Combinatorial immunotherapy and nanoparticle mediated hyperthermia

    Combinatorial immunotherapy and nanoparticle mediated hyperthermia Austin J. Moy, James W. Tunnell PII: DOI: Reference:

S0169-409X(17)30092-3 doi:10.1016/j.addr.2017.06.008 ADR 13134

To appear in:

Advanced Drug Delivery Reviews

Received date: Revised date: Accepted date:

1 March 2017 27 May 2017 13 June 2017

Please cite this article as: Austin J. Moy, James W. Tunnell, Combinatorial immunotherapy and nanoparticle mediated hyperthermia, Advanced Drug Delivery Reviews (2017), doi:10.1016/j.addr.2017.06.008

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ACCEPTED MANUSCRIPT Title

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Combinatorial immunotherapy and nanoparticle mediated hyperthermia

Authors

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Austin J. Moy, James W. Tunnell

Affiliation

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Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA

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Corresponding author

Biomedical Engineering

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107 W. Dean Keaton St., Stop C0800

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The University of Texas at Austin 512-232-2110

[email protected]

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James W. Tunnell

ACCEPTED MANUSCRIPT Abstract

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Immune checkpoint therapy has become the first widely adopted immunotherapy for patients with late stage malignant melanoma, with potential for a wide range of cancers. While some patients can experience long term disease remission, this is limited only to a subset of patients and tumor types. The path forward to expand this therapy to more patients and tumor types is currently thought to be combinatorial treatments, the combination of immunotherapy with other treatments. In this review, the combinatorial approach of immune checkpoint therapy combined with nanoparticle-assisted localized hyperthermia is discussed, starting with an overview of the different nanoparticle hyperthermia approaches in development, an overview of the state of immune checkpoint therapy, recent reports of immune checkpoint therapy and nanoparticle-assisted hyperthermia in a combinatorial approach, and finally a discussion of future research topics and areas to be explored in this new combinatorial approach to cancer treatment.

Keywords

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Nanoparticles, hyperthermia, immunotherapy, cancer, combinatorial treatment, laser, near infrared, radiofrequency, magnetic

Abbreviations

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MM: Metastatic melanoma

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ICP: Immune checkpoint

NPHT: Nanoparticle-assisted hyperthermia

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NIR: Near-infrared

AMF: Alternating magnetic field PTT: Photothermal therapy

MFH: Magnetic field hyperthermia

RFH: Radiofrequency hyperthermia

Table of Contents 1. Introduction 2. Nanoparticle Hyperthermia Platforms 3. Thermal dose dependent physiological response 4. Combinatorial Approaches 5. Future Opportunities

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Immune checkpoint (ICP) therapy [1] has become the first widely adopted and successful immunotherapy for the treatment of metastatic melanoma (MM) and holds great promise for achieving success in a broad range of cancers [2]. In contrast to direct cytotoxic approaches (e.g. chemotherapy, radiation, targeted therapies) that seek to directly kill cancer cells, immunotherapies alter the immune response so that the innate and adaptive systems attack and eradicate the cancer on its own, including induction of a long-term immunity. The first ICP therapy (ipilimumab), which was FDA approved in 2011, demonstrated the use of the first drug ever to significantly improve overall survival benefit in patients with MM. Most importantly, a subset of patients considered “complete responders” (~20%) experienced total tumor remission that included a long term response, remaining cancer free beyond 10 years [3,4]. In 2014, the FDA approved another set of ICP therapies (nivolumab and pembroluzimab) for patients with MM that increased durable response rates to 40% [5–7]. ICP therapies have been the most promising therapeutic to affect MM since the beginning of cancer treatment. In the coming years, we are likely to see additional immunotherapy approaches reach mainstream use, including adoptive T cell transfer, chimeric antigen receptor T cell therapy (CAR-T), and cancer vaccines [8].

