Single Intratumoral Injection Research Articles

Alum-anchored IL-12 combined with cytotoxic chemotherapy and immune checkpoint blockade enhanced antitumor immune responses in head and neck cancer models

BackgroundFirst-line treatment with pembrolizumab plus chemotherapy in recurrent and metastatic head and neck squamous cell carcinomas (HNSCC) has improved survival. However, the overall response rate with this standard of care...

Antitumor effect of the Newcastle disease virus strain NDV/Altai/pigeon/777/2010 on a model of solid Lewis carcinoma

Aim. To evaluate the efficacy of intratumoral administrations of the Newcastle disease virus strain NDV/Altai/pigeon/777/2010, to compare progression of the tumor nodes after virotherapy and to analyse pathomorphological changes in the tumor tissue in vitro and in vivo. Single intratumoral injections of the mesogenic strain of Newcastle disease virus NDV/Altai/pigeon/777/2010, isolated from a rock dove in Siberia, were done into outbred mice of the C57Bl/6 line into solid nodes of Lewis lung carcinoma that were grafted subcutaneously. Then the dynamics of tumor growth and pathomorphological changes in the tumor tissue were assessed and analyzed.It was shown that single intratumoral injections into immunocompetent C57Bl/6 mice with the mesogenic strain of Newcastle disease virus NDV/Altai/pigeon/777/2010 led to an increase in peculiar pathomorphological changes in the tumor tissue of subcutaneously grafted Lewis lung carcinoma and to a decrease in tumor growth compared to the control group of mice in vivo. A direct cytotoxic effect of the NDV strain on the Vero E6 cell line in vitro was noted.The results of this study indicate that the NDV strain NDV/Altai/pigeon/777/2010 has antitumor properties. This may enable clinical trials to prove its effectiveness as an antitumor drug.

Programmable bacteria synergize with PD-1 blockade to overcome cancer cell-intrinsic immune resistance mechanisms.

Interferon-γ (IFN-γ) is a potent cytokine critical for response to immunotherapy, yet conventional methods to systemically deliver this cytokine have been hindered by severe dose-limiting toxicities. Here, we engineered a strain of probiotic bacteria that home to tumors and locally release IFN-γ. A single intratumoral injection of these IFN-γ-producing bacteria was sufficient to drive systemic tumor antigen-specific antitumor immunity, without observable toxicity. Although cancer cells use various resistance mechanisms to evade immune responses, bacteria-derived IFN-γ overcame primary resistance to programmed cell death 1 (PD-1) blockade via activation of cytotoxic Foxp3-CD4+ and CD8+ T cells. Moreover, by activating natural killer (NK) cells, bacteria-derived IFN-γ also overcame acquired resistance mechanisms to PD-1 blockade, specifically loss-of-function mutations in IFN-γ signaling and antigen presentation pathways. Collectively, these results demonstrate the promise of combining IFN-γ-producing bacteria with PD-1 blockade as a therapeutic strategy for overcoming immunotherapy-resistant, locally advanced, and metastatic disease.

Coating Dormant Collagenase-Producing Bacteria with Metal-Anesthetic Networks for Precision Tumor Therapy.

Tumor malignancy highly depends on the stiffness of tumor matrix, which mainly consists of collagen. Despite the destruction of tumor matrix is conducive to tumor therapy, it causes the risk of tumor metastasis. Here, metal-anesthetic network-coated dormant collagenase-producing Clostridium is constructed to simultaneously destruct tumor matrix and inhibit tumor metastasis. By metal-phenolic complexation and π-π stacking interactions, a Fe3+-propofol network is formed on bacterial surface. Coated dormant Clostridium can selectively germinate and rapidly proliferate in tumor sites due to the ability of carried Fe3+ ions to promote bacterial multiplication. Intratumoral colonization of Clostridium produces sufficient collagenases to degrade tumor collagen mesh and the loaded propofol restrains tumor metastasis by inhibiting tumor cell migration and invasion. Meanwhile, the delivered Fe3+ ions are reduced to the Fe2+ form by intracellular glutathione, thereby inducing potent Fenton reaction to trigger lipid peroxidation and ultimate ferroptosis of tumor cells. In addition to a satisfactory safety, a single intratumoral injection of coated dormant Clostridium not only effectively retards the growth of established large primary tumors, but also significantly suppresses distal lung metastasis in two different orthotopic tumor models. This work proposes a strategy to develop advanced therapeutics for malignant tumor treatment and metastasis prevention.

