Direct Intratumoral Injection Research Articles

A Single Direct Intratumoral Injection of TTI-621 (SIRPαFc) Induces Antitumor Activity in Patients with Relapsed/Refractory Mycosis Fungoides and Sézary Syndrome: Preliminary Findings Employing an Immune Checkpoint Inhibitor Blocking the CD47 “Do Not Eat” Signal

BackgroundCD47 is an immune checkpoint that binds to signal regulatory protein alpha (SIRPα) and delivers a “do not eat” signal to suppress macrophage phagocytosis. Tumor cells frequently overexpress CD47 to evade macrophage-mediated destruction. TTI-621 (SIRPαFc) is an immune checkpoint inhibitor consisting of the CD47 binding domain of human SIRPα linked to the Fc region of human IgG1 designed to both: 1) block the CD47 “do not eat” signal, and 2) engage macrophage Fcγ receptors with IgG1 Fc to enhance phagocytosis and antitumor activity. It is hypothesized that direct intratumoral injection of TTI-621 may enhance both local and systemic antitumor activity.MethodsA Phase 1, multicenter, open-label study is ongoing to characterize the safety and tolerability of intratumoral injection of TTI-621 (NCT02890368). Eligible patients (pts) are adults with relapsed or refractory mycosis fungoides/Sézary syndrome or percutaneously-accessible solid tumors who have progressed on standard anticancer therapy or for whom no other approved therapy exists. Pts are enrolled in sequential cohorts to receive a single intratumoral injection of TTI-621 at increasing dose levels to characterize safety, pharmacokinetics, pharmacodynamics, and preliminary evidence of antitumor activity. Repeat TTI-621 intratumoral dosing has recently been initiated. Serial, post-injection biopsies are collected to assess the temporal impact of TTI-621 on the tumor microenvironment.ResultsNine pts (6 M/3F, age 57-85 years) have been enrolled as of July 2017. Diagnoses include mycosis fungoides/Sézary syndrome (N=6), melanoma (N=1), leiomyosarcoma (N=1), and squamous cell carcinoma (N=1). A single intratumoral injection of 1 mg, 3 mg, or 10 mg TTI-621 has been well tolerated by all enrolled pts. The most frequently reported adverse events were fatigue in 5 pts, chills in 4 pts, and decreased appetite in 3 pts. All treatment-related AEs were Grade 1 or 2 in severity. No dose limiting toxicity has been observed to date. All 6 pts with mycosis fungoides/Sézary syndrome experienced decreases in tumor size and/or decreased circulating Sézary cells (3 of 3 pts) as determined by a decrease in a peripheral clonal T-cell populationor CD4:CD8 ratio after a single intratumoral injection of TTI-621. Responses ranged from loco-regional to systemic effects. One pt with localized refractory mycosis fungoides on the dorsum of the foot obtained a complete response of the injected lesion that is ongoing after 17+ weeks of follow up. NanoString analysis of peripheral blood and tumor tissue pre- and post-intratumoral injection of TTI-621 indicated alterations in genes associated with activation of the innate immune system and upregulation of IFN-associated genes.ConclusionsThese preliminary Phase 1 data suggest that mycosis fungoides and its leukemic variant, Sézary syndrome, are highly responsive to a single dose of TTI-621 delivered by intratumoral injection. One pt achieved a complete response of the injected lesion and 5 additional pts experienced decreases in tumor size and/or decreased circulating Sézary cells. Intratumoral injections were well tolerated by all pts. Evidence of innate immune activation was documented, consistent with the proposed function of TTI-621 as an innate immune checkpoint inhibitor. DisclosuresQuerfeld:Soligenix: Research Funding; Elorac: Research Funding; Celgene: Honoraria, Research Funding; MiRagen: Honoraria, Research Funding; Actelion: Honoraria, Research Funding; Medivir: Honoraria; Mallinckrodt: Honoraria; Mindera: Consultancy; Kyowa: Research Funding; Trillium Therapeutics: Research Funding; City of Hope: Employment. Thompson:Trillium Therapeutics: Research Funding. Taylor:Blue Print Medicines: Consultancy, Honoraria; Eisai Inc.: Consultancy, Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Speakers Bureau; Trillium Therapeutics: Consultancy, Honoraria. Pillai:Trillium Therapeutics: Research Funding. Johnson:Trillium Therapeutics: Employment. Catalano:Trillium Therapeutics: Employment; Apotex Inc.: Employment. Petrova:Trillium Therapeutics: Employment, Equity Ownership. Uger:Trillium Therapeutics: Employment, Equity Ownership, Patents & Royalties. Irwin:Hoffmann La Roche: Employment, Equity Ownership; Trillium Therapeutics: Employment, Equity Ownership. Sievers:Trillium Therapeutics: Employment, Equity Ownership. Akilov:Seattle Genetics: Consultancy; Medivir: Consultancy; Actelion Pharmacuticals: Consultancy, Research Funding; Trillium Therapeutics: Research Funding.