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The principle of ICP therapy is based on the expression of proteins on cancer cells that bind checkpoint proteins expressed on T cells. These checkpoint proteins function as “off switches” that inhibit T cell receptor mediated killing of a foreign body. Immune checkpoint inhibitors are antibodies that bind either of the checkpoint proteins on the cancer cell (e.g. PD-L1) or on the T cell (e.g. PD-1 or CTLA-4), allowing T cells to attack and kill cancer cells. The key challenges with current ICP therapy are low patient response rate and the potential for high toxicity [1]. While the reasons for low response rates are not completely understood, the current understanding is that tumors produce numerous immunosuppressive factors creating a “nonimmunogenic” tumor microenvironment. Immunogenic tumors are more likely to respond to ICP therapy as the tumor microenvironment is conducive to tumor cell recognition, containing infiltrating T cells, cytokines such as granzyme B, and memory T cell markers such as CD45RO and PD-L1 [1,9]. A single therapeutic approach will not overcome the numerous, dynamic and evasive immune strategies of the tumor. The path forward is believed to be combinatorial approaches that convert a nonimmunogenic (“cold”) tumor to an immunogenic (“hot”) tumor that will respond to ICP therapy [1,10]. The advent of ICP has led to the investigation of combinatorial clinical treatment strategies combining ICP with well-established treatment approaches, such as radiation [11], chemotherapy [12], oncolytic viral therapy, and targeted therapy [13]. While improvements in treatment outcomes have been observed, there is no rationale or guidance for selecting the best combinatorial approach for an individual patient. This stems from the lack of techniques to monitor the dynamic immune response and a poor understanding of the specific impact of each combinatorial approach on the tumor microenvironment. Importantly, these approaches exhibit significant toxicity (chemotherapy, targeted therapy) and/or disease resistance (chemotherapy, radiation). Therefore, a continued unmet need exists for combinatorial therapies that can enhance ICP therapy with limited toxicity profiles and an understanding of the synergy involved in the combinatorial approaches. Nanoparticle-assisted hyperthermia (NPHT) with ICP therapy has emerged as a potential combinatorial cancer treatment approach. NPHT involves administration of nanoparticle (NP) platforms targeted to the tumor site, followed by irradiation with an external energy source to produce heat and localized

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hyperthermia. An overview of this approach is outlined in Figure 1. The primary advantage of NPHT is the ability to perform well-controlled, targeted volumetric heating specific to tumors. In fact, localized heating provides a relatively benign, low toxicity, outpatient treatment when compared to the systemic toxicity issues associated with molecular targeted therapies (e.g. MEK inhibitors) or chemotherapy and has the potential to open up immunotherapies to a larger population. To date, the vast majority of the research and development of this approach has been directed towards eradication of primary tumors, and many studies have shown the success of NPHT in debulking tumors in preclinical models [14–16]. Several clinical trials have been conducted or are underway utilizing near-infrared (NIR) laser irradiation of gold based nanoparticles [17,18] and alternating magnetic field (AMF) irradiation of magnetic nanoparticles [19].

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While successful in debulking primary tumors, NPHT does not generally address the treatment of metastatic disease, which is responsible for the vast majority of cancer deaths. However, the hyperthermia community has long recognized and documented the systemic immune response due to local hyperthermia, highlighting the opportunity for combining these immune modulatory responses with immunotherapy strategies to treat metastatic disease. Since the late 1990s, several groups have shown enhanced systemic responses of NPHT when used with immune adjuvant treatments [20]. Since ICP therapies are now approved as standalone, first-line treatments for patients with advanced melanoma, combining ICP therapy with NPHT offers a new opportunity as a combinatorial approach. NPHT could be used to stimulate a “cold” tumor microenvironment to become an immunogenic, “hot” tumor microenvironment, working in synergy with immunotherapy to increase patient response rates and lead to successful treatment outcomes.

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In this review, we discuss the current status of combination approaches of immunotherapy and controlled, localized nanoparticle-mediated hyperthermia. We begin with a brief overview of the various nanoparticle-based hyperthermia treatments to induce localized heating of tumors. We then discuss the current understanding of immune system modulation and its dependence on tissue heating and thermal dose. Finally, we discuss current reports in the literature of combining immunotherapy and nanoparticle-mediated hyperthermia for cancer treatment.

