Conditionally Replicating Adenoviruses Research Articles

Triple-regulated conditionally replicating adenovirus for effective and safer treatment of peritoneal carcinomatosis

There is no effective therapy for peritoneal carcinomatosis derived from gastric cancer. An ideal conditionally replicating adenovirus (CRA) that selectively replicates in and kills cancer cells has not been developed for gastric cancer-derived peritoneal carcinomatosis. Using our platform technology of CRA regulated and treating tumors with multiple factors (m-CRA), we generated two types of survivin-responsive m-CRAs, Surv.m-CRA-CMVp and Surv.m-CRA-CEAp, consisting of E1A downstream of the survivin promoter, and the mutated E1B gene downstream of the human cytomegalovirus immediate early gene enhancer/promoter and carcinoembryonic antigen promoter, respectively. Survivin mRNA was expressed at high and undetectable levels in two gastric cancer cells and eleven normal cells, respectively. Carcinoembryonic antigen was expressed at high and very low levels in MKN-45 gastric cancer and normal PrEC cells, respectively, and was not detected in other cell types. While both Surv.m-CRA-CEAp and Surv.m-CRA-CMVp exhibited potent cytotoxic effects on MKN-45 cells in vitro, Surv.m-CRA-CEAp significantly reduced cytotoxicity to normal cells compared to Surv.m-CRA-CMVp. Control mice that received an intraperitoneal injection of MKN-45 cells gradually lost body weight and died of peritoneal carcinomatosis within 98 days. In contrast, all mice receiving Surv.m-CRA-CEAp or Surv.m-CRA-CMVp-infected MKN-45 cells increased their body weight and survived 120 days. In conclusion, the triple-regulated Surv.m-CRA-CEAp enhances cancer specificity (i.e., safety) without reducing the potent therapeutic effect for carcinoembryonic antigen-positive gastric cancer-derived peritoneal carcinomatosis. The modified E1B promoter strategy of CRA facilitates the development of novel CRAs for the effective and safe treatment of a variety of refractory cancers.

The Development and Characterization of a Next-Generation Oncolytic Virus Armed with an Anti-PD-1 sdAb for Osteosarcoma Treatment In Vitro.

Osteosarcoma (OS) is a primary bone malignancy characterized by an aggressive nature, limited treatment options, low survival rate, and poor patient prognosis. Conditionally replicative adenoviruses (CRAds) armed with immune checkpoint inhibitors hold great potential for enhanced therapeutic efficacy. The present study aims to investigate the anti-tumor efficacy of CAV2-AU-M2, a CAV2-based CRAd armed with an anti-PD-1 single-domain antibody (sdAb), against OS cell lines in vitro. The infection, conditional replication, cytopathic effects, and cytotoxicity of CAV2-AU-M2 were tested in four different OS cell lines in two-dimensional (2D) and three-dimensional (3D) cell cultures. CAV2-AU-M2 showed selective replication in the OS cells and induced efficient tumor cell lysis and death. Moreover, CAV2-AU-M2 produced an anti-PD-1 sdAb that demonstrated effective binding to the PD-1 receptors. This study demonstrated the first CRAd armed with an anti-PD-1 sdAb. This combined approach of two distinct immunotherapies is intended to enhance the anti-tumor immune response in the tumor microenvironment.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying a bispecific T cell engager against hepatocellular carcinoma

ABSTRACT We previously established a hepatocellular carcinoma (HCC) targeting system of conditionally replicative adenovirus (CRAd) delivered by human umbilical cord-derived mesenchymal stem cells (HUMSCs). However, this system needed to be developed further to enhance the antitumor effect and overcome the limitations caused by the alpha-fetoprotein (AFP) heterogeneity of HCC. In this study, a bispecific T cell engager (BiTE) targeting programmed death ligand 1 controlled by the human telomerase reverse transcriptase promoter was armed on the CRAd of the old system. It was demonstrated on orthotopic transplantation model mice that the new system had a better anti-tumor effect with no more damage to extrahepatic organs and less liver injury, and the infiltration and activation of T cells were significantly enhanced in the tumor tissues of the model mice treated with the new system. Importantly, we confirmed that the new system eliminated the AFP-negative cells on AFP heterogeneous tumor models efficiently. Conclusion: Compared with the old system, the new system provided a more effective and safer strategy against HCC.

A phase 1 first-in-human study of interferon beta (IFNβ) and membrane-stable CD40L expressing oncolytic virus (MEM-288) in solid tumors including non–small-cell lung cancer (NSCLC).

