Single Intratumoral Injection Research Articles

New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody.

92 Background: Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Methods: We constructed 3 recombinant oHSVs expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). We compared 1) the oncolytic effects of a single or multiple intratumoral injection(s) of T1, T2 and T3 series oHSVs, 2) the effectiveness of intratumoral injection of T3 oHSV with systemic administration of anti PD-1 ab and IL12 alone or in combinations with T1. Results: Insertion of gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). In syngeneic mice the T3 series oHSV expressing murine IL-12 and PD-1 Ab was effective against a variety of murine tumors. Concurrent with enhanced cytolytic activity the T3 induced significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ. Consistent with an earlier report of an inverse correlation between the volume of the tumor and the quantity of retained IFN-γ. The most effective oncolytic effect resulted from the administration of T3855 expressing both IL12 and anti PD-1 antibody or T2850 concurrently with intraperitoneal administration of anti PD-1 antibody alone. The least effective were single intraperitoneal administration of IL12 or PD-1 antibody. Intratumoral injection of T3 oHSV was most effective against murine tumors in comparison of either systemic administration of anti PD-1 ab and IL12 or intratumoral injection of T1 in combinations of systemic administration of anti PD-1 ab and IL12. Conclusions: We report the marriage in a single therapeutic agent of three distinct cancer therapies: the targeting of cancer cells by oncolytic viruses, the stimulation of immune system by IL12, and the production of immunotherapeutic antibodies against PD-1. The significant finding reported in this article is that the anti-tumor responses remain concentrated in the tumor environment.

NBTXR3 for the Treatment of Elderly Frail Patients with Locally Advanced HNSCC

The incidence of head and neck squamous cell carcinoma (HNSCC) increases with age and is expected to increase in the next 20 years. Despite representing 20% of the HNSCC population, elderly patients (pts) cannot be treated like the rest of the population due to their vulnerability to treatment-induced toxicity and thus new therapies are required. NBTXR3 represents a new treatment option and is currently being evaluated clinically. The first in class hafnium oxide nanoparticles (NBTXR3) are activated by radiotherapy and enhance its effects, leading to the physical destruction of cancer cells. A Phase I/II trial [NCT01946867] is underway to evaluate NBTXR3 in elderly pts (>70 years and older) or frail patients with locally advanced HNSCC of the oral cavity and oropharynx ineligible for cisplatin or intolerant to cetuximab. The phase I results are presented here. Elderly or frail pts were treated with a single intratumoral injection of NBTXR3 and intensity modulated radiation therapy (IMRT; 70 Gy/35 fractions/7 weeks) and followed until disease progression, further anticancer therapy or study cut-off date. The study is a 3 + 3 dose escalation to test the NBTXR3 dose equivalent to 5%, 10%, 15%, and 22% of the baseline tumor volume, followed by a dose expansion. Primary endpoints include determination of the Recommended Phase 2 Dose (RP2D) and early dose limiting toxicity (DLT). The presence of NBTXR3 in surrounding healthy tissues and efficacy applying RECIST 1.1 principles were also evaluated. Enrollment was completed at all dose levels: 5% (3 pts), 10% (3 pts), 15% (5 pts), and 22% (8 pts). No early DLT or SAE related to NBTXR3 or the injection procedure were observed. One grade 1 AE (asthenia at 22%) related to NBTXR3 and four AEs (grade 2 oral pain, grade 1 tumor hemorrhage, grade 1 asthenia, and grade 1 injection site hemorrhage) related to the injection procedure were reported. RT-related toxicity was as expected with IMRT. The RP2D has been determined to be 22%. CT-scan assessment between 24h and 7 weeks post injection demonstrated the absence of NBTXR3 leakage in surrounding tissues. Among the 13 evaluable pts treated at doses ≥10%, 9 achieved a complete response of the injected lesion per investigator assessment. The final dose escalation results will be presented herein. NBTXR3 was well tolerated at all tested doses and demonstrated a very good safety profile. With the identification of the RP2D, a dose expansion phase has started. These results of this study highlight the potential of NBTXR3 as a novel treatment option for elderly and/or frail pts with locally advanced HNSCC and address an unmet medical need. NBTXR3 is currently being evaluated in 5 other clinical trials, including a phase II/III trial in soft tissue sarcoma [NCT02379845] and phase I/II trials in prostate [NCT02805894], liver [NCT02721056] and rectal cancers [NCT02465593].