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Figure 1. Combinatorial NPHT and ICP therapy. 1) Systemic administration of nanoparticles that localize to the tumor and 2) irradiation with an external energy source are the main components of NPHT. 3) Tumor hyperthermia initiates the apoptosis responses that upregulate tumor specific antigens (TSA) and expression of heat shock proteins (HSP). Necrosis releases TSA and HSP-TSA complexes that activate antigen presenting dendritic cells (DC). HSP receptors (HSPR) on DCs recognize HSP-TSA complexes, activating natural killer (NK) effector cells and release of cytokines and chemokines. 4) DCs traffic TSA to the lymph nodes (LN) where they activate T cells with T cell receptors (TCR) specific to the TSA. Activated T cells upregulate inhibitory surface receptors (PD-1 and CTLA-4), and 5) traffic back to the primary and distant/metastatic tumors throughout the body, initiating TCR mediated killing of tumor cells. In the absence of ICP therapy, inhibitory ligands on the tumor cells (e.g. PD-L1) would down-regulate and inhibit a full response; however, blocking the immune checkpoints allows for a full, uninhibited immune response, ultimately resulting in tumor cell killing and immune memory.

2. Nanoparticle hyperthermia platforms Immune responses from local tumor hyperthermia have been studied in some detail [21–23], and the cascade of events that lead to immune modulation are well characterized and briefly summarized here. Hyperthermia, which is defined as the heating of tissue to between 39o-45oC, induces a cell stress known as the unfolded protein response (UPR) [24]. Denaturation of cellular proteins follows the Arrhenius rate process, and is a strong function of both time and temperature. The UPR leads to up-regulation of heat shock proteins (HSP) on the cell membrane, specifically HSP70, and tumor specific antigens (TSA) within the tumor cells [23]. Increased HSP70 expression on the tumor cell surface allows recognition by natural killer (NK) effector cells [25]. Necrotic tumor cells release TSAs, TSA-HSP complexes, and TSA-HSP complexes in exosomes, all of which stimulate immune system activity. Antigen presenting cells (APC) acquire TSA and TSA-HSP complexes that aid in expression of antigens on MHC-1 complexes on the APC surface [26]. These cells traffic to the lymph where they activate antigen specific killer T cells that undergo clonal expansion and traffic to all tumors (primary and metastatic) to kill tumor cells directly. In addition, APCs also release cytokines/chemokines in response to TSA uptake, which attracts dendritic cells (DC) and T cells [27,28]. Kobayashi et al. have recently demonstrated that local heating of melanomas induces systemic immune responses, activating T cells directed at a limited number of epitopes in the T cell receptor (TCR) repertoire [20,29]. In essence, localized hyperthermia can alter a

ACCEPTED MANUSCRIPT nonimmunogenic tumor to an immunogenic tumor state that is rich in chemokines/cytokines, activated DC and cytotoxic T lymphocytes (CTLs).

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We highlight here the various nanoparticle-based approaches available to induce local hyperthermia. We do not intend this to be a exhaustive review of this area as numerous platforms have been demonstrated for this purpose, and more thorough reviews can be found elsewhere [30–33]. Our intention is to distinguish the primary approaches being developed in order to discuss their broad tradeoffs directed toward immune modulation. The primary approaches for nanoparticle mediated hyperthermia involve activation with light [30,31], magnetic fields [32], or radiofrequency radiation [33]. For each approach, the general protocol involves administration and localization of the nanoparticles to the tumor, followed by irradiation of the nanoparticles with an external energy source to induce localized hyperthermia.