Injectable hybrid hydrogels enable enhanced combination chemotherapy and roused anti-tumor immunity in the synergistic treatment of pancreatic ductal adenocarcinoma

Chemotherapy and immunotherapy have shown no significant outcome for unresectable pancreatic ductal adenocarcinoma (PDAC). Multi-drug combination therapy has become a consensus in clinical trials to explore how to arouse anti-tumor immunity and meanwhile overcome the poorly tumoricidal effect and the stroma barrier that greatly hinders drug penetration. To address this challenge, a comprehensive strategy is proposed to fully utilize both the ferroptotic vulnerability of PDAC to potently irritate anti-tumor immunity and the desmoplasia-associated focal adhesion kinase (FAK) to wholly improve the immunosuppressive microenvironment via sustained release of drugs in an injectable hydrogel for increasing drug penetration in tumor location and averting systematic toxicity. The injectable hydrogel ED-M@CS/MC is hybridized with micelles loaded with erastin that exclusively induces ferroptosis and a FAK inhibitor defactinib for inhibiting stroma formation, and achieves sustained release of the drugs for up to 12 days. With only a single intratumoral injection, the combination treatment with erastin and defactinib produces further anti-tumor performance both in xenograft and KrasG12D-engineered primary PDAC mice and synergistically promotes the infiltration of CD8+ cytotoxic T cells and the reduction of type II macrophages. The findings may provide a novel promising strategy for the clinical treatment of PDAC.

Early signs of efficacy in patients with anti-PD-1 naïve and anti-PD-1 resistant HNSCC treated with NBTXR3/SBRT in combination with nivolumab or pembrolizumab in the phase I trial Study 1100.

6035 Background: Overcoming resistance to immune checkpoint inhibitors (ICIs) is a major clinical challenge. NBTXR3, a novel radioenhancer composed of functionalized hafnium oxide nanoparticles administered by a single intratumoral injection, locally amplifies radiotherapy (RT) dose without adding toxicity to surrounding healthy tissue. NBTXR3/RT enhances tumor cell death, tumor antigen release, and effectively expands T-cell repertoire in preclinical models, thus potentially triggering local and systemic immune responses to help improve ICI treatment. NBTXR3 has demonstrated favorable local control in pts with LA-HNSCC. Here we report early outcomes from the ongoing Study 1100 trial in pts with locoregionally recurrent (LRR) or metastatic (M+) head & neck squamous cell carcinoma (HNSCC) who were either naïve to or who continued anti-PD-1 therapy combined with NBTRX3/RT after primary or secondary resistance to anti-PD-1 alone. Methods: A phase I dose escalation/expansion trial [NCT03589339] evaluating NBTXR3/Stereotactic Body RT (SBRT) followed by nivolumab or pembrolizumab in 3 cohorts of pts with advanced solid tumors. Pts have H&N lesions either resistant to prior ICI or naïve, or lung, liver, or soft tissue metastases from ICI-resistant advanced solid tumors. SBRT doses were: HN 35 Gy/ 5 fxns; lung 45 Gy/ 5 fxns; liver 45 Gy/ 3 fxns; soft tissue as per investigator. Primary objective is safety of NBTXR3/RT/anti-PD-1 combination and its RP2D. Secondary objectives include efficacy. Results: The RP2D was 33%. There were 55 pts with HNSCC treated in the dose escalation/expansion parts. NBTXR3 injection was feasible and safe. There were 11pts (20%) who experienced G≥3 treatment related AEs, of which 3 pts experienced G≥3 AEs (5.5%) related to NBTXR3. Preliminary results of 33 pts, who were evaluable for efficacy: are reported. 6 pts with LRR disease: (2 ICI resistant, 4 naïve);. 27 pts with M+ disease (18 ICI resistant, 9 naïve). In ICI-resistant pts, objective tumor responses were observed in 25% (5/20), and the disease control rate (DCR) was 75% (15/20). In the ICI-naïve population, objective tumor responses were observed in 54% (7/13) of pts and DCR was 77% (10/13). There were observed improvements in injected lesions as well as in some noninjected target lesions. Updated efficacy data including OS in resistant and naïve pts will be presented. Conclusions: NBTXR3/RT/anti-PD-1 combination was feasible and well tolerated. Early data suggest the addition of NBTXR3/RT to ICI showed evidence of efficacy, including in patients who failed prior ICI, opening up an opportunity to further explore the ability of NBTXR3/RT to mitigate primary or secondary resistance to ICI. Clinical trial information: NCT03589339 .

Matrix Metalloproteinase- and pH-Sensitive Nanoparticle System Enhances Drug Retention and Penetration in Glioblastoma.

Glioblastoma (GBM) is a primary malignant brain tumor with limited therapeutic options. One promising approach is local drug delivery, but the efficacy is hindered by limited diffusion and retention. To address this, we synthesized and developed a dual-sensitive nanoparticle (Dual-NP) system, formed between a dendrimer and dextran NPs, bound by a dual-sensitive [matrix metalloproteinase (MMP) and pH] linker designed to disassemble rapidly in the tumor microenvironment. The disassembly prompts the in situ formation of nanogels via a Schiff base reaction, prolonging Dual-NP retention and releasing small doxorubicin (Dox)-conjugated dendrimer NPs over time. The Dual-NPs were able to penetrate deep into 3D spheroid models and detected at the tumor site up to 6 days after a single intratumoral injection in an orthotopic mouse model of GBM. The prolonged presence of Dual-NPs in the tumor tissue resulted in a significant delay in tumor growth and an overall increase in survival compared to untreated or Dox-conjugated dendrimer NPs alone. This Dual-NP system has the potential to deliver a range of therapeutics for efficiently treating GBM and other solid tumors.