Abstract 4456: Discovery of E7766: A representative of a novel class of macrocycle-bridged STING agonists (MBSAs) with superior potency and pan-genotypic activity

Abstract Introduction STING (stimulator of interferon genes) is an emerging target for cancer immunotherapy. 2’,3'-cGAMP, a natural cyclic dinucleotide (CDN) STING agonist, and its phosphorothioate analogs, have drawn broad attention as lead molecules for STING targeted drug discovery. These CDNs, however, lack efficacy in some common STING genotypes disproportionally represented in non-Caucasians. Moreover, such CDNs have not fully addressed liability in chemical/metabolic stability. Here we report our chemistry approach to control STING agonist conformation to enhance binding affinity across all common STING genotypes and broaden the therapeutic potential of such compounds. Methods Our SBDD approach started with analysis of the binding pocket and key protein-ligand interactions to prioritize a focused set of analogs for chemical synthesis. Systematic SAR was built upon in vitro assays for STING binding affinity and activation of STING genotypes. X-ray single crystal structures were established for STING and diverse analogs, in free and bound states, to provide structural insight for rational analog design. Results Structural modeling was refined to evaluate different binding modes and dynamic conformational changes in the STING-ligand interface. We observed that STING-bound CDNs had the two ancillary nucleobases specifically oriented in close proximity with parallel pi-pi stacking and discovered that covalently linking the nucleobases advantageously pre-organize the bioactive constrained conformation for enhanced STING affinity. Our discovery established a novel class of macrocycle-bridged STING agonists (MBSAs). E7766, a representative of Eisai MBSA platform, shows superior in vitro activity against all the major human STING genotypes over reference CDNs, most distinctly in STINGREF. E7766 co-crystal structures with STINGWT and STINGREF provide structural basis for the added benefit of the topological novelty. The macrocyclic linker bridging the top of nucleobases perturbs the STING lid loop conformation and create new and specific interactions with both genotypes. In twelve subcutaneous tumor models in immune competent mice, single intra-tumoral injections achieved either complete regression or significant tumor growth delay with no serious adverse effect. E7766 also shows excellent chemical and metabolic stability, presumably conferred by conformational rigidity of the unique macrocycle bridge. More biological characterization of E7766 can be found in abstract #. Conclusion Eisai successfully discovered E7766, a representative of a novel class of macrocycle-bridged STING agonist topologically distinct from conventional STING agonists. E7766 demonstrated pan-genotypic STING activation, potent anti-cancer activities and excellent chemical and metabolic stability for further development. Citation Format: Atsushi ENDO, Dae-Shik Kim, Kuan-Chun Huang, Ming-Hong Hao, Steven Mathieu, Hyeong-wook Choi, Utpal Majumder, Xiaojie Zhu, Yongchun Shen, Kristen Sanders, Thomas Noland, Dinesh Chandra, Yu Chen, Karen TenDyke, Kara Loiacono, Donna Kolber-Simonds, Rongrong Jiang, Vaishali Dixit, Janna Hutz, John Wang, Xingfeng Bao, Francis Fang, Nadeem Sarwar. Discovery of E7766: A representative of a novel class of macrocycle-bridged STING agonists (MBSAs) with superior potency and pan-genotypic activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4456.

Sustained co-delivery of gemcitabine and cis-platinum via biodegradable thermo-sensitive hydrogel for synergistic combination therapy of pancreatic cancer

Pancreatic cancer is one of the most devastating cancers with poor prognosis and no significant change in the survival rate over the past decades. Localized targeted drug delivery through interventional endoscopic ultrasonography-guided fine-needle injection (EUS-FNI) is an attractive and minimally invasive strategy for inoperable pancreatic cancer. An injectable in-situ formed long-lasting drug delivery system is a promising alternative for the localized treatment of pancreatic cancer via EUS-FNI. Here, a biodegradable thermo-sensitive copolymer hydrogel for the co-delivery of anticancer agents gemcitabine (GEM) and cis-platinum (DDP) was developed. This hydrogel is a free flowable liquid at room temperature that changes into a semi-solid hydrogel following injection in response to the physiological temperature. Both in vitro and in vivo drug release behaviors indicate sustained drug release of this delivery system. Synergistic cellular proliferation inhibition and desirable apoptosis promotion have been found when pancreatic cancer Bxpc-3 cells were co-cultured with this GEM-DDP/hydrogel system. After a single intratumoral injection, the dual-drug loaded hydrogel formulation exhibited superior anti-tumor efficacy and minimized systemic side effect on pancreatic cancer xenograft mouse model in comparison to the intravenously injected free GEM and DDP combination. In addition, a strong synergistic therapeutic effect of the GEM-DDP/hydrogel system against pancreatic cancer has been found in vitro and in vivo compared to the single-drug loaded hydrogel composites. The obtained findings suggest this developed thermo-sensitive copolymer hydrogel system as a potential universal carrier for the localized targeted delivery of multi-drugs, for use in a variety of inoperable solid tumors.

Evaluation of controlled IL-12 in combination with a PD-1 inhibitor in subjects with recurrent glioblastoma.