A phase I trial evaluating NBTXR3 activated by radiotherapy in combination with nivolumab or pembrolizumab in patients with advanced cancers.

2590 Background: Immune checkpoint inhibitors (ICIs) targeting PD-1 are an effective treatment for a variety of cancers. However, the majority of patients (pts) exhibit resistance to ICIs. Overcoming this resistance represents a major challenge in immuno-oncology. Emerging evidence suggests radiation therapy (RT) produces an immunomodulatory effect that may act synergistically with ICIs. However, RT dose and ultimate efficacy are limited by toxicity to surrounding healthy tissues. NBTXR3, a novel radioenhancer administered by direct intratumoral injection (ITI), is designed at the nanoscale to increase RT dose deposit within tumor cells and subsequent tumor cell killing, without increasing toxicity to surrounding healthy tissue. Preclinical data suggest NBTXR3/RT can trigger a local and systemic anti-tumor immune response and overcome anti-PD-1 resistance. NBTXR3/RT combined with anti-PD-1 may prime the immune system to increase the proportion of ICI responders, or convert ICI non-responders to responders. Methods: This is a multicenter, open-label, phase I trial [NCT03589339] to evaluate NBTXR3/RT/anti-PD-1 in 3 cohorts: (1) Locoregional recurrent or recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) amenable to HN re-irradiation, and metastases from any primary cancer eligible for anti-PD-1 (nivolumab or pembrolizumab) treatment specifically localized in the lung (2) or liver (3), respectively. Stereotactic body RT (SBRT) is delivered at tumor-site selective doses per standard practice. The primary objective is NBTXR3/RT/anti-PD-1 recommended phase 2 dose in each cohort. Secondary objectives are anti-tumor response (objective response rate), safety and feasibility of NBTXR3 injection. Results: Nine pts have been treated: 3 HNSCC, 4 lung, 2 liver. 7/9 pts were anti-PD-1 non-responders. Overall tumor regression was observed in 8/9 pts. NBTXR3/RT/anti-PD-1 resulted in tumor regression in 6/7 pts who had progressed on prior anti-PD-1. A complete response in the injected lymph node lasting over 1 year was observed in 1 anti-PD-1 naïve pt. 2 SAEs related to anti-PD-1 and possibly related to NBTXR3 (G5 pneumonitis, G4 hyperglycemia) were observed in 1 anti-PD-1 naïve HNSCC pt and considered DLTs. This pt also experienced 2 other SAEs related to anti-PD-1 (G4 diabetic ketoacidosis, G4 acute kidney injury). SBRT-related safety profile was as expected. Updated results will be presented. Conclusions: Data from this first-in-human phase I trial evaluating NBTXR3/RT/anti-PD-1 in pts with advanced cancers, show NBTXR3 ITI is feasible and well-tolerated. NBTXR3/RT/anti-PD-1 demonstrated promising signs of efficacy. Of particular interest, NBTXR3/RT can overcome ICI resistance in pts having progressed on prior anti-PD-1, supporting further development of NBTXR3 in combination with anti-PD-1 as well as other ICIs. Clinical trial information: NCT03589339.

Overcoming resistance to anti-PD-1 with tumor agnostic NBTXR3: From bench to bed side.

2591 Background: Despite recent advances, resistance to immune checkpoint inhibitors (ICI), observed in over 80% of treated patients, is currently the main challenge in immuno-oncology. Intense efforts are being made to identify combination therapies that could improve ICI response rates. NBTXR3, a novel radioenhancer administered by direct intratumoral injection (ITI), is designed at the nanoscale to increase radiation therapy (XRT) dose deposit within tumor cells and subsequent tumor cell killing, without increasing toxicity to surrounding healthy tissue. Here we present evidence that NBTXR3 activated by XRT primes the immune system, producing an anti-tumor response, including activation of the cGAS-STING pathway, that overcomes anti-PD-1 resistance both in mice models and patients. Methods: Abscopal assays were conducted in immunocompetent mice. Anti-PD-1 sensitive or resistant tumor cell lines, were injected in both flanks of mice. Intratumoral injection of NBTXR3 (or vehicle) followed by XRT was performed in right flank (primary) tumors only. Some mice also received anti-PD-1 injections. Tumor growth was monitored, and tumor immune cell infiltrates analyzed by immunohistochemistry (IHC). Separately, in the phase II/III randomized Act.in.Sarc [NCT02379845] trial patients with locally advanced soft tissue sarcoma (STS) received either NBTXR3+XRT or XRT alone followed by tumor resection. Pre- and post-treatment tumor samples from patients in both groups were analyzed by IHC and Digital Pathology for immune biomarkers. The safety and efficacy (RECIST 1.1/iRECIST) of NBTXR3 plus stereotactic body radiotherapy (SBRT) in combination with anti-PD-1 is being evaluated in three cohorts of patients with advanced cancers [NCT03589339]. Results: Pre-clinical studies demonstrated that NBTXR3+XRT induces an immune response not observed with XRT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3+XRT treated and untreated tumors compared to XRT alone. Similarly, increased CD8+ T-cell and decreased FOXP3+ Treg density (pre- vs post-treatment) was observed in tumor tissues from STS patients treated with NBTXR3+XRT. Furthermore, NBTXR3+XRT in combination with anti-PD-1 improved local and systemic control in mice bearing anti-PD-1 resistant lung tumors, as well as reduced the number of spontaneous lung metastases. Preliminary efficacy data from the first in human trial of NBTXR3+XRT in combination with anti-PD-1 showed tumor regression in the majority of patients (8/9). Of note, tumor regression was observed in 6/7 pts who had progressed on prior anti-PD-1. Conclusions: The clinical efficacy of NBTXR3+XRT has been demonstrated as a single agent. We now demonstrate that it potentiates anti-PD-1 treatment to overcome resistance mechanisms. These results highlight the potential of NBTXR3+XRT to positively impact the immuno-oncology field. Clinical trial information: NCT03589339.