Tumor-Tailored Ionizable Lipid Nanoparticles Facilitate IL-12 Circular RNA Delivery for Enhanced Lung Cancer Immunotherapy.

The advancement of message RNA (mRNA) -based immunotherapies for cancer is highly dependent on the effective delivery of RNA (Ribonucleic) payloads using ionizable lipid nanoparticles (LNPs). However, the clinical application of these therapies is hindered by variable mRNA expression among different cancer types and the risk of systemic toxicity. The transient expression profile of mRNA further complicates this issue, necessitating frequent dosing and thus increasing the potential for adverse effects. Addressing these challenges, a high-throughput combinatorial method is utilized to synthesize and screen LNPs that efficiently deliver circular RNA (circRNA) to lung tumors. The lead LNP, H1L1A1B3, demonstrates a fourfold increase in circRNA transfection efficiency in lung cancer cells over ALC-0315, the industry-standard LNPs, while providing potent immune activation. A single intratumoral injection of H1L1A1B3 LNPs, loaded with circRNA encoding interleukin-12 (IL-12), induces a robust immune response in a Lewis lung carcinoma model, leading to marked tumor regression. Immunological profiling of treated tumors reveals substantial increments in CD45+ leukocytes and enhances infiltration of CD8+ T cells, underscoring the ability of H1L1A1B3 LNPs to modulate the tumor microenvironment favorably. These results highlight the potential of tailored LNP platforms to advance RNA drug delivery for cancer therapy, broadening the prospects for RNA immunotherapeutics.

Abstract LB024: Permeation enhancer is necessary for the optimized PRV131 mediated cisplatin intratumoral delivery

Abstract PRV131 is an effective and safe polymeric nanoparticle suspension for intratumoral cisplatin delivery. Background: Systemic administration of platinum-based chemotherapy often leads to dose-limiting toxicity, affecting quality of life. While intratumoral delivery may reduce or eliminate the systemic toxicities and adverse side effects associated with intravenous cisplatin chemotherapy, this approach is not commonly adopted, in part due to the difficulty to obtain high drug retention in the tumor and control sustained drug release. PRV131 is a highly-concentrated cisplatin polymeric nanoparticle suspension carefully designed for intratumoral administration. The goal of this study was to assess the safety and efficacy of PRV131 as a monotherapy and in combination with a checkpoint inhibitor in a well-established preclinical murine colorectal adenocarcinoma model. Experimental Methods: MC38 cells were inoculated subcutaneously into the flanks of C57BL/6J female mice and allowed to grow to an average size of 100 mm3. Mice were then randomized into 6 groups of n=10: control (saline injection), 100 µg of cisplatin solution, 400 µg of cisplatin solution, 400 µg of PRV131, 400 µg of PRV131 + 200 µg systemic anti-PD1, and 200 µg systemic anti-PD1. A single injection of PRV131 or cisplatin solution was administered directly into the tumor on Day 1. Intraperitoneal anti-PD-1 was administered on Day 1 and Day 10. Mice were then monitored for efficacy (tumor measurements) and safety (overall health, weight loss and mortality). The durability of a single PRV131 injection was also studied. Efficacy and safety were adjudicated at 28 days post treatment and treatment durability assessment extended through 60 days post inoculation. Results: Our data demonstrates that PRV131 400 µg was significantly more effective than cisplatin solution at an equivalent dose. 80% of PRV131 treated mice survived to day 60 post inoculation, and no detectable tumor was found in 60% of PRV131 treated mice 38 days post administration. The safety profile was acceptable, with no mouse lost more than 20% body weight. Notably, efficacy profile of PRV131 monotherapy did not differ substantially from the addition of anti-PD-1 to the treatment, suggesting a high translational relevance of our drug delivery platform. Conclusion: A single intratumoral injection of PRV131 is well-tolerated in C57BL/6J female mice and elicits a durable antitumor response that lasts for over 30 days, laying the groundwork for its incorporation in clinical trial setting. Citation Format: Manijeh Goldberg, Aaron Manzi, Stefanie Cantin, Peter Conway, Jonathan Shikora, Adelaide McFarland, Matt Cayer, Lillyanna Dunn, Courtney Ball, Alexander T. Pearson, Ari J. Rosenberg, Nishant Agrawal, Evgeny Izumchenko. Permeation enhancer is necessary for the optimized PRV131 mediated cisplatin intratumoral delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB024.

Abstract LB440: PRV131 is an effective and safe polymeric nanoparticle suspension for intratumoral cisplatin delivery