2020 Background: Ad-RTS-hIL-12 (Ad) is a novel gene therapy candidate conditionally expressing IL-12 under the control of veledimex (V) acting via the proprietary RheoSwitch Therapeutic System (RTS) gene switch with a therapeutic window. Intratumoral Ad + oral V monotherapy (Phase 1 study, NCT02026271 ) resulted in a new sustained intra-tumor influx of activated cytotoxic T cells, consistent with an immune-mediated anti-tumor effect improving median overall survival (mOS) of subjects with recurrent glioblastoma (rGBM). This correlated with an increased circulating CD8+/FoxP3+ T cell ratio (“cytoindex”), an emerging biomarker for mOS. PD-1 expression on infiltrating T cells at biopsy after Ad+V, supports combining controlled IL-12 with a PD-1 inhibitor to further augment T-cell-mediated anti-tumor effects. The rationale is also supported by increased OS (100% combo vs 63% for Ad+V vs 40% for anti-PD-1) in mice bearing GL-261 glioma. Methods: An ongoing open label, dose-escalation Phase 1 trial (NCT03636477) is evaluating safety and tolerability of local, controlled IL-12 with nivolumab (nivo) in adult subjects with rGBM. Ad was administered by single intratumoral injection (2 x 1011 viral particles, Day 0) plus V (10-20 mg) PO QD x 15 with nivo (1-3mg/kg) IV on Days -7, 15, then Q2W. Results: Safety data revealed a similar profile as Ad +V monotherapy. Adverse reactions (ARs) during follow-on nivo dosing were consistent with anti-PD-1 reports. ARs were manageable and reversible with no synergistic toxicities. Nivo alone did not alter peripheral IL-12 levels (median baseline (before anti-PD-1) 0.9 pg/mL; Day 0 1 pg/mL) increasing to 5.5 pg/mL on Day 3. Nivo alone increased peripheral T cells (CD3+CD8+ median baseline 23%; Day 0 26%) and Ad+V elevated peripheral CD3+CD8+ to 31% at Day 14. Nivo alone decreased regulatory T cells (FoxP3 baseline 1.5% vs Day 0 0.8%). Ad+V decreased these to 0.3% (Day 14). Combination therapy improved the cytoindex (baseline 15; Day 0 29; Day 14 80). Conclusions: Controlled IL-12 production using Ad + V with nivo is a rational combination with initial data consistent with immune-mediated anti-tumor effects with a favorable safety profile, warranting continued investigation in rGBM. Clinical trial information: NCT03636477.

New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody.

e14250 Background: Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Methods: We constructed 3 families of recombinant oHSVs expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). We compared the oncolytic effects of a single or multiple intratumoral injection(s) of T1, T2 and T3 series oHSVs. We also compared the effectiveness of intratumoral injection of T3 oHSV with systemic administration of anti PD-1 ab and IL12 alone or in combinations with T1. Results: Insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). In syngeneic mice the T3 series oHSV expressing murine IL-12 and PD-1 Ab was effective against a variety of murine tumors. Concurrent with enhanced cytolytic activity the T3 oHSV induced the production and significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ. Consistent with an earlier report we noted an inverse correlation between the volume of the tumor and the quantity of retained IFN-γ. The most effective oncolytic effect resulted from the administration of T3855 expressing both IL12 and anti PD-1 antibody or T2850 concurrently with intraperitoneal administration of anti PD-1 antibody alone. The least effective were single intraperitoneal administration of IL12 or PD-1 antibody. Intratumoral injection of T3 oHSV was most effective against murine tumors in comparison of either systemic administration of anti PD-1 ab and IL12 or intratumoral injection of T1 in combinations of systemic administration of anti PD-1 ab and IL12. Conclusions: We report the marriage in a single therapeutic agent of three distinct cancer therapies: the targeting of cancer cells by oncolytic viruses, the stimulation of immune system by IL12, and the production of immunotherapeutic antibodies against PD-1. The significant finding reported in this article is that the anti-tumor responses remain concentrated in the tumor environment.

Evaluation of controlled IL-12 as monotherapy in subjects with recurrent GBM.

2053 Background: Interleukin-12 (IL-12), a master regulator of the immune system, results in anti-tumor responses in preclinical models, but safe use requires tightly controlled production. It was conditionally produced in Ph1 “main” study (NCT02026271) in subjects with recurrent glioblastoma (rGBM) using a replication-incompetent adenovirus modified to express IL-12 under transcriptional control of the proprietary RheoSwitch Therapeutic System (Ad-RTS-hIL-12, Ad) regulated by dose of veledimex (V). Monotherapy resulted in sustained intra-tumor influx of activated cytotoxic T cells, consistent with immune-mediated anti-tumor effect, improving overall survival (OS). This correlated with increased circulating CD8+/FoxP3+ T-cell ratio (“cytoindex”), an emerging biomarker of OS. While widely used with neurosurgery, dexamethasone (dex) blunts response to immunotherapies, nevertheless median mOS of subjects who received 20mg V of 12.7 mo (n=15) at 13.1 mo follow-up. However, subanalysis (n=6) showed low-dose dex (total ≤20 mg) during V dosing improved mOS (17.8 mo). We report a 36 subject substudy in rGBM with limited dex, total rGBM treated (n=70+). Methods: Ongoing Phase 1 substudy (NCT03679754) assesses safety and tolerability of local, inducible IL-12 by single intratumoral injection of Ad (2 x 1011 viral particles) + V (20 mg PO QD x15 doses Days 0-14) in subjects not receiving dex 4 wks prior to Ad. Results: As of 03Jan19, the majority of new subjects received low-dose dex (total ≤20mg Days 0-14). The initial impact of dex on mOS will be reported. As in the main study, Ad+V 20 mg respectively increased (median) serum IL-12 and downstream IFN-g from Days 0-3: 0.8 to 8.8 pg/mL and 0 to 8.6 pg/mL. Between Days 0-14, there was net increase in cytoindex (from 20 to 46). The safety profile was similar to the main study with the main adverse reaction (AR) being mild to moderate cytokine release syndrome (CRS) characterized by flu-like symptoms. No grade 4 CRS was noted; all ARs were manageable and reversable upon holding V. Conclusions: Local, controlled IL-12 production using the Ad + V platform in subjects with rGBM safely activates the immune system and when dex is limited, appears to further improve mOS, which warrants continued investigation. Clinical trial information: NCT03679754.