2569 Background: MEM-288 is a conditionally-replicative oncolytic adenovirus expressing human IFNβ and a recombinant membrane-stable form of CD40L (MEM40). Preclinical studies show MEM-288 induces robust dendritic cell-mediated systemic T cell responses capable of inhibiting abscopal tumor growth as monotherapy and synergizes with immune checkpoint inhibitors (ICI). Methods: MEM-288 is being evaluated in this Phase 1 open-label trial (NCT05076760) in pts with select solid tumors including NSCLC (a) refractory to standard therapy and (b) with a tumor lesion deemed feasible for biopsy and MEM-288 intratumoral (i.t.) injection. The primary objective is to determine using a BOIN design a recommended phase II dose of MEM-288 across 3 dose levels (DL1-3) spanning 1e10 to 1e11 viral particles by i.t. injection once every 3 weeks. Secondary objectives include efficacy assessment, including response rate of injected and non-injected tumors assessed separately. Tumor biopsies obtained immediately prior to the 1st and 2nd injections are used to explore biomarkers and anti-tumor immune responses. Results: As of February 2023, 12 pts (11 NSCLC and 1 pancreatic cancer; n = 3 DL1, n = 5 DL2, n = 4 DL3) have enrolled. The median number of MEM-288 i.t. injections was 2 (1-3), median age was 63 (39-76), and median prior lines of therapy was 3 (1-5). Radiology-guided injections were administered in superficial/palpable tumors and visceral lesions. No dose limiting toxicities occurred. Treatment-related adverse events observed in > 1 pt include grade 1 injection site reaction (42%) and chills (17%). Of 10 response-evaluable pts, 4 (40%) pts had shrinkage of injected tumor (range -26 to -54%): 3 (30%) PR and 1 (10%) SD. Multiple pts also had stabilization or shrinkage of distal non-injected lesions. Best overall response was 3 (30%) stable disease and 7 (70%) progressive disease. After a single MEM-288 injection, biopsies show decreased tumor cell percentage concomitant with substantial increases in overall CD8+ T cells, increased T clonotype diversity in both tumor and peripheral blood, and increased TCF1+ stem-like CD8+ T cells that are strongly associated with response to ICIs. Plasma cytokine analysis showed increases in IFNg and of IFN-inducible cytokines and chemokines, supportive of stimulation of systemic response after MEM-288. Of note, a pt with strong stimulation of tumor and systemic T cell immunity after MEM-288 had subsequent CR (ongoing > 7 months) to docetaxel + ramucirumab following prior treatment failure with platinum doublet + ICI received before MEM-288. Conclusions: Preliminary safety, antitumor and immune response data are encouraging. Updated results and immune data from this study will be presented. An expansion arm is planned with combination MEM-288 and anti-PD1 antibody in pts with advanced NSCLC refractory to ICI. Clinical trial information: NCT05076760 .

Abstract CT103: A phase 1 first-in-human study of MEM-288 oncolytic virus in solid tumors including non-small cell lung cancer (NSCLC): impact on tumor and systemic T cell immunity