Abstract Background: Systemic administration of platinum-based chemotherapy often leads to dose-limiting toxicity, affecting quality of life. While intratumoral delivery may reduce or eliminate the systemic toxicities and adverse side effects associated with intravenous cisplatin chemotherapy, this approach is not commonly adopted, in part due to the difficulty to obtain high drug retention in the tumor and control sustained drug release. PRV131 is a highly-concentrated cisplatin polymeric nanoparticle suspension carefully designed for intratumoral administration. The goal of this study was to assess the safety and efficacy of PRV131 as a monotherapy and in combination with a checkpoint inhibitor in a well-established preclinical murine colorectal adenocarcinoma model. Experimental Methods: MC38 cells were inoculated subcutaneously into the flanks of C57BL/6J female mice and allowed to grow to an average size of 100 mm3. Mice were then randomized into 6 groups of n=10: control (saline injection), 100 µg of cisplatin solution, 400 µg of cisplatin solution, 400 µg of PRV131, 400 µg of PRV131 + 200 µg systemic anti-PD1, and 200 µg systemic anti-PD1. A single injection of PRV131 or cisplatin solution was administered directly into the tumor on Day 1. Intraperitoneal anti-PD-1 was administered on Day 1 and Day 10. Mice were then monitored for efficacy (tumor measurements) and safety (overall health, weight loss and mortality). The durability of a single PRV131 injection was also studied. Efficacy and safety were adjudicated at 28 days post treatment and treatment durability assessment extended through 60 days post inoculation. Results: Our data demonstrates that PRV131 400 µg was significantly more effective than cisplatin solution at an equivalent dose. 80% of PRV131 treated mice survived to day 60 post inoculation, and no detectable tumor was found in 60% of PRV131 treated mice 38 days post administration. The safety profile was acceptable, with no mouse lost more than 20% body weight. Notably, efficacy profile of PRV131 monotherapy did not differ substantially from the addition of anti-PD-1 to the treatment, suggesting a high translational relevance of our drug delivery platform. Conclusion: A single intratumoral injection of PRV131 is well-tolerated in C57BL/6J female mice and elicits a durable antitumor response that lasts for over 30 days, laying the groundwork for its incorporation in clinical trial setting. Citation Format: Manijeh Goldberg, Aaron Manzi, Stefanie Cantin, Peter Conway, Jonathan Shikora, Adelaide McFarland, Matt Cayer, Lillyanna Dunn, Courtney Ball, Alexander T. Pearson, Ari J. Rosenberg, Nishant Agrawal, Evgeny Izumchenko. PRV131 is an effective and safe polymeric nanoparticle suspension for intratumoral cisplatin delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB440.

The NDV-MLS as an Immunotherapeutic Strategy for Breast Cancer: Proof of Concept in Female Companion Dogs with Spontaneous Mammary Cancer.

The absence of tumor-infiltrating lymphocytes negatively impacts the response to chemotherapy and prognosis in all subtypes of breast cancer. Therapies that stimulate a proinflammatory environment may help improve the response to standard treatments and also to immunotherapies such as checkpoint inhibitors. Newcastle disease virus (NDV) shows oncolytic activity, as well as immune modulating potential, in the treatment of breast cancer in vitro and in vivo; however, its potential to enhance tumor-infiltrating immune cells in breast cancer has yet to be evaluated. Since spontaneous canine mammary tumors represent a translational model of human breast cancer, we conducted this proof-of-concept study, which could provide a rationale for further investigating NDV-MLS as immunotherapy for mammary cancer. Six female companion dogs with spontaneous mammary cancer received a single intravenous and intratumoral injection of oncolytic NDV-MLS. Immune cell infiltrates were evaluated by histology and immunohistochemistry in the stromal, intratumoral, and peritumoral compartments on day 6 after viral administration. Increasing numbers of immune cells were documented post-viral treatment, mainly in the peritumoral compartment, where plasma cells and CD3+ and CD3-/CD79- lymphocytes predominated. Viral administration was well tolerated, with no significant adverse events. These findings support additional research on the use of NDV-MLS immunotherapy for mammary cancer.

Abstract B002: Phase I study of endoscopic ultrasound (EUS)-guided NBTXR3 delivery activated by radiotherapy (RT) for locally advanced or borderline resectable pancreatic cancer (LAPC or BRPC)