Intratumoral Injection of Anlotinib Hydrogel Combined With Radiotherapy Reduces Hypoxia in Lewis Lung Carcinoma Xenografts: Assessment by Micro Fluorine-18-fluoromisonidazole Positron Emission Tomography/Computed Tomography Hypoxia Imaging.

Hypoxia is a common feature of solid tumors that increases tumor invasiveness and resistance to radiotherapy (RT) and chemotherapy. Local application of anlotinib (AL) might increase the regulation of new blood vessel growth and improve tumor hypoxia in RT. Therefore, it is essential to fully understand the drug delivery system of AL. Herein, we applied hypoxia imaging using micro fluorine-18-fluoromisonidazole positron emission tomography/computed tomography (micro 18F-FMISO PET/CT) to assess responses to intratumoral injections of an AL hydrogel (AL-HA-Tyr) combined with RT in mice bearing Lewis lung carcinoma (LLC). We formed AL-HA-Tyr by encapsulating AL with hyaluronic acid-tyramine (HA-Tyr) conjugates via the oxidative coupling of tyramine moieties catalyzed by H2O2 and horseradish peroxidase. AL-HA-Tyr restrained the proliferation of human umbilical endothelial cells (HUVECs) in colony formation assays in vitro (p < 0.001). We established a subcutaneous LLC xenograft model using C57BL/6J mice that were randomly assigned to six groups that were treated with AL, HA-Tyr, AL-HA-Tyr, RT, and RT+AL-HA-Tyr, or untreated (controls). Tumor volume and weight were dynamically measured. Post treatment changes in hypoxia were assessed in some mice using micro 18F-FMISO PET/CT, and survival was assessed in others. We histopathologically examined toxicity in visceral tissues and Ki-67, VEGF-A, γ-H2AX, and HIF-1α expression using immunohistochemistry. Direct intratumoral injections of AL-HA-Tyr exerted anti-tumor effects and improved hypoxia like orally administered AL (p > 0.05), but reduced visceral toxicity and prolonged survival. The uptake of 18F-FMISO did not significantly differ among the AL, AL-HA-Tyr, and RT+AL-HA-Tyr treated groups. Compared with the other agents, RT+AL-HA-Tyr decreased HIF-1α, Ki67, and VEGF-A expression, and increased γ-H2AX levels in tumor cells. Overall, compared with AL and AL-HA-Tyr, RT+AL-HA-Tyr improved tumor hypoxia, enhanced anti-tumor effects, and prolonged the survival of mice bearing LLC.

Single injection and multiple treatments: An injectable nanozyme hydrogel as AIEgen reservoir and release controller for efficient tumor therapy

• AIEgens loaded thermo-sensitive hydrogel was used to achieve the goal of multiple intratumoral treatment with one injection. • Prussian blue nanoparticles were used to improve the hypoxic microenvironment of tumor. • Prussian blue nanoparticles enhance the therapeutic effect of AIEgen based PDT. Scheme 1. Schematic illustration of an injectable nanozyme hydrogel as AIEgen release controller for efficient tumor therapy. Direct intratumoral injection of aggregation-induced emission luminogen (AIEgen) enable direct photodynamic therapy (PDT) of tumors, but these injections may cause localized pain and a range of postoperative complications during frequent injections. In the present study, we developed a novel injectable nanozyme hydrogel that functions as AIEgens reservoir and release controller (ARC) and thereby facilitates more efficient tumor therapy. The ARC system was developed by simultaneously encapsulating Prussian blue (PB) nanoparticles and an AIEgen (CQu) in agarose hydrogels. PB converts near-infrared laser into heat, resulting in agarose degradation and consequent CQu release. Then PB nanozyme can drive the decomposition of endogenous hydrogen peroxide, then yielding oxygen (O 2 ) generation. Following subsequent low-power white light exposure, AIEgens can generate high levels of reactive oxygen species (ROS) with sufficient O 2 , thereby inducing tumor cell cytotoxicity. As hydrogels can persist within tumors for a minimum of 48 h, they can facilitate multiple rounds of treatment following a single injection. The present study is the first attempt to utilize hydrogel for AIEgens delivery, and the ARC system developed herein offers a novel means of supporting the clinical development of AIEgens treatment strategies for cancer patients.