Multifunctional Chitosan/Porous Silicon@Au Nanocomposite Hydrogels for Long-Term and Repeatedly Localized Combinatorial Therapy of Cancer via a Single Injection.

Considering the future clinical applications of localized cancer therapy, it is of great importance to construct injectable biodegradable nanocomposite hydrogels with combinatorial therapeutic efficacy. Here, porous silicon nanoparticles (PSiNPs) as host matrix were chosen to fabricate PSiNPs@Au nanocomposites via in situ reductive synthesis of gold nanoparticles. Then PSiNPs@Au nanocomposites were further incorporated into thermosensitive chitosan (CS) hydrogels to construct CS/PSiNPs@Au nanocomposite hydrogels, which showed in situ gelation at physiological temperature, excellent biodegradability, and biocompatibility. Especially with the encapsulation of CS hydrogels, PSiNPs@Au nanocomposites had a long-term stable photothermal effect with higher local temperature under near-infrared (NIR) laser irradiation, whether in vitro or in vivo. Besides, assisted by NIR laser irradiation, CS/PSiNPs@Au nanocomposite hydrogels exhibited a long-term sustained release of anticancer drugs (doxorubicin hydrochloride, DOX) in acidic tumor environments. Finally, DOX/CS/PSiNPs@Au precursors were administrated into tumor-bearing mice via a single intratumoral injection, which presented a significant synergistic chemo-photothermal therapeutic efficacy under repeated NIR laser irradiation during long-term cancer treatments. Accordingly, we developed a novel strategy to prepare multifunctional CS/PSiNPs@Au nanocomposite hydrogels and also demonstrated their potential applications in localized cancer therapy in future clinics.

Anti‐tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first-line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti-tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase-dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra-tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus-induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.

In Situ Growth of a Cationic Polymer from the N-Terminus of Glucose Oxidase To Regulate H2O2 Generation for Cancer Starvation and H2O2 Therapy.

Hydrogen peroxide (H2O2)-generating enzymes (HGEs) are potentially useful for tumor therapy, but the potential is limited by the challenge in regulating H2O2 production. Herein, we present site-specific in situ growth of a cationic polymer poly( N, N'-dimethylamino-2-ethyl methacrylate) (PDMA) from the N-terminus of glucose oxidase (GOX) to generate a site-specific and cationic GOX-PDMA conjugate with well-retained activity and enhanced stability to regulate H2O2 generation for cancer starvation and H2O2 therapy. Notably, the efficiency of endocytosis of the conjugate was 4-fold higher than that of free GOX. As a result, relative to free GOX, the conjugate showed 1.5-fold increased cytotoxicity, 2-fold enhanced tumor retention, and 5-fold increased tolerability after intratumoral injection. Importantly, a single intratumoral injection of the conjugate completely abolished colon tumors without detectable side effects, whereas free GOX was ineffective and systemically toxic. This chemistry may provide a new, simple, general, and efficient solution to regulate H2O2 production and thereby to dramatically improve the antitumor efficacy of HGEs while reducing side effects.

Abstract A011: First-in-man clinical trial of intratumoral injection of Clostridiumnovyi-NT spores in patients with treatment-refractory advanced solid tumors: Safety, activity, and immune responses