Abstract Patients with LAPC or BRPC often receive radiation therapy (RT) after chemotherapy (chemo) if no metastatic progression has occurred. Historically, CA19-9 normalizes in ~20 to 30% of patients after all therapy. While escalated dose radiotherapy (EDR) has shown promising survival in selected patients, extending use of EDR to all patients is challenging due to RT dose limitations of the bowel. To overcome this limitation, we are investigating NBTXR3, composed of functionalized hafnium oxide nanoparticles administered by a single intratumoral injection, locally amplifying the RT dose. In this phase 1 trial, the primary objective is to determine the NBTXR3 recommended phase 2 dose (RP2D). Secondary objectives are to measure anti-tumor effects. The study uses a Bayesian optimal interval design (BOIN) and has two parts: dose-finding (NBTXR3 at [level 1] 33% of gross tumor volume [GTV] or [level 2] at 42% of GTV) and cohort expansion at RP2D. In part 1, eligibility criteria included only LAPC patients with no evidence of metastatic disease after 2-6 months of chemo, part 2 allows BRPC. NBTXR3 was administered once prior to RT via an EUS-guided intratumoral injection. All patients received 45 Gy in 15 fractions with intensity modulated radiation therapy (IMRT). Patients were followed up to 1 year. Dose-limiting toxicities (DLTs) were defined as any grade ≥ 3 adverse events definitely or possibly related to NBTXR3 and/or RT. Target tumor response was per RECIST v1.1 criteria. CA19-9 was measured serially. We defined CA19-9 normalization as a value of 37 U/ml after finishing NBTXR3/RT. We compared rates of CA19-9 normalization in this phase 1 trial to a historical cohort of patients who received modern chemo/RT without NBTXR3. As of July 12, 2023, 15 LAPC patients have been treated with NBTXR3/RT. The median age was 63 years [range 43-81], 8 males, 7 females. The first patient (level 1) and subsequent 14 patients (level 2) had no injection complications. Level 2 had 1 non-serious DLT related to RT. At 4 weeks post RT, 13 had stable disease (SD) locally (2 had progressive disease [PD] overall), 1 had PD locally, and 1 had radiographic complete response (CR). At 3 months post RT, 11 had SD locally (1 PD overall), 1 PD locally, and 1 had surgery with negative margins and no residual viable tumor. 9 patients had elevated CA19-9 before NBTXR3/RT, 7 of these 9 (78%) had a decrease in CA19-9 subsequently, and 2 of these 9 (22%) had CA19-9 normalization eventually. 12 patients had elevated CA19-9 at diagnosis, and 5 out of 12 patients (42%) had normalization of CA19-9 after all therapy. We reviewed 455 patients with LAPC/BRPC between 2015-2019 who were treated at our institution as a comparison cohort for CA19-9 normalization rates. Out of 307 patients with elevated CA19-9 at diagnosis and normal bilirubin, 95 normalized (31%) and 212 did not normalize (69%) within 6 months of receiving cytotoxic therapy. The RP2D of NBTXR3 is 42% of GTV. The responses and CA19-9 results in comparison to historical data suggest promising anti-tumor efficacy of NBTXR3/RT. Citation Format: Gabriela Fuentes, Maria J. Rodriguez, Matthew H.G. Katz, Naruhiko Ikoma, Ching-Wei D. Tzeng, Michael Overman, Shubham Pant, Michael S. Lee, Robert A. Wolff, Milind Javle, Cullen M. Taniguchi, Emma B. Holliday, Ethan B. Ludmir, Prajnan Das, Albert C. Koong, Omar I. Vivar, Suyu Liu, Eric P. Tamm, Leonard A. Farber, Manoop S. Bhutani, Eugene J. Koay. Phase I study of endoscopic ultrasound (EUS)-guided NBTXR3 delivery activated by radiotherapy (RT) for locally advanced or borderline resectable pancreatic cancer (LAPC or BRPC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B002.

Oxygen‐Supplying Piezocatalytic Therapy of Hypoxic Tumors by Intratumoral Delivery of pH‐Responsive Multicompartmental Carriers with Sequential Drug Release Capability

AbstractPiezocatalytic cancer therapy, in which piezoelectric nanomaterials generate reactive oxygen species (ROS) via piezocatalytic redox reactions under mechanical stress, has emerged as an effective strategy for cancer treatment. However, the inherent hypoxia in tumor microenvironments enormously restricts its efficacy. To address this issue, acid‐degradable Janus‐type multicompartmental carriers able to separately encapsulate piezocatalytic gold nanoparticle‐coated poly(ethylene glycol)‐modified zinc oxide nanorods (Au@P‐ZnO NRs) and O2‐generating catalase (CAT) are fabricated in this study using stop‐flow lithography (SFL). The CAT and Au@P‐ZnO NRs are sequentially released by modulating the composition ratios of acid‐cleavable monomers in the precursor solution during the SFL. The sequential release by the Janus carriers significantly increased the intracellular ROS levels under hypoxia conditions upon ultrasound irradiation owing to the O2 supplied by the CAT. An in vivo study showed that a single intratumoral injection of Janus particles encapsulating the CAT and Au@P‐ZnO NRs efficiently alleviated tumor hypoxia and substantially suppressed tumor growth. This study demonstrates that pH‐responsive, O2‐generating, and piezocatalytic Janus carriers have high potential for piezocatalytic therapy of hypoxic tumors and offers insights into using pH‐responsive Janus carriers for efficient hypoxia‐relieving piezocatalytic cancer therapy via the cascade of oxygenation and ROS generation.

BETA prime: a first-in-man phase 1 study of AdAPT-001, an armed oncolytic adenovirus for solid tumors

AdAPT-001 is an oncolytic adenovirus (OAV) with a transforming growth factor beta (TGF-ß) trap, which neutralizes the immunosuppressive and profibrotic cytokine, TGF-ß. The aim or purpose of this phase 1 study was to assess the safety and tolerability and, secondarily, the efficacy of AdAPT-001 after single intratumoral injection (IT) (Part 1) and multidose IT injection (Part 2) in patients with superficially accessible, advanced refractory solid tumors. Part 1 enrolled 9 patients with a 3 + 3 single dose-escalation safety run-in involving 2.5 × 1011, 5.0 × 1011, 1.0 × 1012 viral particles (vps). No dose-limiting toxicities or treatment-related serious adverse events (SAEs) were seen. In Part 2, a dose-expansion phase, 19 patients received AdAPT-001 at 1.0 × 1012 vps until disease progression according to Response Evaluation Criteria in Solid Tumors or RECIST 1.1. The overall responses to treatment included confirmed partial responses (3), durable stable disease ≥ 6 months (5), and progressive disease (13). AdAPT-001 is well tolerated. Evidence of an anti-tumor effect was seen in both injected and uninjected lesions. The recommended Phase 2 dose was 1.0 × 1012 vp administered by intratumoral injection once every 2 weeks. Combination of AdAPT-001 with a checkpoint inhibition is enrolling.