Paclitaxel-loaded polymeric nanoparticles based on α-tocopheryl succinate for the treatment of head and neck squamous cell carcinoma: in vivo murine model

: The prognosis of patients with recurrent or metastatic head and neck squamous cell cancer (HNSCC) is generally poor. New treatments are required to supplement the current standard of care. Paclitaxel (PTX), an effective chemotherapeutic for HNSCC, has serious side effects. A polymeric nanocarrier system was developed for the delivery of PTX to improve HNSCC treatment. This study aimed to evaluate the antitumor efficacy of PTX-loaded polymeric nanoparticles based on α-TOS (PTX-NPs) administered by direct intratumoral injection into a Hypopharynx carcinoma squamous cells (FaDu) tumor xenograft mouse model. The nanocarrier system based on block copolymers of polyethylene glycol (PEG) and a methacrylic derivative of α-TOS was synthesized and PTX was loaded into the delivery system. Tumor volume was measured to evaluate the antitumor effect of the PTX-NPs. The relative mechanisms of apoptosis, cell proliferation, growth, angiogenesis, and oxidative and nitrosative stress were detected by Western blotting, fluorescent probes, and immunohistochemical analysis. The antitumor activity results showed that compared to free PTX, PTX-NPs exhibited much higher antitumor efficacy and apoptosis-inducing in a FaDu mouse xenograft model and demonstrated an improved safety profile. Ki-67, EGFR, and angiogenesis markers (Factor VIII, CD31, and CD34) expression were significantly lower in the PTX-NPs group compared with other groups (p < .05). Also, PTX-NPs induced oxidative and nitrosative stress in tumor tissue. Direct administration of PTX-loaded polymeric nanoparticles based on α-Tocopheryl Succinate at the tumor sites, proved to be promising for HNSCC therapy.

NBTXR3 Radiation Enhancing Hafnium Oxide Nanoparticles Activated By Radiotherapy In Combination With Anti-PD-1 Therapy: A Phase I Study

Immune checkpoint inhibitors (ICIs) are being increasingly used to improve patient outcomes across different cancer types. However, the response rate to ICIs remains low (∼15%), indicating the need for novel strategies to improve treatment outcome. Emerging evidence suggests that radiation therapy (RT) could potentially enhance the antitumor response and provide synergy with ICIs. RT dose and ultimate efficacy are however limited by toxicity related to exposure of healthy tissues. The first-in-class radioenhancer NBTXR3, administered by direct intratumoral injection, is designed at the nanoscale to increase RT dose deposition within tumor cells and RT-dependent tumor cell killing, without increasing toxicity to surrounding normal tissue. Preclinical and early clinical data suggest NBTXR3 activated by RT can trigger an anti-tumor immune response, producing both local and systemic (abscopal) effects. We hypothesize that NBTXR3 activated by RT, in combination with anti-PD-1 therapy (R3/RT/PD-1), will act synergistically to maximize the local RT effect and produce a systemic response sufficient to increase the proportion of ICI responders or convert ICI non-responders to responders. This multicenter, open-label, phase I trial [NCT03589339] will evaluate safety and tolerability of R3/RT/PD-1 in three cohorts: (1) Locoregional recurrent or recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) amenable to re-irradiation of the HN field, (2) Lung metastases, or (3) Liver metastases, both from any primary cancer eligible for anti-PD-1 treatment. Approximately two-thirds of patients in each cohort will be anti-PD-1 non-responders. NBTXR3 injected volume is based on a percentage of baseline gross tumor volume (GTV). The primary objective is to determine the R3/RT/PD-1 recommended phase 2 dose in each cohort. Secondary objectives are to evaluate anti-tumor response (objective response rate; ORR), safety and feasibility of NBTXR3 injection, and NBTXR3 body kinetic profile. Exploratory objectives will assess biomarkers of R3/RT/PD-1 response, including PD-L1 status by IHC, as well as mRNA and cytokine immune marker profiling. Recruitment is ongoing. To date, three patients have been treated, one in cohort 1 and two in cohort 2. NBTXR3 activated by RT induces an anti-tumor immune response which may convert immunologically “cold” tumors into “hot” tumors. In combination R3/RT/PD-1 holds the potential to increase the proportion of ICI responders or convert ICI non-responders to responders.