Abstract Bacteriolytic strategies offer unique advantages to combat a broad range of cancers often refractive to conventional chemotherapies and/or radiotherapies. C. novyi-NT is an attenuated strain of Clostridium novyi, a spore-forming, gram-positive, obligate anaerobe that lacks a lethal alpha- toxin, expressed by the parental strain. When administered intravenously or intratumorally with percutaneous injection, C. novyi-NT colonizes and replicates within the hypoxic region of the tumors, eliciting robust, tumor-confined cell lysis. In this first-in-man phase 1 study, patients with injectable treatment-refractory solid tumors received a single intratumoral injection of C. novyi-NT spores across 6 dose cohorts (spore concentrations of 104, 3x104, 105, 3x105, 106, 3x106) using a 3+3 trial design. The primary endpoints were to assess the safety profile, the dose limiting toxicity, and the maximum tolerated dose of C. novyi-NT. Key secondary endpoints included assessments of the preliminary antitumor activity of the injected tumor, an overall response evaluated by RECIST v. 1.1, and the host immune and inflammatory response to C. novyi-NT. Twenty-four patients were enrolled between November, 2013 and April, 2017. A single intratumoral injection of C. novyi-NT led to germination and resultant tumor lysis of injected tumor masses in 46% of patients across all dosing cohorts. The cohort 5 dose of 106 spores was defined as the maximum tolerated dose. Dose-limiting toxicities were grade 4 sepsis and grade 4 gas gangrene (n=1), all in patients with germination. In the 22 evaluable patients, 21 (95%) had stable disease (SD) as the best response for the injected lesion (tumor shrinkage of > 10% was observed in 21% of patients) and 19 (86%) had overall SD as the best response per RECIST 1.1. C. novyi-NT injection resulted in transient serum cytokine responses consistent with inflammasome activity, activation of innate immunity, tissue remodeling, and angiogenesis. Tumor antigen specific ELISPOT assays pre- and post- treatment documented enhanced secretion of IFNγ and TNFα by circulating T-cells, indicating improved systemic tumor specific T-cell responses. Multiparametric in situ immunostaining of core biopsies suggested improved immune cell infiltration in metastatic lesions. These early signs of improved antitumor activity in patients with advanced solid tumors have encouraged a new trial of C. novyi-NT in combination with immune checkpoint inhibitors. Citation Format: Filip Janku, Mrinal Gounder, Abdul Mohammad Pezeshki, Ravi Murthy, Andrea Wang-Gillam, Dale Shepard, David S. Hong, Sarina A. Piha-Paul, Anjali Raina, Alexey A. Leontovich, Gary DeCrescenzo, Brent L. Kreider, David Tung, Mary Varterasian, Halle H. Zhang, Khashayarsha Khazaie. First-in-man clinical trial of intratumoral injection of Clostridiumnovyi-NT spores in patients with treatment-refractory advanced solid tumors: Safety, activity, and immune responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A011.

Altered function of the glutamate-aspartate transporter GLAST, a potential therapeutic target in glioblastoma.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

ATIM-24. INTERIM RESULTS OF A PHASE II MULTICENTER STUDY OF THE CONDITIONALLY REPLICATIVE ONCOLYTIC ADENOVIRUS DNX-2401 WITH PEMBROLIZUMAB (KEYTRUDA) FOR RECURRENT GLIOBLASTOMA; CAPTIVE STUDY (KEYNOTE-192)

BACKGROUND DNX-2401 (tasadenoturev) is a replication-competent, tumor-selective, oncolytic adenovirus. A dose-escalating, Phase II study of a single intratumoral injection of DNX-2401 with pembrolizumab to determine optimal dose, safety and efficacy is ongoing and enrolling up to 48 patients with recurrent glioblastoma. Key inclusion criteria include patients with a single lesion at first or second recurrence for which resection is not possible or planned.

Elderly Patients: NBTXR3 as a Novel Treatment Option in Locally Advanced HNSCC

As the incidence of head and neck squamous cell carcinoma (HNSCC) increases with age, elderly patients represent 25% of the affected population with an expected increase in the next 20 years. Being more vulnerable to treatment-induced toxicities, the elderly cannot be treated like younger patients, hence requiring innovative therapies. A new treatment option is currently clinically evaluated, NBTXR3. These first-in-class hafnium oxide nanoparticles are activated by radiotherapy and can physically destroy cancer cells. A phase I study [NCT01946867] was implemented for the treatment of locally advanced HNSCC of the oral cavity and oropharynx, focused on elderly patients (65 years and older) not eligible for cisplatin, the non-surgical standard of care, or intolerant to cetuximab. Currently, 14 patients (pts) eligible for exclusive radiotherapy were included. A single intratumoral (IT) injection of NBTXR3 was followed by intensity-modulated radiation therapy (IMRT; 70 Gy/35 fractions/7 weeks), and a follow-up period until disease progression or study cut-off date. The study was designed as a 3 + 3 escalation dose with tested dose levels at 5%, 10%, 15% and 22% of baseline tumor volume. Primary endpoints included the determination of recommended dose and early dose limiting toxicity (DLT). Presence of NBTXR3 in the surrounding healthy tissues and efficacy per RECIST 1.1 response are also evaluated. No early DLT nor SAE related to NBTXR3 or injection procedure were observed for volume dose levels 5% (3 pts), 10% (3 pts), 15% (5 pts) and 22% (3 pts). So far, one AE (asthenia, grade 1) related to NBTXR3 was reported. Additionally, three AE related to the injection procedure (tumor hemorrhage, grade 1; oral pain, grade 2; asthenia, grade 1) occurred at 15% and 22%. The injection feasibility was confirmed for all patients, and persistence of NBTXR3 over time and absence of leakage in the surrounding tissues were verified by CT scan comparison between post IT injection and post radiation images in evaluable patients. For 5 out of 8 patients treated with NBTXR3 at doses >10%, the best response observed was a complete response (RECIST 1.1, local assessment). Even at the highest doses, NBTXR3 is associated with a positive safety profile. Recruitment in the fourth and last dose level at 22% is ongoing. Preliminary results tend to highlight NBTXR3 benefit as a novel treatment option for elderly patients in locally advanced HNSCC. Considering the challenges brought by the population of interest, this study could lead to new perspectives in the support of frail patients with advanced age. This study answers a major medical need, as most HNSCC clinical trials do not focus on elderly patients. In parallel, NBTXR3 is evaluated in 5 other clinical trials, including a phase II/III in soft tissue sarcoma (STS) [NCT02379845] and phases I/II for prostate [NCT02805894], liver [NCT02721056] and rectum cancers [NCT02465593].

