Replication-competent Adenovirus Research Articles

P53-armed oncolytic virotherapy induces abscopal effect in osteosarcoma by promoting immunogenic cell death

Osteosarcoma (OS), the most frequent primary malignant tumor of bone in children and adolescents, is refractory to immune checkpoint inhibitors due to poor antitumor immune response. Chemotherapy and virotherapy induce immunogenic cell death (ICD) and antitumor immune responses, leading to the abscopal effect in untreated tumors. We previously demonstrated the antitumor activity of the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 in human OS cells. Here, we show the therapeutic potential of chemotherapeutic drugs (doxorubicin, cisplatin) and telomerase-specific oncolytic adenoviruses (OBP-301, p53-armed OBP-702) to induce ICD in human OS cells (U2OS, MNNG/HOS, SaOS-2) and murine OS cells (NHOS). OBP-702 induced more profound ICD via secretion of adenosine triphosphate (ATP) and high-mobility group box protein B1 (HMGB1) compared with chemotherapy and OBP-301 in human OS cells. Murine NHOS cells were also more sensitive to OBP-702 than OBP-301. Subcutaneous NHOS tumor models demonstrated that intratumoral injection of OBP-702 significantly increased the tumor infiltration of cytotoxic CD8+ T cells and induced the abscopal effect against non-treated tumors compared with OBP-301. Our results suggest that OBP-702 is a promising antitumor reagent to induce ICD with secretion of ATP and HMGB1 and the abscopal effect against OS.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

P53-Armed Oncolytic Virotherapy Improves Radiosensitivity in Soft-Tissue Sarcoma by Suppressing BCL-xL Expression.

Soft-tissue sarcoma (STS) is a heterogeneous group of rare tumors originating predominantly from the embryonic mesoderm. Despite the development of combined modalities including radiotherapy, STSs are often refractory to antitumor modalities, and novel strategies that improve the prognosis of STS patients are needed. We previously demonstrated the therapeutic potential of two telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in human STS cells. Here, we demonstrate in vitro and in vivo antitumor effects of OBP-702 in combination with ionizing radiation against human STS cells (HT1080, NMS-2, SYO-1). OBP-702 synergistically promoted the antitumor effect of ionizing radiation in the STS cells by suppressing the expression of B-cell lymphoma-X large (BCL-xL) and enhancing ionizing radiation-induced apoptosis. The in vivo experiments demonstrated that this combination therapy significantly suppressed STS tumors' growth. Our results suggest that OBP-702 is a promising antitumor reagent for promoting the radiosensitivity of STS tumors.

Antiviral activity of adenoviral vector expressing human interferon lambda-4 against influenza virus.

Interferon lambda (IFNλ), classified as a type III IFN, is a representative cytokine that plays an important role in innate immunity along with type I IFN. IFNλ can elicit antiviral states by inducing peculiar sets of IFN-stimulated genes (ISGs). In this study, an adenoviral vector expression system with a tetracycline operator system was used to express human IFNλ4 in cells and mice. The formation of recombinant adenovirus (rAd-huIFNλ4) was confirmed using immunohistochemistry assays and transmission electron microscopy. Its purity was verified by quantifying host cell DNA and host cell proteins, as well as by confirming the absence of the replication-competent adenovirus. The transduction of rAd-huIFNλ4 induced ISGs and inhibited four subtypes of the influenza virus in both mouse-derived (LA-4) and human-derived cells (A549). The antiviral state was confirmed in BALB/c mice following intranasal inoculation with 109 PFU of rAd-huIFNλ4, which led to the inhibition of four subtypes of the influenza virus in mouse lungs, with reduced inflammatory lesions. These results imply that human IFNλ4 could induce antiviral status by modulating ISG expression in mice.

Fluorescence-guided assessment of bone and soft-tissue sarcomas for predicting the efficacy of telomerase-specific oncolytic adenovirus.