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2.1 Photothermal therapy

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Light induced hyperthermia, or photothermal therapy (PTT), involves irradiation of nanoparticles with near infrared (NIR) light in the 700-900 nm range. First reported in 2003 in a preclinical model of cancer [14], several types of nanoparticles have been reported for use in PTT [34], comprising different materials and geometries/shapes. Commonly reported PTT nanoparticle materials include gold [16,35– 38], copper [39,40], and graphene [41]. In addition, use of polymeric nanoparticles [42,43], carbon nanotubes [44], silver [45,46] and platinum [47] nanoparticles are being investigated for PTT. Commonly used nanoparticle geometries include nanoshells [15,16], nanorods [35,48], nanostars [49,50], and nanocages [51]. Nanoparticles are typically delivered to the treatment site by systemic intravenous injection or by direct intratumor injection. Additionally, antibody conjugated nanoparticles that target tumor specific markers, such as EGFR [35] or HER2 [52], to increase nanoparticle localization to tumors have been widely reported. Biocompatibility of PTT is mostly dictated by the nanoparticle chemistry; most reports of in vivo PTT use nanoparticles coated with poly(ethylene) glycol, or PEG, an inert polymer that is nonimmunogenic. Since PTT is performed with NIR wavelengths, PTT is limited to penetration depths of roughly 1 cm, requiring that tumors are either subcutaneous, surgically exposed, or accessible via catheter. Additionally, the penetration depth of NIR light limits treatment to tumors that are less than 1 cm in diameter or linear size; larger tumors will not be treated throughout the entire volume. While PTT continues to be studied primarily in preclinical in vivo models of cancer [37,53–55] (Figure 2), PTT has begun to be evaluated in clinical trials for cancer treatment [17,18].

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Figure 2: PTT on a preclinical model of squamous cell carcinoma. Nanoparticles were administered systemically (tail vein, green) or by direct injection (intratumor, red) and irradiated with NIR light. Tumor volume decreased after PTT (red and green), as compared to the untreated control (blue). Reproduced with permission from Cancer Lett. 2008 Sep 28; 269(1): 57–66. [37]

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2.2 Magnetic fluid hyperthermia

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Magnetic fluid hyperthermia (MFH) involves the use of magnetic nanoparticles that generate heat when in the presence of an alternating magnetic field. First reported in 1957 [56], iron oxide (Fe2O3) nanoparticles are currently FDA approved for use as a contrast agent in MRI [57] and are typically used for magnetic hyperthermia due to their superparamagnetic properties. As the magnetic nanoparticles are typically 2-20 nm, delivery to the tumor by either intravenous or direct intratumor injection is in the form of a magnetic fluid. Iron oxide nanoparticles are coated with a polymer, such as dextran [58], polymer combinations [59], chitosan [60], or silica [44,61] to prevent aggregation of the nanoparticles, and then functionalized with PEG to enhance circulation. Additionally, targeting molecules, such as folic acid [62,63] and other antibodies [64], have been conjugated to magnetic nanoparticles for molecular targeting of tumor cells. Iron oxide nanoparticles have been found to accumulate rapidly in the liver, spleen, and lymph nodes [32], demonstrating rapid clearance from the human body, however, have also been shown the potential to be cytotoxic after accumulation in the tissue [32,65]. Since MFH relies on alternating magnetic fields to induce hyperthermia, there is no limit to the penetration depth in vivo; however, while this makes MFH seemingly ideal for cancer treatment with hyperthermia, an important consideration is that unwanted heating of healthy tissue may occur by nanoparticles that have not localized to the tumor. MFH continues to be studied in depth in preclinical models of cancer [66–69] (Figure 3) and has begun to be explored in clinical studies for cancer treatment [19].

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Figure 3. MFH treatment of preclinical models of breast cancer. Tumors were injected intratumorally with magnetic nanoparticles and then irradiated either once (MFH1), twice (MFH2), or three times (MFH3) with subsequent treatments in MFH2 and MFH3 groups occurring 24 hours after the previous treatment. Tumor growth rate was initially reduced in the first 15 days for the MFH2 and MFH3 groups before reverting to the rate of the control groups. Reproduced under the Creative Commons Attribution License from Oncol Lett. 2014 May; 7(5): 1370–1374 [69]

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2.3 Radiofrequency hyperthermia