99mTc-citrate-gold nanoparticles as a tumor tracer: synthesis, characterization, radiolabeling and in-vivo studies

Abstract Targeted drug delivery system can reduce the side effects of high drug concentration by improving drug pharmacokinetics at lower doses. Citrate-gold nanoparticles (AuNPs) as a drug delivery system were synthesized via green nanotechnology technique to be used as a new imaging platform for tumor targeting. Citrate-AuNPs were synthesized with core size of 10 nm. Citrate-AuNPs were labeled with technetium-99m (99mTc) with radiochemical yield of 95.20 ± 2.70% with good in-vitro stability in both saline and human serum and well in-vivo studied in both normal and solid tumor bearing mice. The in-vivo biodistribution study of [99mTc]Tc-citrate-AuNPs in solid tumor bearing mice (as preliminary study) showed a high accumulation in tumor site with tumor/muscle of 4.35 ± 0.22 after 30 min post injection. The direct intratumoral (I.T) injection of [99mTc]Tc-citrate-AuNPs showed that this complex was retained in the tumor up to 77.86 ± 1.90 % at 5 min and still around 50.00 ± 1.42 % after 30 min post injection (p.i.). The newly presented nano-platform could be presented as a new potential radiopharmaceutical tumor imaging probe.

Photodynamic treatment with purpurin 18 effectively inhibits triple negative breast cancer by inducing cell apoptosis.

This study aims to evaluate the photodynamic efficacy of purpurin 18 (pu-18) on triple negative breast cancer both in vitro and in vivo. Two states of 4T1 cells, 2D culture and 3D spheroids, were used to evaluate the photodynamic action of pu-18 in vitro. The in vitro study results indicated that for the 4T1 2D cell culture, the photodynamic therapy (PDT) treatment showed significant photocytotoxicity at low pu-18 concentrations following light irradiation. Pu-18 was found to distribute on the lysosomes, mitochondria, Golgi apparatus, and endoplasmic reticulum. After irradiation, pu-18 can generate ROS to destroy the mitochondrial membrane potential (MMP) and eventually induce apoptosis in the 2D 4T1 cells. Light-activated pu-18 could also induce the destruction of the 3D 4T1 cell spheroids. The in vivo study was conducted by using a subcutaneous 4T1 breast cancer animal model. The results demonstrated that pu-18 could remain in the tumor for more than 4 days by direct intra-tumoral injection. The PDT treatment was performed every 2 days for a total of 3 times. The results showed that PDT treatment could significantly inhibit tumor growth in vivo, indicating a good photodynamic efficacy of pu-18 in the mouse breast cancer model, without influencing weight and major organ function. The survival pattern results showed that PDT treatment could largely extend the survival time of mice with breast cancer. The preliminary conclusion is that photodynamic treatment using pu-18 is effective at preventing the growth of triple negative breast cancer cells both in vitro and in vivo. A combination of light irradiation and pu-18 could therefore be a worthwhile approach for the treatment of triple negative breast cancer.

Bronchoscopic intratumoural therapies for non-small cell lung cancer.

The past decade has brought remarkable improvements in the treatment of non-small cell lung cancer (NSCLC) with novel therapies, such as immune checkpoint inhibitors, although response rates remain suboptimal. Direct intratumoural injection of therapeutic agents via bronchoscopic approaches poses the unique ability to directly target the tumour microenvironment and offers several theoretical advantages over systemic delivery including decreased toxicity. Increases in understanding of the tumour microenvironment and cancer immunology have identified many potential options for intratumoural therapy, especially combination immunotherapies. Herein, we review advances in the development of novel bronchoscopic treatments for NSCLC over the past decade with a focus on the potential of intratumoural immunotherapy alone or in combination with systemic treatments.

Ad-CD40L mobilizes CD4 T cells for the treatment of brainstem tumors.

BackgroundDiffuse midline glioma, formerly DIPG (diffuse intrinsic pontine glioma), is the deadliest pediatric brainstem tumor with median survival of less than one year. Here, we investigated (i) whether direct delivery of adenovirus-expressing cluster of differentiation (CD)40 ligand (Ad-CD40L) to brainstem tumors would induce immune-mediated tumor clearance and (ii) if so, whether therapy would be associated with a manageable toxicity due to immune-mediated inflammation in the brainstem.MethodsSyngeneic gliomas in the brainstems of immunocompetent mice were treated with Ad-CD40L and survival, toxicity, and immune profiles determined. A clinically translatable vector, whose replication would be tightly restricted to tumor cells, rAd-Δ24-CD40L, was tested in human patient–derived diffuse midline gliomas and immunocompetent models.ResultsExpression of Ad-CD40L restricted to brainstem gliomas by pre-infection induced complete rejection, associated with immune cell infiltration, of which CD4+ T cells were critical for therapy. Direct intratumoral injection of Ad-CD40L into established brainstem tumors improved survival and induced some complete cures but with some acute toxicity. RNA-sequencing analysis showed that Ad-CD40L therapy induced neuroinflammatory immune responses associated with interleukin (IL)-6, IL-1β, and tumor necrosis factor α. Therefore, to generate a vector whose replication, and transgene expression, would be tightly restricted to tumor cells, we constructed rAd-Δ24-CD40L, the backbone of which has already entered clinical trials for diffuse midline gliomas. Direct intratumoral injection of rAd-Δ24-CD40L, with systemic blockade of IL-6 and IL-1β, generated significant numbers of cures with readily manageable toxicity.ConclusionsVirus-mediated delivery of CD40L has the potential to be effective in treating diffuse midline gliomas without obligatory neuroinflammation-associated toxicity.