Bone and soft-tissue sarcomas are rare malignancies with histological diversity and tumor heterogeneity, leading to the lack of a common molecular target. Telomerase is a key enzyme for keeping the telomere length and human telomerase reverse transcriptase (hTERT) expression is often activated in most human cancers, including bone and soft-tissue sarcomas. For targeting of telomerase-positive tumor cells, we developed OBP-301, a telomerase-specific replication-competent oncolytic adenovirus, in which the hTERT promoter regulates adenoviral E1 gene for tumor-specific viral replication. In this study, we present the diagnostic potential of green fluorescent protein (GFP)-expressing oncolytic adenovirus OBP-401 for assessing virotherapy sensitivity using bone and soft-tissue sarcomas. OBP-401-mediated GFP expression was significantly associated with the therapeutic efficacy of OBP-401 in human bone and soft-tissue sarcomas. In the tumor specimens from 68 patients, malignant and intermediate tumors demonstrated significantly higher expression levels of coxsackie and adenovirus receptor (CAR) and hTERT than benign tumors. OBP-401-mediated GFP expression was significantly increased in malignant and intermediate tumors with high expression levels of CAR and hTERT between 24 and 48 h after infection. Our results suggest that the OBP-401-based GFP expression system is a useful tool for predicting the therapeutic efficacy of oncolytic virotherapy on bone and soft-tissue sarcomas.

Initial safety assessment of the endoscopically injected oncolytic virus OBP-301 in medically inoperable esophageal cancer: NRG-GI007.

341 Background: Definitive chemoradiation (CRT) is a standard-of-care for patients (Pts) with medically inoperable esophageal cancer (EC). The NRG/RTOG 0436 study of cisplatin/paclitaxel and RT (+/- cetuximab) as definitive therapy showed that about half of Pts have locally persistent disease. OBP-301 is a conditionally restricted, replication-competent adenovirus derived from human adenovirus type 5 that adds a human Telomerase Reverse Transcriptase (hTERT) gene promoter, replicating only in tumor cells to cause lysis. It may cause immunogenic cell death, enhance RT, and improve local control. OBP-301 was previously studied with RT in Japanese esophageal cancer Pts and shown to be safe and has promising activity.1 Methods: In NRG-GI007, a phase 1 study (NCT04391049), OBP-301 is added to weekly carboplatin/paclitaxel and RT (50.4 Gy/28 fractions) for Pts with medically inoperable EC. Pts receive 1-2mL of intratumoral OBP-301 1×1012virus particles/ml via endoscopy 3 days prior to and then at days 12 and 26 of CRT. The primary endpoint is dose-limiting toxicity (DLT), defined as adverse events (AEs, by CTCAE v5) definitely or probably attributed to OBP-301 that meet either of the following: (1) leading to a >14-day cumulative delay in CRT or (2) any grade ≥ 3 AE EXCEPT: grade 3 nausea/vomiting, grade 3 esophagitis or dehydration, first occurrence of grade 3/4 neutropenia, or grade 3/4 lymphopenia. Initially, 6 evaluable Pts (eligible and started protocol treatment) are enrolled. If protocol defined DLT occurs in ≤1 of 6 Pts, the dose will be deemed safe and an expansion cohort of 9 more will be enrolled to further assess safety and obtain a preliminary assessment of clinical complete response. If ≥2 Pts have a DLT then one de-escalated OBP-301 dose will be assessed. Results: From June 2020 to April 2023, 6 evaluable Pts were enrolled. Median age was 73.5 years, all male. Five Pts had adenocarcinoma and 1 had squamous cell carcinoma; 3 Pts had node-positive disease. All Pts received all planned OBP-301 injections, along with 50.4 Gy RT. Four out of 6 Pts received all 5 weekly doses of chemo for > 85% of planned total dose, and 2 got 4 weekly doses for > 70% of planned total dose. No Pt experienced ³7 day treatment delay. The following treatment-related (attributed as definitely, probably, or possibly related to CRT and/or OBP-301) grade 3 AEs were reported across 5 Pts: decreased neutrophil count (3 Pts), decreased lymphocyte count (2 Pts), decreased WBC (2 Pts), and fever and fatigue (1 Pt each). There were 4 Pts with AEs reported as definitely or probably related to OBP-301, all grade ≤2. No DLT occurred. Conclusions: No DLTs in the 6 evaluable Pts were observed and it is concluded that the initial OBP-301 dose level is safe. NRG-GI007 was reopened on August 7, 2023 to the expansion cohort. Full toxicity and treatment data will be presented. 1. Eur J Cancer 2021;153:98. Clinical trial information: NCT04391049 .