Hafnium Oxide Nanoparticles Activated By Radiation Therapy for the Treatment of Solid Tumors

To improve radiotherapy (RT) in terms of tumor response and to reduce irradiation of healthy tissues, innovative therapeutic approaches are needed. In response, NBTXR3, injectable hafnium oxide nanoparticles, was developed for the treatment of solid tumors. Once injected intratumorally, NBTXR3 can deposit high energy within tumors only when activated by an ionizing radiation source, like current standard RTs. Upon activation, the high energy radiation physically kills the tumor cells by triggering DNA damage and cell destruction improving clinical outcomes. Since its first successful clinical evaluation in a completed phase I trial in patients with locally advanced soft tissue sarcoma, NBTXR3 is currently evaluated in numerous indications worldwide (EU, Asia, US). NBTXR3 was the object of numerous in vitro and in vivo tumor models to determine its mechanism of action, performance and biocompatibility profile. Once they were assessed, NBTXR3 entered clinical development and was administered as a single intratumoral (IT) injection activated by RT. NBTXR3 is clinically evaluated in head and neck [NCT01946867; NCT02901483], prostate [NCT02805894], liver [NCT02721056] and rectum cancers [NCT02465593] with the scope of determining the Recommended Dose or observing any Dose Limiting Toxicities (DLTs) in each indication. A phase II/III trial in soft tissue sarcoma (STS) of the trunk and extremities [NCT02379845] is about to be finalized. Preclinically, in vitro results showed an increase of cancer cells death and in vivo results demonstrated antitumor efficacy with NBTXR3 + RT compared to RT alone. This physical cell killing could open a potential systemic activity through immune response by triggering immunogenic cell death, reinforcing local effect. Clinically, across the 7 clinical trials and 6 indications, NBTXR3 demonstrated an overall positive safety profile. The numerous types of tumors and different body locations involved in these trials confirmed feasibility of IT injection and persistence of NBTXR3 in the tumor, with no leakage in the surrounding healthy tissues. NBTXR3 antitumor activity is currently evaluated in its first phase II/III in patients with STS. Besides, analysis of tumor biopsies collected pre- and post-radiotherapy suggested a release of tumor antigens during cancer cell death and stimulation of local immunological effects. NBTXR3 have shown promising results in non-clinical studies with marked antitumor efficacy and in clinical development in terms of safety and preliminary evaluations of efficacy. Considering the preliminary results of the 145 patients injected across all clinical trials, these first-in-class nanoparticles have already proven to be an encouraging innovative treatment in various types of tumors.

Laser Immunotherapy in Combination with Perdurable PD-1 Blocking for the Treatment of Metastatic Tumors.

A convenient and feasible therapeutic strategy for malignant and metastatic tumors was constructed here by combining photothermal ablation (PTA)-based laser immunotherapy with perdurable PD-1 blockade immunotherapy. Hollow gold nanoshells (HAuNS, a photothermal agent) and AUNP12 (an anti PD-1 peptide, APP) were co-encapsulated into poly(lactic- co-glycolic) acid (PLGA) nanoparticles. Unlike monoclonal PD-1/PD-L1 antibodies, PD-1 peptide inhibitor shows lower cost and immunotoxicity but needs frequent administration due to its rapid clearance in vivo. Our data here showed that the formed HAuNS- and APP-loaded PLGA nanoparticles (AA@PN) could maintain release periods of up to 40 days for the peptide, and a single intratumoral injection of AA@PN could replace the frequent administration of free APP. After the administration of AA@PN and irradiation with a near-infrared laser at the tumor site, an excellent killing effect on the primary tumor cells was achieved by the PTA. The nanoparticles also played a vaccine-like role under the adjuvant of cytosine-phospho-guanine (CpG) oligodeoxynucleotide and generated a localized antitumor-immune response. Furthermore, sustained APP release with laser-dependent transient triggering could induce the blockage of PD-1/PD-L1 pathway to activate T cells, thus subsequently generating a systemic immune response. Our data demonstrated that the PTA combined with perdurable PD-1 blocking could efficiently eradicate the primary tumors and inhibit the growth of metastatic tumors as well as their formation. The present study provides a promising therapeutic strategy for the treatment of advanced cancer with metastasis and presents a valuable reference for obtaining better outcomes in clinical cancer immunotherapy.

Magnetosomes Extracted from Magnetospirillum gryphiswaldense as Theranostic Agents in an Experimental Model of Glioblastoma.