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Radiofrequency hyperthermia (RFH) originates from radiofrequency ablation of tissue, where radiofrequency radiation is applied via a contact electrode to thermally ablate the target tissue. Applications of radiofrequency ablation include treating cancer [70] and in correcting cardiac arrhythmia [71]. RFH differs in that radiofrequency radiation is delivered noninvasively (i.e. without an electrode) to heat localized nanoparticles for targeted thermal treatment of tissue. Nanoparticles used in RFH include those made of gold [72,73], silicon [74], and carbon nanotubes [75]. Similar to the nanoparticles used for PTT, nanoparticles for RFH are PEGylated, administered via intravenous or intratumor injection, and are commonly conjugated to antibodies for tumor targeting [76]. The radiofrequency radiation used in RFH does not have any limitations on penetration depth, similar to MFH, but has a similar drawback in that unintentional tissue heating caused by nanoparticles that do not localize to the tumor can occur. Additionally, RFH requires a specialized radiofrequency radiation source, which is analogous to the alternating magnetic field source needed for MFH. RFH has been studied in preclinical models of cancer [76,77], though has not yet progressed to human clinical trials.

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Figure 4. RFH treatment of preclinical models of lung cancer. Tumors treated with RFH (red, green) show a reduction in volume over a 2 week period, as compared to control (black) and untreated (blue) tumors. Reproduced with permission from Sci Rep. 2014 Nov 13;4:7034. [74]

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A summary comparison of the three methods of nanoparticle mediated hyperthermia is presented in Table 1. An important consideration in future clinical application is the cost of each method. The nanoparticles used have straightforward synthesis procedures and similar costs. Of more significance to the cost is the hyperthermia activation sources for each method. MFH and RFH require an alternating magnetic field generator and radiofrequency radiation generator, respectively, both of which are specialized hyperthermia activation sources and are more expensive than the diode laser light sources used in PTT. Another important consideration is the useful treatment depth of each method. The limited penetration depth of PTT requires direct access to the tumor and therefore limits its utility to superficial or surgically/catheter accessed tumors. Since each of the NPHT platforms utilizes systemic administration and distribution of nanoparticles, the limited penetration depth of PTT prevents unwanted hyperthermia of nontumor tissues, which by contrast can occur in both MFH and RFH and must be carefully considered. Selection of the appropriate nanoparticle mediated hyperthermia must therefore be performed after careful consideration of all of these aspects. Table 1: Summary of nanoparticle mediated hyperthermia cancer treatments

Energy Source Biocompatibility

Penetration depth

Photothermal Therapy (PTT) Near-infrared Light (700-900 nm) PEG-coated, antibody conjugated NPs for molecular targeting

Magnetic Fluid Hyperthermia (MFH) Alternating magnetic field Polymer coated, dextran coated, antibody conjugated

~1 cm

Accumulation in liver, spleen, lymph nodes Through body

Radiofrequency Hyperthermia (RFH) Radiofrequency radiation Similar particles used as in PTT

Through body

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Nanoshells (~120 nm diameter), nanorods (50 nm length), nanostars, nanocages Gold, copper, silicon, polymers, carbon nanotubes Clinical, preclinical

Materials

Clinical, preclinical

Gold, carbon nanotubes, silicon Preclinical

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Developmental Progress

Iron oxide

Primary cost is radiofrequency radiation source Nanospheres (25-30 nm diameter)

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Primary cost is alternating magnetic field source Magnetic nanoparticle fluid (~2-20 nm)

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Primary cost is light source