Construction of recombinant adenovirus-5 vector to prevent replication-competent adenovirus occurrence

Human adenovirus-5 (hAd5) is an important gene delivery vector, which has been widely used in various fields of biomedicine, such as gene therapy, cancer therapy, and vaccine development. However, replication-competent adenovirus (RCA) generated when adenoviral vectors are prepared in HEK293 cells has remained a concern. In this study, the human adenovirus-5 was modified to shorten the length of homologous sequence between the adenovirus and HEK293 genomic DNA, thereby reducing the production of RCA. The recombinant hAd5 was amplified and serially passaged 12 times in HEK293 cells. The amounts of RCA at passage 2, 4, 6, 8, 10, and 12 were detected by quantitative real-time PCR. The results demonstrated that the modification of adenoviral vector could effectively reduce the production of RCA during serial passages in HEK293 cells.

A New HEK293 Cell with CR2 Region of E1A Gene Deletion Prevents the Emergence of Replication-Competent Adenovirus.

To eliminate the contaminants of Replication-Competent Adenovirus (RCA) during high titer recombinant oncolytic adenovirus production. At first, we detected E1A copy numbers of different sources of 293 cells using Q-PCR, and we screened a subclone JH293-C21 of the JH293 cell line (purchased from ATCC) with lower early region 1A (E1A) copy numbers and higher adenovirus production ability. Then, we deleted the conserved region (CR)2 of the E1A gene in this subclone using the CRISPR-Cas9 system and obtained a stable cell clone JH293-C21-C14 with lower E1A expression, but the RCA formation had no significant reduction. Then, we further deleted the CR2 of JH293-C21-C14 cells with the CRISPR-Cas9 system and obtained a strain of cells named JH293-C21-C14-C28. Finally, we detected the capacity for cell proliferation, adenovirus production, and RCA formation in the production of recombinant adenovirus. The JH293-C21-C14-C28 cells had a similar cell proliferation ability and human adenovirus production as JH293-C21 cells. Most importantly, RCA production in JH293-C21-C14-C28 cells was lower than in JH293-C21 cells. Human adenovirus producer cell clone JH293-C21-C14-C28 with CR2 deletion can effectively prevent the RCA production of replication-competent oncolytic adenovirus; this will provide significant advantages in utility and safety in gene therapy.

Adenoviral Gene Therapy Vectors in Clinical Use-Basic Aspects with a Special Reference to Replication-Competent Adenovirus Formation and Its Impact on Clinical Safety.

Adenoviral vectors are commonly used in clinical gene therapy. Apart from oncolytic adenoviruses, vector replication is highly undesired as it may pose a safety risk for the treated patient. Thus, careful monitoring for the formation of replication-competent adenoviruses (RCA) during vector manufacturing is required. To render adenoviruses replication deficient, their genomic E1 region is deleted. However, it has been known for a long time that during their propagation, some viruses will regain their replication capability by recombination in production cells, most commonly HEK293. Recently developed RCA assays have revealed that many clinical batches contain more RCA than previously assumed and allowed by regulatory authorities. The clinical significance of the higher RCA content has yet to be thoroughly evaluated. In this review, we summarize the biology of adenovirus vectors, their manufacturing methods, and the origins of RCA formed during HEK293-based vector production. Lastly, we share our experience using minimally RCA-positive serotype 5 adenoviral vectors based on observations from our clinical cardiovascular gene therapy studies.

P53-armed oncolytic adenovirus induces autophagy and apoptosis in KRAS and BRAF-mutant colorectal cancer cells.

Colorectal cancer (CRC) cells harboring KRAS or BRAF mutations show a more-malignant phenotype than cells with wild-type KRAS and BRAF. KRAS/BRAF-wild-type CRCs are sensitive to epidermal growth factor receptor (EGFR)-targeting agents, whereas KRAS/BRAF-mutant CRCs are resistant due to constitutive activation of the EGFR-downstream KRAS/BRAF signaling pathway. Novel therapeutic strategies to treat KRAS/BRAF mutant CRC cells are thus needed. We recently demonstrated that the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 exhibit therapeutic potential against KRAS-mutant human pancreatic cancer cells. In this study, we evaluated the therapeutic potential of OBP-301 and OBP-702 against human CRC cells with differing KRAS/BRAF status. Human CRC cells with wild-type KRAS/BRAF (SW48, Colo320DM, CACO-2), mutant KRAS (DLD-1, SW620, HCT116), and mutant BRAF (RKO, HT29, COLO205) were used in this study. The antitumor effect of OBP-301 and OBP-702 against CRC cells was analyzed using the XTT assay. Virus-mediated modulation of apoptosis, autophagy, and the EGFR-MEK-ERK and AKT-mTOR signaling pathways was analyzed by Western blotting. Wild-type and KRAS-mutant CRC cells were sensitive to OBP-301 and OBP-702, whereas BRAF-mutant CRC cells were sensitive to OBP-702 but resistant to OBP-301. Western blot analysis demonstrated that OBP-301 induced autophagy and that OBP-702 induced autophagy and apoptosis in human CRC cells. In BRAF-mutant CRC cells, OBP-301 and OBP-702 suppressed the expression of EGFR, MEK, ERK, and AKT proteins, whereas mTOR expression was suppressed only by OBP-702. Our results suggest that p53-armed oncolytic virotherapy is a viable therapeutic option for treating KRAS/BRAF-mutant CRC cells via induction of autophagy and apoptosis.