Magnetic fluid hyperthermia (MFH) with chemically synthesized nanoparticles is currently used in clinical trials as it destroys tumor cells with an extremely localized deposition of thermal energy. In this paper, we investigated an MFH protocol based on magnetic nanoparticles naturally produced by magnetotactic bacteria: magnetosomes. The efficacy of such protocol is tested in a xenograft model of glioblastoma. Mice receive a single intratumoral injection of magnetosomes, and they are exposed three times in a week to an alternating magnetic field with concurrent temperature measurements. MRI is used to visualize the nanoparticles and to monitor tumor size before and after the treatment. Statistically significant inhibition of the tumor growth is detected in subjects exposed to the alternating magnetic field compared to control groups. Moreover, thanks to magnetosomes high transversal relaxivity, their effective delivery to the tumor tissue is monitored by MRI. It is apparent that the efficacy of this protocol is limited by inhomogeneous delivery of magnetosomes to tumor tissue. These results suggest that naturally synthesized magnetosomes could be effectively considered as theranostic agent candidates for hyperthermia based on iron oxide nanoparticles.

Abstract CT051: Preliminary correlative and clinical data from a first-in-human (FIH) study of the intratumoral (IT) oncolytic virotherapy, Voyager-V1, in patients with solid tumors

Abstract Introduction Voyager-V1TM is derived from VSV, a bullet-shaped negative sense RNA virus with very low human seroprevalence; it is engineered to selectively replicate in and kill human cancer cells. Voyager-V1 encodes the human IFNβ gene to boost antitumoral immune responses and the thyroidal sodium iodide symporter NIS gene to permit noninvasive imaging of virus spread. FIH studies are underway via both IV and IT routes. Here we report safety and preliminary correlative data from the FIH IT study. Methods and objectives This is a classical 3+3 phase 1 design, using escalating single IT viral doses from 3 x 106 to 3 x 109 TCID50 into one target lesion. The primary objective is safety and tolerability, monitored by committee. Other objectives include PK by RT-PCR for viral genomes, serum IFNβ levels, Tc-99m SPECT/CT imaging to monitor virus infection in injected lesions, peripheral blood immunophenotyping with 11-color flow cytometry for activation markers on T cells, T-regs, NK cells, and MDSCs, and serial biopsies to assess the tumor microenvironment (TME). IHC was performed on tumor biopsies for CD3, CD8, CD4, FoxP3, CD68, PD-1 and PDL-1 pre and post treatment in non-injected and injected lesions. CD45 staining is ongoing. Results Dose level 3 is ongoing (n=8+). No DLTs have been observed to date. Most patients were male (75%), white (100%), with ECOG PS1 (75%) and a median of >6 lines of prior systemic therapy. AEs (in 63% patients) reported as related to study drug were mild-moderate, short-lived, and consisted of G1 fever, chills, hot flashes, nausea, vomiting, hyperhidrosis and G2 fatigue. Most patients had some mild AEs (pain, bruising, subclinical pneumothorax) related to biopsy and IT injection procedures and one had an SAE related to biopsy (G2 pneumothorax). There was no difference in incidence, intensity or duration of related AEs between dose levels so far. In patients analyzed to date, at the first three dose levels of 3 x 106, 1 x 107 and 3 x 107 TCID50 (n=7), viremia and serum IFNβ were below levels of detection after IT injection. There is evidence in all patients of an increase in PD-1 expression on CD4 and CD8 T cells, suggesting T cell activation post- single virus injection. One patient at dose level 2 had increased CD4 and CD8 T cells in the peripheral blood. TME analysis revealed increased infiltration of CD3 and CD8 cells in tumors of some patients. In addition, there are changes in numbers of FoxP3 and CD68 positive cells in the injected lesions compared to the pre-treatment and non-injected lesion. Two patients at dose level 3, one with an injected adrenal metastasis of colorectal cancer and another with a chest wall lesion from a head of pancreas primary, have positive SPECT/CTs on days 3-15 showing viral replication in tumor plus concomitant lymphocyte/neutrophil trafficking in the periphery. Conclusions IT injection of a single dose of the novel oncolytic virotherapy Voyager-V1 has proven safe at doses up to 3 x 107 TCID50. There was evidence of T cell activation with increased PD-1 expression in peripheral blood lymphocytes after a single IT injection. Tumor biopsies indicate increased lymphocyte infiltration. Two patients at the highest dose tested show signs of viral replication in the injected lesion plus evidence of systemic impact on leukocyte populations. Citation Format: Steven Powell, Manish R. Patel, Jaime R. Merchan, Timothy P. Cripe, James Strauss, Rosa M. Diaz, Nandakumar Packiriswamy, Bethany A. Brunton, Deepak Upreti, Rehan Khan, Lukkana Suksanpaisan, Rianna Vandergaast, Stephen J. Russell, Alice S. Bexon, Kah Whye Peng. Preliminary correlative and clinical data from a first-in-human (FIH) study of the intratumoral (IT) oncolytic virotherapy, Voyager-V1, in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT051.