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Cost

3. Thermal dose dependent physiological response

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The immune modulation response is strongly dependent on the thermal dose (which is a function of temperature and exposure time), and the hyperthermia community has recognized the strong need for better understanding and control [23,78]. Nanoparticle-assisted hyperthermia offers a means for controlling the thermal dose and treatment specificity. Thermal dose in the context of NPHT is not well understood, likely because most treatments in the literature have focused on eradicating the primary tumor instead of systemic immune modulation. There have been, however, a few studies of thermal dose immune modulation. Our group has demonstrated in vitro [79] the impact of thermal dose on driving the cell death pathway to either apoptosis or necrosis. Brown et al. illustrated the importance of dose in localized heating of melanomas using magnetic nanoparticles stimulated by an external magnetic field. Tumors heated at 43oC for 30 min. elicit a CD8+ T cell response that affects distant, untreated tumors and rejects tumors upon rechallenge (i.e. an “in vitro vaccine”); however, this does not occur in tumors heated at 45oC for 30 min. [80]. In another recent study, Bear et al. used PTT to treat primary tumors and found that irradiation with a laser set at a fixed power (without taking into account the tissue temperature) for 6 min. resulted in decreased size of the primary tumor, but observed no effect on distant, untreated tumors. In fact, the PTT treatment stimulated inflammatory cytokines and initiated inhibitory immune mechanisms mediated by myeloid derived suppressor cells (MDSC) [81]. Each of these three dosing examples, all of which used B16 tumor models, illustrate the strong dependence of dose on treatment efficacy and the elicited immune response, while also highlighting the gap in understanding between proper thermal dose and enhancement of immunotherapies. A unique aspect of NPHT is the ability to localize subcellular heating and injury using molecularly targeted NPs. Numerous in vitro and in vivo evidence indicates that nanoparticle surface chemistry (i.e. “active targeting”) strongly dictates biodistribution to and within the tumor as well as intracellularly. Nanoparticles reach the tumor site primarily using “passive targeting” via the enhanced permeability and retention effect (EPR) to escape the vasculature and enter the tumor microenvironment. Antibody labeling of the nanoparticle surface has shown to increase retention of nanoparticles once they reach the tumor site. Gold nanocages targeting the melanocortin type 1 receptor (MC1R) using the melanocyte-stimulating hormone (MHS) peptide have shown increased retention in melanomas in mice [82]. Targeting the human melanoma-specific antigen CSPG4 using the Ep1 monoclonal antibody

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showed an increase of 25-fold in tumor uptake in CSPG4+ melanoma vs. CSPG4- breast carcinomas [83]. A first-in-human study on patients with melanoma by Davis et al. showed cellular uptake of nanoparticles targeting the transferrin receptor [84]. Nanoparticles targeting the tumor neovasculature receptor v 3 have been extensively studied [85,86] and have been shown to enhance nanoparticle uptake in melanoma tumors [87]. These studies demonstrate that antibody labeling of nanoparticles enhances nanoparticle retention in tumors and the ability to target specific tumor tissue compartments (i.e. extracellular, tumor vasculature, or intracellular).

4. Combinatorial approaches

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Our own work has demonstrated that nanoparticle location (extracellular vs. intracellular) strongly impacts the cytotoxic effectiveness of PTT and cell death pathway [79]. The bulk of the current literature investigating the impact of nanoparticle targeting has addressed the effectiveness of the direct cytotoxic effect under investigation (e.g. whether targeting increased tumor cell killing or reduced tumor burden). However, the intratumor particle location likely impacts the cell types that are treated and the cell death pathways that are initiated, which in turn impacts the immune response initiated. The impact of active targeting on the immune response is not well understood, and further work will lead to optimized nanoparticle designs to induce cell and tissue damage that stimulates an immune response.