A Method to Generate and Rescue Recombinant Adenovirus Devoid of Replication-Competent Particles in Animal-Origin-Free Culture Medium.

Adenoviruses are promising vectors for vaccine production and gene therapy. Despite all the efforts in removing animal-derived components such as fetal bovine serum (FBS) during the production of adenovirus vector (AdV), FBS is still frequently employed in the early stages of production. Conventionally, first-generation AdVs (E1 deleted) are generated in different variants of adherent HEK293 cells, and plaque purification (if needed) is performed in adherent cell lines in the presence of FBS. In this study, we generated an AdV stock in SF-BMAdR (A549 cells adapted to suspension culture in serum-free medium). We also developed a limiting dilution method using the same cell line to replace the plaque purification assay. By combining these two technologies, we were able to completely remove the need for FBS from the process of generating and producing AdVs. In addition, we demonstrated that the purified AdV stock is free of any replication-competent adenovirus (RCA). Furthermore, we demonstrated that our limiting dilution method could effectively rescue an AdV from a stock that is highly contaminated with RCA.

Protease-deleted adenovirus as an alternative for replication-competent adenovirus vector

For cancer therapy and vaccination an amplified expression of the therapeutic gene is desired. Previously, we have developed a single-cycle adenovirus vector (SC-AdV) by deleting the adenovirus protease (PS) gene. In order to keep the E1 region intact within the PS-deleted adenoviruses, we examined the insertion of two transgenes under the control of a constitutive or inducible promoters. These were inserted between E4 and the right inverted terminal repeat in a wide variety of backbones with various combinations of PS, E3 and E4 deletion. Our data showed that PS-deleted adenoviruses, expressed transgenes as strongly as replication-competent AdVs in HEK293A and a variant of HeLa cells. In a head-to-head comparison in four human cell lines, we demonstrated that SC-AdV, was comparable for transgene expression efficacy with its replication-competent counterpart. However, the SC-AdV expresses its transgene 10 to 16,000 times higher than its replication-defective counterpart.

Viral Shedding in Mice following Intravenous Adenovirus Injection: Impact on Biosafety Classification.

There have been numerous advances in gene therapy and oncolytic virotherapy in recent years, especially with respect to cutting-edge animal models to test these novel therapeutics. With all of these advances, it is important to understand the biosafety risks of testing these vectors in animals. We performed adenovirus-based viral shedding studies in murine models to ascertain when it is appropriate to downgrade the animals from Biosafety Level (BSL) 2 to BSL 1 for experimental handling and transport. We utilized intravenous injections of a replication-competent adenovirus and analyzed viral shedding via the collection of buccal and dermal swabs from each animal, in addition to obtaining urine and stool samples. The adenovirus hexon copy number was determined by qPCR, and plaque formation was analyzed to assess the biologic activity of viral particles. Our results demonstrate that after 72 h following viral inoculation, there is no significant quantity of biologically active virus shedding from the animals. This observation suggests that on day 4 following adenovirus injection, mice can be safely downgraded to BSL 1 for the remainder of the experiment with no concern for hazardous exposure to laboratory personnel.