Abstract CT122: Phase I study of potentially "best-in-class" survivin-responsive conditionally replicating adenovirus for advanced sarcoma actually demonstrates potent and long-term efficacy and high safety

Abstract [Background] Recent approvals of oncolytic virus (OV) by FDA and EMA attract the worldwide oncologists and anticancer market. We developed the first platform technology to efficiently construct diverse types of a next-generation conditionally replicating adenovirus (CRA), i.e., CRAs that target and/or treat tumor cells with multiple factors (m-CRAs), which can increase tumor-specificity (i.e., safety) and efficacy (Gene Ther 2005). Using this platform technology, we have generated and tested numbers of m-CRAs as anticancer agents, and found that one of the best was survivin-responsive m-CRAs (Surv.m-CRAs). In preclinical studies, Surv.m-CRAs induced more potent cytotoxic effects against most of malignant tumors than other competing CRAs, and exhibited not only stronger but also more cancer-selective phenotypes than telomerase reverse transcriptase (Tert)-responsive m-CRAs (Cancer Res 2005). Moreover, Surv.m-CRAs induced increased effectiveness against cancer stem cells, which are resistant to conventional therapies (J Trans Med 2014). Preclinical analyses suggested that Surv.m-CRA is the “best-in class” CRA. [Trial protocol] We submitted an IND in March, 2016 and have been performing the first-in-human investigator-initiated ICH-GCP clinical trial for refractory malignant bone and soft tissue tumors (Phase I). Three doses (low: 1×10^10 viral particle [vp], mid: 1×10^11 vp, high: 1×10^12 vp) are planned. Adverse effects were graded according to CTCAE v4.0. Viral shedding into blood, urine and saliva was monitored by PCR. Efficacy was assessed by CT according to RECIST and Choi criteria. [Results] Three patients underwent a single intratumoral injection of low dose Surv.m-CRA-1. Treatment-related adverse events included asymptomatic and temporal leukopenia (grade 3, n=1), CPK elevation (grade 1, n=1), fever (grade 1, n=1) or other mild events (no grade 4 events). Viral shedding into saliva was found in the first case only at 3 hours after injection and was negative in other two cases. Clinical efficacy for target lesion was observed in all three cases (PR or better). First case showed PR by Choi criteria at 4 weeks and PR by RECIST (reduction rate, 50%) at 28 weeks; surprisingly the antitumor efficacy continued for more than one year. In the second case, tumor necrosis (PR by Choi criteria) was found at 4 weeks and subsequently, bone formation (beneficial remodeling) was observed at 12 weeks after the injection. Since no DLT was observed in the low dose cohort, we started mid dose treatment. [Conclusions] First-in-human trial of Surv.m-CRA-1 demonstrated drastic and long-term antitumor effects only by a sinle injection of only 1/100 of the predicted maximal dose. Thus, the potential of “best-in-class” Surv.m-CRA-1 is, at least in part, being reproduced in clinical trial. After finishing the phase I study, we will proceed Phase I/II study of multiple injections for advanced solid tumors. Citation Format: Satoshi Nagano, Toshitaka Futagawa, Eriko Sumi, Nobuhiro Ijichi, Munekazu Yamaguchi, Masanori Nakajo, Teruto Hashiguchi, Yasuo Takeda, Takashi Yoshiura, Akira Shimizu, Muneo Takatani, Setsuro Komiya, Ken-ichiro Kosai. Phase I study of potentially "best-in-class" survivin-responsive conditionally replicating adenovirus for advanced sarcoma actually demonstrates potent and long-term efficacy and high safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT122.

An oncolytic measles virus-sensitive Group 3 medulloblastoma model in immune-competent mice.

Oncolytic measles virus (MV) is effective in xenograft models of many tumor types in immune-compromised mice. However, no murine cell line exists that is tumorigenic, grows in immune-competent mice, and is killed by MV. The lack of such a model prevents an examination of the effect of the immune system on MV oncotherapy. Cerebellar stem cells from human CD46-transgenic immunocompetent mice were transduced to express Sendai virus C-protein, murine C-Myc, and Gfi1b proteins. The resultant cells were injected into the brain of NSG mice, and a cell line, called CSCG, was prepared from the resulting tumor. CSCG cells are highly proliferative, and express stem cell markers. These cells are permissive for replication of MV and are killed by the virus in a dose- and time-dependent manner. CSCG cells form aggressive tumors that morphologically resemble medulloblastoma when injected into the brains of immune-competent mice. On the molecular level, CSCG tumors overexpress natriuretic peptide receptor 3 and gamma-aminobutyric acid type A receptor alpha 5, markers of Group 3 medulloblastoma. A single intratumoral injection of MV‒green fluorescent protein resulted in complete tumor regression and prolonged survival of animals compared with treatments with phosphate buffered saline (P = 0.0018) or heat-inactivated MV (P = 0.0027). This immune-competent model provides the first platform to test therapeutic regimens of oncolytic MV for Group 3 medulloblastoma in the presence of anti-measles immunity. The strategy presented here can be used to make MV-sensitive murine models of any human tumor for which the driving mutations are known.