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Given the strong immunomodulatory response of NPHT, a number of immune adjuvant strategies have been explored since the late 1990s using both magnetic field and laser induced hyperthermia. “Laser immunotherapy” (LIT) was first proposed, in which an immunoadjuvant, a substance that induces an immune response, is used in conjunction with PTT to treat tumors. The initial report of LIT utilized indocyanine green (ICG) mediated PTT for laser activated hyperthermia of the tumor in combination with the immunoadjuvant glycated chitosan (GC) [88,89]and demonstrated that the combination of all three components showed substantial reduction in tumor volume increased survival in preclinical animal models. Other approaches include using imiquomod, another immunoadjuvant [90] and GC in an in situ cancer vaccine, termed InCVAX [91]. Safety of these agents was even demonstrated in phase I clinical trials in melanoma [90] and breast [92]. In parallel, Kobayashi et al. extended their localized hyperthermia approach using magnetite cationic liposomes in combination with interleukin-2 (IL-2) and granulocyte macrophage-colony stimulating factor (GM-CSF), illustrating in a preclinical melanoma model reduced tumor burden and increased survival for the combination treatments over monotherapies alone [20]. These pioneering studies demonstrated the synergy between immune response from local heating and immune modulatory agents; however, the immunotherapies studied had not yet achieved widespread clinical use. In 2011 and 2014, the FDA approved the use of two immune checkpoint inhibitors (anti-CTLA-4 and antiPD-1, respectively) for the treatment of malignant melanoma, and they have since become the first-line treatment for this disease. The widespread use of these immune checkpoint inhibitors provides a new combinatorial platform for enhancing NPHT. Hyperthermia is administered first to initiate the cascade of events that leads to T cell receptor specific clonal expansion (Figure 1), which occurs over the following 4-5 days. As these CTLs traffic to distant metastatic tumor sites, they encounter an immunosuppressive microenvironment where tumor cells express PD-L1 and CD80/86, which work to inhibit CTL killing of tumor cells. Administration of ICP antibodies after the NPHT block these inhibitory signals, reinvigorate exhausted T cells, and allow for tumor specific antigen cell killing (Figure 1).

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Several recent studies have demonstrated the strong synergy of this combinatorial approach. Wang et al. demonstrated the first combination of anti-CTLA-4 therapy with PTT using single wall carbon nanotubes (SWNT) [93]. Using two models of metastasis, they demonstrated that the combination treatment resulted in slowed growth of the untreated tumors. Lung metastases were reduced from an average of thirty to just one, and mice exhibited prolonged survival measured at 50 days (57% for the combination group as compared to 25% for the anti-CTLA-4 only group). Similar long term survival statistics (55.5% combination group vs. 12.5% anti-CTLA-4 only group at 100 days) were later observed using a Prussian blue nanoparticle based PTT approach in a preclinical neuroblastoma model [94]. In addition, all of the mice that survived past 100 days also rejected a tumor rechallenge (as compared to only 12-14 days total survival for naïve mice), illustrating an immune memory response. Finally, in a recent and extensive study using a PLGA-ICG platform with anti-CTLA-4 therapy, Chen et al. demonstrated significant to complete regression of untreated, distant tumors in breast (4T1) and colon (CT26) subcutaneous models of metastasis [95]. In lung metastasis rechallenge models, combinatorial treated mice developed almost no tumors and demonstrated long term survival (70% at 70 days) compared to no survival for the anti-CTLA-4 only treated mice. They also extended this work to an orthotopic breast cancer model where they showed 90% long term survival of combinatorial treated mice as compared to only 50% for anti-CTLA-4 only treated mice (Figure 5). Interestingly, immune memory was not induced unless hyperthermia was used. This latter platform is particularly significant in that all components have been previously FDA approved for various indications. These three studies clearly demonstrate the synergy between ICP therapies and PTT, all illustrating drastically improved preclinical outcomes for models of melanoma, neuroblastoma, and breast cancer. In addition to these demonstrations using combinations with ICP therapy, Bear et al. demonstrated strong synergy of PTT with another emerging immunotherapy approach, adoptive T cell transfer (ATCT) [81]. Using hollow gold nanoshells to induce PTT, they demonstrated reduced recurrence of primary tumors and slowed growth of distant, untreated tumors when using a combined approach. Interestingly, PTT alone promoted lung metastases, but the addition of ATCT abrogated metastatic tumor growth. This opposing response highlights the need to understand the immune modulatory mechanisms of PTT and how best to combine PTT with immunotherapies.