Phase Ib neoadjuvant CG0070 and nivolumab (N) for cisplatin (C)-ineligible muscle invasive bladder cancer (MIBC).

e16613 Background: CG0070 is a replication-competent oncolytic adenovirus engineered to target RB deficient tumor cells and to express GM-CSF. CG0070 has previously demonstrated safety and efficacy against BCG-exposed high risk non-muscle invasive bladder cancer through tumor lysis and anti-tumor immune activation. We tested the safety and efficacy of CG0070 in combination with N as neoadjuvant therapy for MIBC in C-ineligible patients in this study (NCT04610671). Methods: C-ineligible pts with MIBC (cT2-4a, N≤1) were enrolled. Pts received 6 weekly intravesical CG0070 (1x1012vp) and 2 doses of N at wks 2 and 6, followed by radical cystectomy (RC). The primary objective is safety of CG0070+N as measured by CTCAEv5.0. Secondary objective is to assess pathologic response (PaR) (pT0N0) and 1yr event free survival (EFS). Exploratory objectives include the assessment of correlation between PaR with baseline 1) E2F expression; 2) immune infiltration; 3) PD-L1 expression; and 4) TLS expression. Results: Between Nov 2020 and Feb 2023, 21 pts were enrolled with median age 75yrs, 81% male, 33% > cT2; 1 pt refused RC but was included in the ITT population. Treatment was stopped after 1 infusion of CG0070 in 1 pt due to treatment unrelated worsening of renal function. The overall rate of >grade 3 AEs was 12/20 (60%), and the vast majority were related to RC (83%). Immune related AEs were seen in 1 pt, who had grade 2 autoimmune thyroiditis. There was no delay in time to RC and no unexpected surgical complications from treatment. Overall, 1 pt was unevaluable for efficacy, 2 pts progressed prior to RC, PaR was observed in 8/15 (53%) evaluable pts, and an additional pt had negative post-treatment biopsy but refused RC. On exploratory analysis, treatment response correlated with increased maturation of tertiary lymphoid structures and immunogenicity to bespoke tumor neoantigens. Conclusions: Neoadjuvant CG0070+N is safe and effective in C-ineligible pts with MIBC. This combination was well tolerated without any delays in RC and induced an overall response rate of 50%. Clinical trial information: NCT04610671 .

Generation and Characterization of a Replication-Competent Human Adenovirus Type 55 Encoding EGFP.

Human adenovirus 55 (HAdV-55) has recently caused outbreaks of acute respiratory disease (ARD), posing a significant public threat to civilians and military trainees. Efforts to develop antiviral inhibitors and quantify neutralizing antibodies require an experimental system to rapidly monitor viral infections, which can be achieved through the use of a plasmid that can produce an infectious virus. Here, we used a bacteria-mediated recombination approach to construct a full-length infectious cDNA clone, pAd55-FL, containing the whole genome of HadV-55. Then, the green fluorescent protein expression cassette was assembled into pAd55-FL to replace the E3 region to obtain a recombinant plasmid of pAd55-dE3-EGFP. The rescued recombinant virus rAdv55-dE3-EGFP is genetically stable and replicates similarly to the wild-type virus in cell culture. The virus rAdv55-dE3-EGFP can be used to quantify neutralizing antibody activity in sera samples, producing results in concordance with the cytopathic effect (CPE)-based microneutralization assay. Using an rAdv55-dE3-EGFP infection of A549 cells, we showed that the assay could be used for antiviral screening. Our findings suggest that the rAdv55-dE3-EGFP-based high-throughput assay provides a reliable tool for rapid neutralization testing and antiviral screening for HAdV-55.

Vector-Mediated Cancer Gene Therapy Reduces Toxicity and Inhibition of Lung Carcinoma Growth in Nude Mice.

Replication-competent oncolytic adenovirus (TOA2) gene therapy is a recently introduced anti-tumor treatment regimen with superior results. The biodistribution studies of virus vector-based medicine seem more cautious and have been given much attention recently in terms of its quality and safety in preclinical trials. The current study determined the biodistribution and safety of a replication-competent adenovirus in different organs to predict its toxicity threshold. The present study has used TOA2, while biodistribution analysis was performed in human lung carcinoma A549-induced tumor-bearing nude mice model. Intratumoral injection was applied onto tumor-bearing mice with the adenovirus (3×1010 VP per mouse). Mice were sacrificed at the end of the experiment and the organs were dissected. Biodistribution analysis was done with complete hexon gene detection in each organ using quantitative real-time polymerase chain reaction (qRT-PCR). The biodistribution and concentration profiles showed that the TOA2 is well distributed in the entire tumor tissue. After dose 3 at day 11, the concentration of the virus has increased in the tumor tissue from 2240.54 (± 01.69) copies/100 ng genome to 13,120.28 (± 88.21) copies/100 ng genome on the 18th day, which eventually approached 336.45 (± 23.41) copies/100ng genome on the day 36. On the contrary, the concentration of the same decreased in the order of the liver, kidney, spleen, lung, and heart over time but no distributional traces in gonads. But the concentration found decreased dramatically in blood and other organs, while at the end of the experiment no detectable distribution was seen besides tumor tissue. The study confirms that adenovirus-based tumor therapy using conditionally replicating competent oncolytic TOA2 exhibited great efficiency with no toxicity at all.