Phase I study of NBTXR3 activated by radiotherapy in patients with advanced cancers treated with an anti-PD-1 therapy.

TPS3173 Background: Despite the past decade of transformative advances in immuno-oncology, the response rate to checkpoint inhibitors (ICIs) remains low (~15%). There is significant interest in developing strategies to overcome resistance to these treatments, thus increasing response rate. Emerging evidence suggests that radiation therapy (RT) could potentially augment the antitumor response to ICIs through synergic effect. However, RT dose and ultimate efficacy are limited by toxicity related to exposure of healthy tissues. NBTXR3 is a first-in-class radioenhancer administered by direct intratumoral injection, designed at the nanoscale to increase RT dose deposition within tumor cells and RT-dependent tumor cell killing, without increasing surrounding normal tissue toxicity. Preclinical and early clinical data suggest NBTXR3 activated by RT can trigger an anti-tumor immune response, producing both local and systemic (abscopal) effects. We hypothesize that NBTXR3 activated by RT, in combination with anti-PD-1 therapy (R3/RT/PD-1), will act synergistically to maximize the local RT effect and produce a systemic response sufficient to increase the proportion of ICI responders or convert ICI non-responders to responders. Methods: This trial [NCT03589339] is a multicenter, open-label, phase I study to evaluate safety and tolerability of R3/RT/PD-1 in three cohorts: (1) Locoregional recurrent or recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) amenable to re-irradiation of the HN field, (2) Lung metastases, or (3) Liver metastases, both from any primary cancer eligible for anti-PD-1 treatment. Approximately two-thirds of patients in each cohort will be anti-PD-1 non-responders. NBTXR3 injected volume is based on a percentage of gross tumor volume (GTV). The primary objective is to determine the R3/RT/PD-1 recommended phase 2 dose in each cohort. Secondary objectives are to evaluate anti-tumor response (objective response rate; ORR), safety and feasibility of NBTXR3 injection, and NBTXR3 body kinetic profile. Exploratory objectives will assess biomarkers of R3/RT/PD-1 response, including PD-L1 status by IHC, as well as mRNA and cytokine immune marker profiling. To date, three patients have been treated, one in cohort 1, two in cohort 2. Clinical trial information: NCT03589339 .

69 NanoPac-2017-01 Mid-Study Report: Safety, Tolerability, and Preliminary Efficacy of Intracystic Submicron Particle Paclitaxel (SPP) for the Treatment of Mucinous Cysts

INTRODUCTION: Pancreatic Mucinous cystic lesions (MCL) have significant potential for malignant transformation; delivery of SPP as a direct intratumoral injection confirmed SPP present at the delivery site for at least 100 days in a prior study; intracystic therapy with SPP may prevent progression to cancer without corresponding systemic toxicities. METHODS: Aim: To determine safety and preliminary efficacy of SPP for treatment of MCLs using endoscopic ultrasound Fine Needle Injection (EUS-FNI). Materials and Methods: Subjects with confirmed MCL, based on elevated intracystic CEA and cytology, receive intracystic SPP via EUS FNI at volumes equal to the aspirated cyst fluid volume in sequential cohorts at 6, 10, and 15 mg/mL in a standard “3+3” dose-escalation protocol. The highest dose with acceptable safety and tolerability profile, as determined by a DSMB, will proceed into the second phase of the study; 9 additional subjects will receive two injections of SPP (same dose) 12 weeks apart. Subjects are followed for 6 months for clinical endpoints including: safety and tolerability evaluations, physical examination findings and vital signs; pharmacokinetic analysis of systemic paclitaxel drug levels; and cyst volume response as reported by imaging at 3 and 6 months. RESULTS: Nine subjects have been enrolled to date; 5 have completed the study (1 injection). EUS, imaging, CEA and amylase concentration were consistent with branched duct IPMN cyst (BD-IPMN) or MCNs (Table 1). To date, no dose limiting toxicities, serious adverse events related to treatment, or clinically significant blood work (chemistry; hematology; coagulation) or urinalysis have been reported. Adverse events potentially related to SPP include mild, transient abdominal pain/discomfort in 4 subjects and nausea in 2 subjects. Systemic paclitaxel concentration did not exceed 1 ng/mL, and was undetectable at 2 weeks supporting retention of SPP at delivery site. Cyst volumes in 3 of 5 completed subjects reduced by month 6, 2 remained stable (Table 2). CONCLUSION: Intracystic SPP appears safe and tolerable when administered at 6, 10, and 15 mg/mL in a volume equal to that of aspirated cyst fluid. Minimal systemic study drug exposure is experienced. No patients developed pancreatitis. Intracystic SPP appears to prevent continued growth of the cysts and may reduce total cyst volume. Subject enrollment to the second phase (2 injections 12 weeks apart) is underway at 15 mg/mL.