IMMU-29. A CHIMERIC ONCOLYTIC ADENOVIRUS CIRCUMVENTS NEUTRALIZING ANTIBODIES DEVELOPED BY PATIENTS WITH RECURRENT GLIOBLASTOMA TREATED WITH DELTA-24-RGD

Abstract Oncolytic virotherapy holds great potential to treat high-grade gliomas by harnessing virus-mediated oncolysis to stimulate anti-tumor immune responses. However, the efficacy of oncolytic adenoviruses can be hampered by neutralizing antibodies. In this study, we examined sera obtained from a phase 1 clinical trial using oncolytic adenovirus Delta-24-RGD to treat patients with recurrent malignant gliomas. We found that a single intratumoral injection of Delta-24-RGD induced the development of neutralizing antibodies in less than half of the patients but demonstrated remarkable outcomes, with 20% of the patients surviving beyond three years. Conversely, multiple injections of the oncolytic adenovirus induced neutralizing antibodies in 80% of patients (P=0.05) but resulted in no long-term survivors. This inverse correlation led us to further investigate the role of neutralizing antibodies during glioma virotherapy. Examination of Delta-24-RGD-treated murine brain tumors using immunofluorescence displayed colocalization of antibodies and viral proteins. To counteract virus neutralization and maximize oncolysis, we engineered a chimeric virus, Delta-24-RGD-H43m, by replacing the hypervariable regions of the hexon protein from the prevalent serotype 5-based Delta-24-RGD with those from the rare serotype 43. The infectivity, replication, and oncolytic capabilities of Delta-24-RGD-H43m were similar to those of the parental virus. Using clinical trial patient sera, we observed that Delta-24-RGD-H43m displayed significant resilience against the inhibitory effects of neutralizing sera from patients (P=0.01). In murine models with pre-existing immunity to adenovirus, Delta-24-RGD-H43m treatment led to a significantly extended median survival and resulted in a higher percentage of long-term survivors than the parental virus (P=0.005). These results underscore the effectiveness of hexon swapping as a strategy to overcome the dominant immune response against oncolytic viruses, provide valuable insights into the interaction between oncolytic viruses and the host immune system, and lay the foundation to translate to the clinical setting immune-evading virotherapies for glioma patients.

Novel Radioenhancer NBTXR3 Activated by Radiotherapy in Cisplatin-Ineligible Locally Advanced HNSCC Patients: Final Results of a Phase I Trial

New approaches are needed for frail or elderly patients with locally advanced head and neck squamous cell carcinoma (LA HNSCC) who are unfit to receive cisplatin with concurrent radiotherapy (RT). NBTXR3 is a first-in-class radioenhancer, composed of functionalized hafnium oxide nanoparticles, administered by a single intratumoral (IT) injection and activated by RT. NBTXR3 locally amplifies the anti-tumoral response of RT without adding toxicity to surrounding healthy tissue as shown in a randomized trial in soft tissue sarcoma. This two-part study: dose-escalation followed by the dose-expansion part reported here, evaluated the safety and preliminary efficacy for NBTXR3 activated by RT in elderly or frail patients ineligible to cisplatin. This trial enrolled patients who had previously untreated AJCC 8th Stage III-IVA or T3, T4 SCC of the oral cavity or oropharynx (OPC) ineligible to cisplatin. Eligible patients received a single IT injection of NBTXR3 at the recommended dose (22% of the baseline tumor volume) followed by RT (IMRT 70 Gy in 35 fractions). The primary objectives of the dose expansion part were to test the recommended dose, to confirm its safety, and obtain preliminary evidence of efficacy. The secondary objectives included the evaluation of progression-free survival (PFS) and overall survival (OS). Fifty-sixpatients in the dose expansion part were treated from April 2019-January 2022; 44 patients were evaluable for objective tumor response. In the all-treated population, median age was 71.9 years. 64.3% had age-adjusted Charlson Comorbidity Index scores ≥4, 55.4% had OPC (45.2% HPV+) and 80% had T3-4. Median injected volume of NBTXR3 was 13.6 [0.5-57.1] mL. Grade ≥ 3 adverse events reported as potentially related to NBTXR3 or to injection procedure were 1.2% and 0.4% of all AEs reported, respectively. In the evaluable population, the best objective response rate of the NBTXR3 injected lesion was 81.8% with a complete response rate of 63.6%. The best overall response rate (injected and non-injected lesions) was 79.5%. Final analyses on PFS and OS with long-term follow-up will be presented. NBTXR3 IT injection followed by activation with RT was confirmed to be feasible and well tolerated in elderly or frail patients with LA HNSSC and significant comorbidities. The high rate of best overall response suggests that NBTXR3+RT is effective in this elderly population ineligible to cisplatin with a high unmet medical need. These results support our ongoing phase III study comparing NBTXR3/RT ± cetuximab vs. RT ± cetuximab in platinum-based chemotherapy ineligible elderly patients with LA-HNSCC: NANORAY 312 (NCIT04892173).