Abstract CT196: A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer

Abstract Introduction: Men with locally recurrent prostate cancer, after definitive radiotherapy, have few therapeutic options. Oncolytic adenovirus-mediated cytotoxic gene therapy is an investigational cancer therapy. Delivery of the suicide gene to the tumor is by direct intratumoral or systemic injection of a viral vector containing the suicide gene. Our approach incorporates yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK), to confer sensitivity to 5-fluorocytosine (5-FC) and Valganciclovir (vGCV), respectively. The pro-drugs are converted into active drugs that inhibit DNA damage repair. Here we report the safety of oncolytic adenovirus-mediated suicide gene therapy that incorporates an interleukin-12 (IL12) gene for treatment of recurrent prostate cancer. Methods: In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL12) was injected into tumors of 15 subjects with recurrent prostate cancer (T1c-T2) at escalating doses (1 × 1010, 3 × 1010, 1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-FC and vGCV for 7 days. The study endpoint was toxicity through day 30. Experimental endpoints included measurements of serum IL12, interferon gamma (IFNγ), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells (PBMC) and proliferation markers were analyzed by flow cytometry. Results and Conclusions: Fifteen patients received Ad5-yCD/mutTKSR39rep-hIL-12 and oral 5-FC and vGCV. Approximately 92% of the 115 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39rep-hIL-12 DNA was detected in the blood of only two patients. Elevated serum IL12, IFNγ, and CXCL10 levels were detected in 57%, 93%, and 79% of subjects, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39rep-hIL-12 administration in cohort 5. The study did not detect a significant difference in the PSA doubling time (PSADT) between pre and post treatment by paired Wilcoxon rank test (p=0.17). There was no correlation between adenoviral dose and PSADT in each cohort separately or pooled (cohorts1-3 and cohorts 4-5). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39 rep-hIL-12) was well tolerated when administered locally to prostate tumors. Citation Format: Shyam Nyati, Hans Stricker, Kenneth L. Barton, Piln Li, Mohamed Elshaikh, Haythem Ali, Stephen L. Brown, Clara Hwang, James Peabody, Svend O. Freytag, Benjamin Movsas, Farzan Siddiqui. A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT196.

Abstract PD2-03: Targeting TGF-β and over-expressing GM-CSF in the Tumor Microenvironment (TME) with AMUN-003 Inhibits Tumor Growth and Metastases and Augments Immune Checkpoint Inhibitor (ICI) Response in Triple Negative Breast Cancer (TNBC)