Abstract Background: MEM-288 is a conditionally-replicative oncolytic adenovirus expressing human IFNβ and a recombinant membrane-stable form of CD40L (MEM40). Preclinical studies show MEM-288 induces robust dendritic cell-mediated systemic T cell responses capable of inhibiting abscopal tumor growth as monotherapy and synergizes with immune checkpoint inhibitors (ICI). Methods: Safety, antitumor and immunologic activity of MEM-288 are being evaluated in this Phase 1, multicenter, open-label trial (NCT05076760). Pts with select solid tumors including NSCLC (a) refractory to standard therapy and (b) with a tumor lesion deemed feasible for biopsy and MEM-288 intratumoral injection are eligible. The primary objective is to determine a recommended phase 2 dose of MEM-288 monotherapy across 3 dose levels (DL1-3) spanning 1e10 to 1e11 viral particles by intratumoral injection once every 3 weeks, for up to 6 injections, using a BOIN design. Secondary objectives include efficacy assessment (including response rate of injected and non-injected tumors assessed separately). Tumor biopsies immediately prior to the 1st and 2nd injections, and peripheral blood at serial timepoints, are used to explore biomarkers and anti-tumor immune responses. Results: As of Jan 2023, the study is ongoing with the DL3 high-dose cohort completing accrual. 11 pts (10 NSCLC and 1 pancreatic) enrolled and received ≥1 dose of MEM-288 (n=3 DL1, n=5 DL2, n=3 DL3). No dose limiting toxicities have been reported to date and no pts have discontinued treatment due to toxicity. Treatment-related adverse events observed in >1 pt include grade 1 injection site reaction (45%) and chills (18%). Of 7 pts evaluable for response, 3 had shrinkage of injected tumor (range -40 to -54%), and multiple pts also had stabilization or shrinkage of distal non-injected lesions with best RECIST response in uninjected tumors as 2 SD and 5 PD. After a single MEM-288 injection, biopsies show decreased tumor cell percentage concomitant with substantial increases in overall CD8+ T cells, increased T clonotype diversity in both tumor and peripheral blood, and increased TCF1+ stem-like CD8+ T cells that are strongly associated with response to ICIs. Plasma cytokine analysis showed increases in IFNγ and of IFN-inducible cytokines and chemokines, supportive of stimulation of systemic response after MEM-288. Of note, a pt with strong stimulation of tumor and systemic T cell immunity after MEM-288 had a subsequent CR (ongoing >6 months) to docetaxel + ramucirumab following prior treatment failure with platinum doublet + ICI received before MEM-288. Conclusions: Preliminary safety, antitumor and immune response data are encouraging. Updated results and immune data will be presented. An expansion arm is planned with combination MEM-288 and anti-PD1 antibody in pts with advanced NSCLC refractory to ICI. Citation Format: Andreas N. Saltos, Christy Arrowood, Georgia Beasley, James Ronald, Ghassan El-Haddad, Uzma Khan, Luiziane Guerra-Guevara, Steven Wolf, Lin Gu, Xiaofei F. Wang, Dana Foresman, Xiaoqing Yu, Mark J. Cantwell, Scott J. Antonia, Amer A. Beg, Neal E. Ready. A phase 1 first-in-human study of MEM-288 oncolytic virus in solid tumors including non-small cell lung cancer (NSCLC): impact on tumor and systemic T cell immunity [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 CT103.

Oncolytic adenoviruses and the treatment of pancreatic cancer: a review of clinical trials.

Pancreatic ductal adenocarcinoma (PDAC) remains a common and difficult cancer to treat. Surgical resection and chemotherapy are standard of care and clinical outcomes remain poor. Oncolytic adenoviruses are a unique approach to the treatment of this challenging cancer, aiming to overcome the features of this disease that pose the key obstacles to standard therapies. This paper provides a detailed review of the clinical trials of conditionally-replicative adenoviruses in pancreatic cancer to date, with a brief summary of the past preclinical literature and future prospects of this therapy. MEDLINE, Embase, and clinicaltrials.gov were searched from inception to December 23rd 2022 for clinical trials of conditionally-replicative adenoviruses used in patients with pancreatic ductal adenocarcinoma. Primary features for review included patient demographics, treatment protocol including dose and administration route, adverse events, patient responses and survival rates. The six published clinical trials suggest that objective clinical responses can be achieved with a tolerable level of side effects, even at high viral doses. The more clinically adaptable intravenous route of administration also appears to be as well tolerated as the more challenging intratumoural injections. Published clinical trials provide data of the safety and some signs of oncolytic activity of conditionally-replicative adenoviruses in patients with pancreatic cancer. Importantly, on the latest trials, the easier intravenous route of administration seems to be well tolerated and safe, providing the opportunity for further clinical evaluation. It is hoped that the ongoing clinical trials will yield more promising results of this therapeutic approach against a currently intractable disease.

Infectivity-Enhanced, Conditionally Replicative Adenovirus for COX-2-Expressing Castration-Resistant Prostate Cancer.

The development of conditionally replicative adenoviruses (CRAds) for castration-resistant prostate cancer (CRPC), particularly neuroendocrine prostate cancer (NEPC), has two major obstacles: choice of control element and poor infectivity. We applied fiber-modification-based infectivity enhancement and an androgen-independent promoter (cyclooxynegase-2, COX-2) to overcome these issues. The properties of the COX-2 promoter and the effect of fiber modification were tested in two CRPC cell lines (Du-145 and PC3). Fiber-modified COX-2 CRAds were tested in vitro for cytocidal effect as well as in vivo for antitumor effect with subcutaneous CRPC xenografts. In both CRPC cell lines, the COX-2 promoter showed high activity, and Ad5/Ad3 fiber modification significantly enhanced adenoviral infectivity. COX-2 CRAds showed a potent cytocidal effect in CRPC cells with remarkable augmentation by fiber modification. In vivo, COX-2 CRAds showed an antitumor effect in Du-145 while only Ad5/Ad3 CRAd showed the strongest antitumor effect in PC3. COX-2 promoter-based, infectivity-enhanced CRAds showed a potent antitumor effect in CRPC/NEPC cells.