Arylboronate prodrugs of doxorubicin as promising chemotherapy for pancreatic cancer

This study describes the synthesis of arylboronate-based ROS-responsive prodrugs of doxorubicin and their biological evaluation as anticancer agents. The determination of the most sensitive cancer type toward arylboronate prodrugs is crucial for further consideration of these molecules in clinical phase. To address this goal, an arylboronate-based profluorescent probe was used to compare the capacity of various cancer cell lines to efficiently convert the precursor into the free fluorophore. On the selected MiaPaCa-2 pancreatic cancer cells, a benzeneboronate prodrug exhibited 67% of the cytotoxicity obtained with the free doxorubicin. The prodrug was also able to induce tumor regression on MiaPaCa-2 pancreatic tumor model in ovo. Using this model, the amount of free doxorubicin liberated from this prodrug into the tumor was equivalent to the quantity measured after direct intratumoral injection of the same concentration of doxorubicin.

Targeting hypoxia downstream signaling protein, CAIX, for CAR T-cell therapy against glioblastoma.

Glioblastoma survival remains unchanged despite continuing therapeutic innovation. Herein, we aim to (i) develop chimeric antigen receptor (CAR) T cells with a specificity to a unique antigen, carbonic anhydrase IX (CAIX), which is expressed in the hypoxic microenvironment characteristic of glioblastoma, and (ii) demonstrate its efficacy with limited off-target effects. First we demonstrated expression of CAIX in patient-derived glioblastoma samples and available databases. CAR T cells were generated against CAIX and efficacy was assessed in 4 glioblastoma cell lines and 2 glioblastoma stem cell lines. Cytotoxicity of anti-CAIX CAR T cells was assessed via interferon gamma, tumor necrosis factor alpha, and interleukin-2 levels when co-cultured with tumor cells. Finally, we assessed efficacy of direct intratumoral injection of the anti-CAIX CAR T cells on an in vivo xenograft mouse model using the U251 luciferase cell line. Tumor infiltrating lymphocyte analyses were performed. We confirm that CAIX is highly expressed in glioblastoma from patients. We demonstrate that CAIX is a suitable target for CAR T-cell therapy using anti-CAIX CAR T cells against glioblastoma in vitro and in vivo. In our mouse model, a 20% cure rate was observed without detectable systemic effects. By establishing the specificity of CAIX under hypoxic conditions in glioblastoma and highlighting its efficacy as a target for CAR T-cell therapy, our data suggest that anti-CAIX CAR T may be a promising strategy to treat glioblastoma. Direct intratumoral injection increases anti-CAIX CAR T-cell potency while limiting its off-target effects.

Recombinant human insulin as a solid tumor potential imaging agent: Radio-synthesis and biological evaluation

The aim of this study was to synthesis an imaging agent for tumor targeting. New recombinant insulin analogue was successfully produced from E. coli by recombinant DNA technique, and was well labeled with Technetium-99m with a high radiochemical yield of 93.3 ± 2.1 %. Moreover, it showed good in-vitro stability in both saline and human serum. Preclinical evaluation of Technetium-99m [99mTc] Tc-insulin in solid tumor-bearing mice showed high accumulation in tumor tissues. The T/NT (target to non-target ratio) was of 5.4, after 60 min. of post injection (p.i). The direct intra-tumoral (I.T) injection of [99mTc] Tc-insulin showed good retention in tumor tissues with a ratio more than 50 % after 15 min. As a result of the promising bio-distribution studies; the newly recombinant insulin showed good uptake in tumor site, which assured high concentration of insulin receptor on tumor cell surface, accompanied with high cell density of tumor cells as well. This work affords a potential radio-carrier that could be used as a good tumor marker and imaging probe via SPECT (Single Photon Emission Computed Tomography) technique, after further preclinical studies.

Biocompatible Nanoclusters with High Heating Efficiency for Systemically Delivered Magnetic Hyperthermia.

Despite its promising therapeutic potential, nanoparticle-mediated magnetic hyperthermia is currently limited to the treatment of localized and relatively accessible cancer tumors because the required therapeutic temperatures above 40 °C can only be achieved by direct intratumoral injection of conventional iron oxide nanoparticles. To realize the true potential of magnetic hyperthermia for cancer treatment, there is an unmet need for nanoparticles with high heating capacity that can efficiently accumulate at tumor sites following systemic administration and generate desirable intratumoral temperatures upon exposure to an alternating magnetic field (AMF). Although there have been many attempts to develop the desired nanoparticles, reported animal studies reveal the challenges associated with reaching therapeutically relevant intratumoral temperatures following systemic administration at clinically relevant doses. Therefore, we developed efficient magnetic nanoclusters with enhanced heating efficiency for systemically delivered magnetic hyperthermia that are composed of cobalt- and manganese-doped, hexagon-shaped iron oxide nanoparticles (CoMn-IONP) encapsulated in biocompatible PEG-PCL (poly(ethylene glycol)- b-poly(ε-caprolactone))-based nanocarriers. Animal studies validated that the developed nanoclusters are nontoxic, efficiently accumulate in ovarian cancer tumors following a single intravenous injection, and elevate intratumoral temperature up to 44 °C upon exposure to safe and tolerable AMF. Moreover, the obtained results confirmed the efficiency of the nanoclusters to generate the required intratumoral temperature after repeated injections and demonstrated that nanocluster-mediated magnetic hyperthermia significantly inhibits cancer growth. In summary, this nanoplatform is a milestone in the development of systemically delivered magnetic hyperthermia for the treatment of cancer tumors that are difficult to access for intratumoral injection.