Abstract Introduction: Despite several therapeutic advancements, metastatic TNBC remains incurable for nearly all patients and is a frequent cause of cancer-related deaths worldwide. We tested the hypothesis that inhibiting suppressive signals sustained by TGF-β and concurrently stimulating recruitment of inflammatory cells with GM-CSF within the TME would result in improved anti-tumor responses. We report here pre-clinical single agent and ICI combination data for a newly developed oncolytic adenovirus rAd.sT.GM (AMUN-003) that expresses both sTGFβRIIFc (a TGF-β protein decoy), and GM-CSF tested in an immunocompetent mouse model. Methods: In addition to manufacturing Amun-003 which is a replication competent adenovirus encoding both sTGFβRIIFc and GM-CSF transgenes, we generated multiple replication competent control viruses coding for sTGFβRIIFc alone, GM-CSF alone or no transgene. We implanted 4T1 cells in the flanks of BALB/c mice, a syngeneic tumor model that is immune competent. On day 7 and day 10 post tumor cell inoculation, we performed saline or single agent virus injections into the tumor. We monitored the health of the animals, performed periodic tumor caliper measurements and collected sera for analysis of cytokines and markers of tissue injury. Animals were sacrificed on Day 25 and tissues collected. Similarly, with the same animal model and using reduced dose Amun-003, we tested combinations with systemic anti-CTLA-4, anti-PD-1 monoclonal antibodies, or both. Animals were sacrificed on Day 25 with specimens collected as above but also to report the metastatic burden in distant tissues. Results: In single agent experiments, all adenovirus constructs were similarly tolerated by the animals with no notable differences in animal weights. General safety as assessed by serum LDH and IL-6 demonstrated no difference between animal groups treated with the different virus constructs. All adenovirus resulted in delay of cancer growth and on day 25, excised tumors from sacrificed animals weighed less than tumors from animals treated with buffer. However, a single dose of Amun-003 was the most effective at delaying tumor progression than any other tested constructs (Day 21 and Day 25: P < 0.0001 vs the buffer group by two-way ANOVA analysis). Next, we conducted experiments evaluating a single dose of Amun-003 alone or in combination with anti-CTLA4, anti-PD1 or both ICI. Combinations of Amun-003 with ICI delayed progression better than ICI treatments alone or Amun-003 alone. Moreover, the combination with both ICI and Amun-003 was most effective at delay of tumor progression (Day 22 and Day 25: P < 0.0001 vs the buffer group; D25: P=0.0119 vs dual ICI therapy). Lung surface metastatic nodule counts and lung tissue luciferase assay for 4T1-luc2 cells both showed treatment consisted of Amun003 and both ICI was most potent in inhibiting lung metastasis (Lung nodules: P < 0.0001 vs the buffer group; Relative luminescence: P=0.0036 vs the buffer group, both by one-way ANOVA analysis). Finally, the metastatic burden in the lung was least in animals treated with Amun-003 and both ICI (42% metastases free) (P = 0.0373 vs the buffer group by fisher’s exact test). Conclusions: In syngeneic immune competent animals harboring an aggressive TNBC tumor, single agent Amun-003 appeared safe and was more effective at controlling tumor progression following an intratumoral injection when compared to other tested adenovirus constructs. In the same model, combinations of ICI with Amun-003 resulted in delayed tumor growth and control of metastatic spread. These results with Amun-003 support advancement to human clinical testing. Citation Format: Sooncheon Shin, Beniamin Filimon, Yuefeng Yang, Zebin Hu, Prem Seth, Vijayakrishna K. Gadi, Weidong Xu. Targeting TGF-β and over-expressing GM-CSF in the Tumor Microenvironment (TME) with AMUN-003 Inhibits Tumor Growth and Metastases and Augments Immune Checkpoint Inhibitor (ICI) Response in Triple Negative Breast Cancer (TNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD2-03.

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

Oncolytic virus-mediated reducing of myeloid-derived suppressor cells enhances the efficacy of PD-L1 blockade in gemcitabine-resistant pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is often refractory to treatment with gemcitabine (GEM) and immune checkpoint inhibitors including anti-programmed cell death ligand 1 (PD-L1) antibody. However, the precise relationship between GEM-resistant PDAC and development of an immunosuppressive tumor microenvironment (TME) remains unclear. In this study, we investigated the immunosuppressive TME in parental and GEM-resistant PDAC tumors and assessed the therapeutic potential of combination therapy with the telomerase-specific replication-competent oncolytic adenovirus OBP-702, which induces tumor suppressor p53 protein and PD-L1 blockade against GEM-resistant PDAC tumors. Mouse PDAC cells (PAN02) and human PDAC cells (MIA PaCa-2, BxPC-3) were used to establish GEM-resistant PDAC lines. PD-L1 expression and the immunosuppressive TME were analyzed using parental and GEM-resistant PDAC cells. A cytokine array was used to investigate the underlying mechanism of immunosuppressive TME induction by GEM-resistant PAN02 cells. The GEM-resistant PAN02 tumor model was used to evaluate the antitumor effect of combination therapy with OBP-702 and PD-L1 blockade. GEM-resistant PDAC cells exhibited higher PD-L1 expression and produced higher granulocyte-macrophage colony-stimulating factor (GM-CSF) levels compared with parental cells, inducing an immunosuppressive TME and the accumulation of myeloid-derived suppressor cells (MDSCs). OBP-702 significantly inhibited GEM-resistant PAN02 tumor growth by suppressing GM-CSF-mediated MDSC accumulation. Moreover, combination treatment with OBP-702 significantly enhanced the antitumor efficacy of PD-L1 blockade against GEM-resistant PAN02 tumors. The present results suggest that combination therapy involving OBP-702 and PD-L1 blockade is a promising antitumor strategy for treating GEM-resistant PDAC with GM-CSF-induced immunosuppressive TME formation.