Figure 5. a) Schematic illustration of a PTT and anti-CTLA-4 combinatorial approach to prevent tumor relapse. (b) Growth curves of tumors reinoculated 40 days after elimination of initial tumor (eight mice per group). The treatment groups (1-6) are as follows: (1) Surgery only; (2) Surgery + anti-CTLA-4; (3) Surgery + Nanoparticles only (no laser) + anti-CTLA-4; (4) PTT; (5) PTT + anti-CTLA-4 (pre); (6) PTT + anti-CTLA-4 (pre + post). Treatment groups (5) and (6) are combinatorial approaches showing diminished tumor volume compared to the non-combinatorial approaches. Reproduced under the Creative Commons Attribution License from Nat Commun. 2016 Oct 21;7:13193 [95]

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The mechanisms behind the success of these combination approaches appears to be linked to a combination of factors related to the hyperthermia response and specifics of each nanoparticle platform. As discussed above, hyperthermia upregulates cytokine expression, tumor specific antigen expression, and heat shock proteins leading to maturation of dendritic cells and activation of T cells. Multiple studies measured increased infiltration of lymphocytes after PTT and determined that CD4+ and CD8+ T cells are essential for a positive response [93,94]. PTT without anti-CTLA-4 therapy resulted in an increase in immunosuppressive regulatory T cells within secondary tumor. ICP therapy was needed to increase effector T cells to regulatory T cell ratios and cytotoxic T lymphocyte to regulatory T cell ratios [95]. Dendritic cell maturation was reported using PTT alone [81] as well as with individual NP platforms alone (SWNTs [93] and imiquimod [95]), indicating that combining not only hyperthermia but other immunoadjuvants may also enhance the approach. Lu et al. demonstrated enhanced immune modulation with chitosan coated hollow copper sulfide nanoparticles in the context of PTT [96,97]. Therefore, moving forward, it will be important to understand the mechanisms of the interplay between hyperthermia and nanoparticle design.

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Table 2: Summary of Hyperthermia and Immunotherapy Combinatorial Approaches Type of Hyperthermia Photothermal (ICG)

InCVAX

Photothermal (ICG)

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Laser Immunotherapy

Immunotherapy Immunoadjuvant (glycated chitosan [88,89], imiquomod [90]) Immunoadjuvant (cancer vaccine) [91]

Developmental stage Clinical, preclinical

Preclinical

Preclinical

Hyperthermia + Cytokines

Magnetic (liposome)

Hyperthermia + Checkpoint inhibitor

Photothermal (carbon nanotubes)

Interleukin-2 + Granulocyte macrophage colony stimulating factor [20] Checkpoint inhibitor (α-CTLA-4) [93]

Photothermal (Prussian blue nanoparticles)

Checkpoint inhibitor (α-CTLA-4) [94]

Preclinical

Photothermal (ICG embedded nanoparticle) Photothermal (gold nanoshells)

Checkpoint inhibitor (α-CTLA-4) [95]

Preclinical

Adoptive T cell transfer [81]

Preclinical

Hyperthermia + Adoptive T cell transfer

Preclinical

5. Future opportunities Given this body of work, a clear synergy exists between NPHT and immunotherapies. More work is needed to understand the full scope of the mechanisms between the two approaches. For example, T

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cell receptor (TCR) repertoire has been found to expand with radiation that works in concert with antiCTLA4 and anti-PD-1 treatments [11]. More mechanistic studies are needed to understand this impact for hyperthermia. In addition, other checkpoint inhibitors need to be studied, including anti-PD-1 therapy, which has shown lower immune related toxicity over anti-CTLA4 therapy, and anti-PD-L1, which is expressed on the tumor cells and currently in clinical trials. Because NPHT heats the tumor cells directly, an interesting issue will be the effect of blocking antibodies present on CTLs or tumor cells. In addition, all current approaches reported delivery of the blocking antibodies after NPHT; however, hyperthermia induced inflammatory responses within the tumor microenvironment may work to dampen the CTL response. Therefore, the timing of the NPHT and antibody delivery should be investigated in more detail. All the studies to date have utilized intra-tumoral delivery of the nanoparticle agent; thus, a need exists for studying the impact of systemic delivery and molecular targeting to enhance tissue localization and response. The thermal dose response on immune modulation is largely understudied, and its impact on combination with ICP therapies presents new opportunities and research areas to be explored.

ACCEPTED MANUSCRIPT Acknowledgements

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This work was supported by the Cancer Prevention and Research Institute of Texas [RP130702]; and the Texas Health Catalyst Program of the Dell Medical School at The University of Texas at Austin

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Graphical abstract