Intratumoral Localization of Pattern Recognition Receptor Signaling Informs CAR T Cell Design

Abstract Immune therapies have significantly improved outcomes for cancer patients with poor prognosis, but mechanisms that underlie response or resistance to therapy remain elusive. Type I and Type II interferon signaling is required for immune activation but is also known to drive programs of tumor-intrinsic resistance. We propose that activation of pattern recognition receptor (PRR) signaling in specific cellular compartments underlies this seemingly contradictory observation. We have recently identified the highly structured RNA RN7SL1 (7SL) as a novel intratumoral PAMP (pathogen associated molecular pattern) capable of activating PRR signaling. When over-expressed in tumor cells, the resulting tumors are resistant to immune checkpoint blockade (ICB) therapy. However, 7SL is stimulatory to primary human DCs, and improves T cell priming in vitro. Furthermore, direct intratumoral injection of 7SL improves immune activation and response to ICB in vivo. Thus, we propose the localization of PAMP recognition and PRR signaling may represent an important determinant of response to ICB therapy. Based on this finding, we have developed a CAR T cell capable of producing 7SL in the tumor microenvironment, as well as a fully syngeneic B16 solid tumor model for CAR T cell therapy. Murine 7SL-CAR T cells control tumors more robustly than controls and synergize with ICB. Importantly, this effect is dependent on endogenous T cells, indicating that 7SL-CAR T cells are jump-starting an endogenous polyclonal anti-tumor immune response. In total, this represents a novel insight into the role of PRR signaling in tumors and leverages the findings to create a therapy with potential to serve patients that are currently refractory to CAR-T or ICB therapies alone.

Translating Nanomedicine to Comparative Oncology-the Case for Combining Zinc Oxide Nanomaterials with Nucleic Acid Therapeutic and Protein Delivery for Treating Metastatic Cancer.

The unique anticancer, biochemical, and immunologic properties of nanomaterials are becoming a new tool in biomedical research. Their translation into the clinic promises a new wave of targeted therapies. One nanomaterial of particular interest are zinc oxide (ZnO) nanoparticles (NPs), which has distinct mechanisms of anticancer activity including unique surface, induction of reactive oxygen species, lipid oxidation, pH, and also ionic gradients within cancer cells and the tumor microenvironment. It is recognized that ZnO NPs can serve as a direct enzyme inhibitor. Significantly, ZnO NPs inhibit extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) associated with melanoma progression, drug resistance, and metastasis. Indeed, direct intratumoral injection of ZnO NPs or a complex of ZnO with RNA significantly suppresses ERK and AKT phosphorylation. These data suggest ZnO NPs and their complexes or conjugates with nucleic acid therapeutic or anticancer protein may represent a potential new strategy for the treatment of metastatic melanoma, and potentially other cancers. This review focuses on the anticancer mechanisms of ZnO NPs and what is currently known about its biochemical effects on melanoma, biologic activity, and pharmacokinetics in rodents and its potential for translation into large animal, spontaneously developing models of melanoma and other cancers, which represent models of comparative oncology.

Schwannoma gene therapy by adeno-associated virus delivery of the pore-forming protein Gasdermin-D.

Schwannomas are peripheral nerve sheath tumors associated with three genetically distinct disease entities, namely sporadic schwannoma, neurofibromatosis type-2, and schwannomatosis. Schwannomas are associated with severe disability and in cases lead to death. The primary treatment is operative resection that itself can cause neurologic damage and is at times contra-indicated due to tumor location. Given their homogenous Schwann-lineage cellular composition, schwannomas are appealing targets for gene therapy. In the present study, we have generated an adeno-associated serotype 1 virus (AAV1)-based vector delivering N-terminal of the pyroptotic gene Gasdermin-D; (GSDMDNterm) under the control of the Schwann-cell specific promoter, P0. we have demonstrated that AAV1-P0-GSDMDNterm injection into intra-sciatic schwannomas reduces the growth of these tumors and resolves tumor-associated pain without causing neurologic damage. This AAV1-P0-GSDMDNterm vector holds promise for clinical treatment of schwannomas via direct intra-tumoral injection.