic disease and not depleted rejected the subcutaneous rechallenge (n=4). Conclusions: The elimination of established orthotopic tumors in response to chitosan/IL-12 therapy involves multiple immune cells types. As expected CD8+ T-cells appear to be the primary effectors with CD4+ and NK+ cells playing lesser, but still important roles. Interestingly, the relative importance of CD8+ and CD4+ T-cells seems to reverse for the systemic memory response to tumor rechallenge; almost all subcutaneously rechallenged mice depleted of CD4 grew tumors, but nearly all CD8 depleted mice rejected tumors. Further mechanistic studies are warranted to explain and build upon these results. Citation Format: Sean Smith, Jack Baltz, Bhanu Koppolu, Sruthi Ravindranathan, Khue Nguyen, David Zaharoff. Effector cells in chitosan/interleukin-12 immunotherapy of bladder tumors in mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B020.

Particle-based vaccine delivery systems are under exploration to enhance antigen-specific immunity against safe but poorly immunogenic polypeptide antigens. Chitosan is a promising biomaterial for antigen encapsulation and delivery due to its ability to form nano- and microparticles in mild aqueous conditions thus preserving the antigenicity of loaded polypeptides. In this study, the influence of chitosan encapsulation on antigen uptake, activation and presentation by antigen presenting cells (APCs) is explored. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) and ovalbumin (OVA) were used as model protein antigens and encapsulated in chitosan particles via precipitation-coacervation at loading efficiencies >89%. Formulation conditions were manipulated to create antigen-encapsulated chitosan particles (AgCPs) with discrete nominal sizes (300 nm, 1 μm, and 3 μm). Uptake of AgCPs by dendritic cells and macrophages was found to be dependent on particle size, antigen concentration and exposure time. Flow cytometry analysis revealed that uptake of AgCPs enhanced upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Lastly, antigen-specific T cells exhibited higher proliferative responses when stimulated with APCs activated with AgCPs versus soluble antigen. These data suggest that encapsulation of antigens in chitosan particles enhances uptake, activation and presentation by APCs.

Vaccines based on recombinant proteins require adjuvant systems in order to generate Th1-type immune responses. We have developed a vaccine adjuvant system using a viscous chitosan solution and interleukin (IL)-12, a Th1-inducing cytokine. The chitosan solution is designed to create a depot of antigen and IL-12 at a subcutaneous injection site. We measured the in vivo immune response of a vaccine containing 0.25, 1, or 4 μg murine IL-12 and 75 μg ovalbumin (OVA), formulated in a 1.5% chitosan glutamate solution. The chitosan/IL-12/OVA vaccine, in comparison to chitosan/OVA, IL-12/OVA, or OVA alone, elicited greater antigen-specific CD4(+) and CD8(+) T-cell responses, as determined by CD4(+) splenocyte proliferation, Th1 cytokine release, CD8(+) T-cell interferon-γ release, and MHC class I peptide pentamer staining. The combination of chitosan and IL-12 also enhanced IgG2a and IgG2b antibody responses to OVA. Co-formulation of chitosan and IL-12 thus promoted the generation of a Th1 immune response to a model protein vaccine.

Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent TH1 cytokine with robust antitumor activity and the ability to potentiate immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan, a biocompatible, mucoadhesive polysaccharide, could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88% to 100% of mice bearing orthotopic bladder tumors were cured after four intravesical treatments with chitosan/IL-12. In contrast, only 38% to 60% of mice treated with IL-12 alone and 0% treated with BCG were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple TH1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer. [Cancer Res 2009;69(15):6192–9]