Abstract PR05: Changing the radiation and immune-oncology paradigm in patients with head and neck squamous cell carcinoma (HNSCC) with the radioenhancer NBTXR3: From bench to bedside

Abstract Purpose/Objectives: Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, yet most patients (pts) ultimately develop ICI resistance. Overcoming this resistance is a major clinical challenge. Radiation therapy (RT) has emerged as a promising combination with ICIs since in some settings it may produce an immunomodulatory effect. NBTXR3, a novel radioenhancer composed of functionalized hafnium oxide nanoparticles, is administered by a single intratumoral injection. NBTXR3 increases RT energy deposition inside tumor cells, subsequent tumor cell death, and tumor antigen release and thus in combination with ICI may overcome resistance. Materials/Methods: Pre-Clinical: Abscopal assays were conducted in immunocompetent mice. Anti-PD-1 sensitive or resistant lung tumor cell lines were injected in both legs. Intratumoral NBTXR3 injection (or vehicle) followed by RT was performed in right leg (primary) tumors only. Some mice also received anti-PD-1 injections alone or in combination with anti-LAG3 and anti-TIGIT. Tumor growth was monitored, and immune cell infiltrates analyzed by IHC. Clinical: A multicenter phase I trial [NCT01946867] is evaluating NBTXR3/RT in pts with AJCC 8th stage III-IVA or T3, T4 SCC of the oral cavity or oropharynx ineligible to cisplatin. Pts received NBTXR3 at the recommended dose (22% of baseline tumor volume) followed by RT (IMRT 70 Gy in 35 fractions). A multicenter phase I trial [NCT03589339] is evaluating NBTXR3/RT/anti-PD-1 in 3 cohorts of pts with advanced solid tumors eligible for anti-PD-1: 1) Locoregional recurrent or recurrent and metastatic HNSCC, 2) Lung metastases, or 3) Liver metastases. NBTXR3 was administered by intratumoral injection. Stereotactic Body RT (SBRT) was delivered at tumor site-specific doses per standard practice. Results: Pre-clinical studies demonstrated that NBTXR3/RT induces an immune response not observed with RT alone and enhances systemic control. IHC showed significant increase of CD8+ T-cell infiltrates in both NBTXR3/RT-treated and untreated tumors. NBTXR3/RT/anti-PD-1 improved local and systemic control in mice bearing anti-PD-1 resistant tumors, produced long-term memory, and reduced spontaneous lung metastases. NBTXR3/RT/anti-PD-1/anti-LAG3/anti-TIGIT improved tumor control, extended survival and produced a robust long-term memory anti-tumor immune response. Clinically, NBTXR3 intratumoral injection was feasible and safe. Preliminary signs of efficacy observed in pts with locally advanced HNSCC treated with NBTXR3/RT will be presented. Preliminary signs of efficacy were observed in pts treated with NBTXR3/RT/anti-PD-1 including in pts resistant to anti-PD-1, as well as in remote non-irradiated lesions. Responses in pts with HNSCC will be presented. Conclusions: With demonstrated radioenhancement as well as immune system priming, NBTXR3 positions itself to be a paradigm shift catalyst in the treatment of pts with head and neck cancers. These data support further development of NBTXR3/RT alone as well as in combination with anti-PD-1 and/or other ICIs. Citation Format: Colette J. Shen, Christophe Le Tourneau, Zoltán Takacsi-Nagy, Xavier Liem, Jessica Frakes, Jiaxin Niu, Jared Weiss, Jimmy Caudell, Yun Hu, James W. Welsh, Sebastien Paris, Anais Debard, Patricia Said, Pavel Tyan, Tanguy Y. Seiwert, Zsuzsanna Papai, Omar I. Vivar, Leonard A. Farber, Ari Rosenberg. Changing the radiation and immune-oncology paradigm in patients with head and neck squamous cell carcinoma (HNSCC) with the radioenhancer NBTXR3: From bench to bedside [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR05.

P17.15.A THANK PHASE I TRIAL OF DNX-2440 ONCOLYTIC ADENOVIRUS IN PATIENTS WITH FIRST OR SECOND RECURRENCE OF GLIOBLASTOMA: PRELIMINARY RESULTS

Abstract BACKGROUND Oncolytic immunovirotherapy is emerging as a potential therapeutic approach in neuro-oncology. The oncolytic adenovirus DNX-2401, genetically engineered for selective replication and enhanced infectivity in tumor cells, has shown efficacy in adult patients with recurrent glioblastoma (GBM). In order to overcome the immunosuppressive brain-tumor microenvironment and thus enhance its antiglioma effect, DNX-2440 is a new armed virus which contains the same modifications but additionally incorporates OX40 ligand to induce the expression of such molecule in tumor cells and so increase the antitumor immune response. MATERIAL AND METHODS We are conducting a phase I open-label single-center study to investigate the safety and efficacy of a single intratumoral injection of DNX-2440 in patients with first or second recurrence of GBM. This is a dose escalation study, with a 3 + 3 design, evaluating an initial dose of 4x10e9 viral particles (vp) and a target dose of 4x10e10 vp. A single measurable lesion bigger than 10 mm in two perpendicular diameters and smaller than 25 cc is required for inclusion in the study. RESULTS To date 16 patients (6 [37.5%] female, 10 [62.5%] male) have been enrolled and treated with DNX-2401. Median age is 55 years (range, 39-80) and median KPS is 80 (range 70-90). As per protocol, surgery consisted of biopsy in all patients followed by intratumoral injection of DNX-2440. No dose limiting toxicity has been registered during the dose escalation phase (first cohort of 3 patients receiving 4x10e9 vp, and second cohort of 3 patients receiving 4x10e10 vp), and the remainder patients have received the target dose of 4x10e10 vp in the dose expansion cohort. Safety profile is acceptable with no serious adverse events related to DNX-2440 reported to date. Clinical benefit with tumor size reduction and prolonged survival have already been confirmed in 3 patients. Correlative analyses evaluating immune response in blood samples are ongoing. CONCLUSION Our current data suggest that intratumoral injection of DNX-2440 is feasible and safe, and seems to provide a clinical benefit in some patients with recurrent GBM. Furthermore, this trial illustrates the potential of oncolytic virus platforms to reshape the brain-tumor microenvironment and its interesting role in combination strategies with other immune-oncology agents for treating this disease.

Improvement of the Effectiveness of HER2+ Cancer Therapy by Use of Doxorubicin and Trastuzumab Modified Radioactive Gold Nanoparticles.

In the present article, we describe a multimodal radiobioconjugate that contains a chemotherapeutic agent (doxorubicin, DOX), a β-emitter (198Au), and a guiding vector (trastuzumab, Tmab) for targeted therapy of cancers overexpressing HER2 receptors. To achieve this goal, radioactive gold nanoparticles (198AuNPs) with a mean diameter of 30 nm were synthesized and coated with a poly(ethylene glycol) (PEG) linker conjugated to DOX and monoclonal antibody (Tmab) via peptide bond formation. In vitro experiments demonstrated a high affinity of the radiobioconjugate to HER2 receptors and cell internalization. Cytotoxicity experiments performed using the MTS assay showed a significant decrease in the viability of SKOV-3 cells. A synergistic cytotoxic effect due to the simultaneous presence of DOX and 198Au was revealed after 48 h of treatment with 2.5 MBq/mL. Flow cytometry analysis indicated that DOX-198AuNPs-Tmab mainly induced cell cycle arrest in the G2/M phase and late apoptosis. Dose-dependent additive and synergistic effects of the radiobioconjugate were also shown in spheroid models. Ex vivo biodistribution experiments were performed in SKOV-3 tumor-bearing mice, investigating different distributions of the 198AuNPs-DOX and DOX-198AuNPs-Tmab after intravenous (i.v.) and intratumoral (i.t.) administration. Finally, in vivo therapeutic efficacy studies on the same animal model demonstrated very promising results, as they showed a significant tumor growth arrest up to 28 days following a single intratumoral injection of 10 MBq. Therefore, the proposed multimodal radiobioconjugate shows great potential for the local treatment of HER2+ cancers.

Combination of PD-1 Checkpoint Blockade and Botulinum Toxin Type A1 Improves Antitumor Responses in Mouse Tumor Models of Melanoma and Colon Carcinoma

ABSTRACT Background Tumor innervation has been shown to be utilized by some solid cancers to support tumor initiation, growth, progression, and metastasis, as well as confer resistance to immune checkpoint blockade through suppression of antitumor immunologic responses. Since botulinum neurotoxin type A1 (BoNT/A1) blocks neuronal cholinergic signaling, its potential use as an anticancer drug in combination with anti-PD-1 therapy was investigated in four different syngeneic mouse tumor models. Methods Mice implanted with breast (4T1), lung (LLC1), colon (MC38), and melanoma (B16-F10) tumors were administered a single intratumoral injection of 15 U/kg BoNT/A1, repeated intraperitoneal injections of 5 mg/kg anti-PD-1 (RMP1-14), or both. Results Compared to the single-agent treatments, anti-PD-1 and BoNT/A1 combination treatment elicited significant reduction in tumor growth among B16-F10 and MC38 tumor-bearing mice. The combination treatment also lowered serum exosome levels in these mice compared to the placebo control group. In the B16-F10 syngeneic mouse tumor model, anti-PD-1 + BoNT/A1 combination treatment lowered the proportion of MDSCs, negated the increased proportion of Treg cells, and elicited a higher number of tumor-infiltrating CD4+ and CD8+ T lymphocytes into the tumor microenvironment compared to anti-PD-1 treatment alone. Conclusion Our findings demonstrate the synergistic antitumor effects of BoNT/A1 and PD-1 checkpoint blockade in mouse tumor models of melanoma and colon carcinoma. These findings provide some evidence on the potential application of BoNT/A1 as an anticancer drug in combination with immune checkpoint blockade and should be further explored.