Improving the Transduction Efficiency and Antitumor Effect of Conditionally Replicative Adenovirus by Application of 6-cyclohexyl Methyl-β-D-maltoside

As a tumor-targeting oncolytic adenovirus (Ad), conditionally replicating adenovirus (CRAd) can access the cell interior by binding to coxsackievirus-Ad receptors (CARs) and specifically replicate and destroy cancer cells without lethal effects on normal cells. The transduction efficiency of CRAd is highly dependent on the number of CARs on the cell membrane. However, not all tumor cells highly express CARs; therefore, improving the transduction efficiency of CRAd is beneficial for improving its antitumor effect. In this study, 6-cyclohexyl methyl-β-D-maltoside (6-β-D), as maltoside transfection agent, showed several advantages, including high transfection efficiency, low toxicity, and potential for intensive use and easy operation. With pretreatment of cancer cells with low concentration of 6-β-D (≤5 μg/mL), the transduction efficiency of "model" Ad (eGFP-Ad) was improved 18-fold compared to eGFP-Ad alone. 6-β-D improved the antitumor effect of CRAd while being safe for normal cells, in which treatment with 6-β-D helped the lethal effects of CRAd at a multiplicity-of-infection ratio of 10 (MOI 10) achieve the oncolytic outcomes of MOI 50. This means that if CRAd is combined with 6-β-D, the amount of CRAd used in clinical practice could be greatly reduced without diminishing its curative effect or exposing patients to the potential side effects of high-titer CRAd. Finally, the underlying mechanism of antitumor effect of CRAd + 6-β-D was primarily investigated, and we found that 6-β-D increased the virus's replication in cancer cells at the early stage of infection and activated the apoptosis signaling pathway at the late stage of the cell cycle. This research will provide an effective technical reference for further improving Ad-mediated cancer gene therapy in clinical practice.

Modulating Oncolytic Adenovirus Immunotherapy by Driving Two Axes of the Immune System by Expressing 4-1BBL and CD40L.

Oncolytic viruses (OVs) can have utility for direct killing of cancer cells, but may also serve to activate the immune system against cancer cells. While viruses alone can serve as immune stimulators, there is great interest in arming OVs with genes encoding immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads, murine CD40L (mCD40L) or 4-1BBL (m4-1BBL), in an immune competent mouse model of melanoma. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival compared to empty CRAd657. However, combined injection of both CRAd-m4-1BBL and CRAd-mCD40L mediated significantly better control of tumor growth. All of the payloads increased immune cell infiltration into tumors and notably reduced expression of PD-1 exhaustion marker on T cells. Tumor volumes were negatively associated with total infiltrating T cell population. We found that the payloads increased immune cell infiltration into tumors with some specificities: recruitment of CD8+ T cells was higher with m4-1BBL expression, while mCD40L expression induced more CD4+ T cell infiltration. Importantly, the combination of CRAd657-m4-1BBL and CRAd657-mCD40L induced the highest immune cells/T cell infiltration and the highest anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vector. This combination also caused depigmentation in areas adjacent to the tumor sites in more animals. These data indicate that driving two axes of the immune system with combined immune stimulatory payloads can lead to improved anticancer immune responses and better tumor control in an immune competent model of cancer.

Concepts in Oncolytic Adenovirus Therapy.

Oncolytic adenovirus therapy is gaining importance as a novel treatment option for the management of various cancers. Different concepts of modification within the adenovirus vector have been identified that define the mode of action against and the interaction with the tumour. Adenoviral vectors allow for genetic manipulations that restrict tumour specificity and also the expression of specific transgenes in order to support the anti-tumour effect. Additionally, replication of the virus and reinfection of neighbouring tumour cells amplify the therapeutic effect. Another important aspect in oncolytic adenovirus therapy is the virus induced cell death which is a process that activates the immune system against the tumour. This review describes which elements in adenovirus vectors have been identified for modification not only to utilize oncolytic adenovirus vectors into conditionally replicating adenoviruses (CRAds) that allow replication specifically in tumour cells but also to confer specific characteristics to these viruses. These advances in development resulted in clinical trials that are summarized based on the conceptual design.

Interferon Alpha-Expressing Oncolytic Adenovirus for Treatment of Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) has increased in incidence in Western countries, and its poor prognosis necessitates the development of novel therapeutics. We previously reported the potential of conditionally replicative adenoviruses (CRAd) as a novel therapeutic treatment for this disease. To further augment the therapeutic effectiveness of our cyclooxygenase-2 (Cox2) controlled CRAd in EAC, we inserted an interferon alpha (IFN) transgene into the viral genome that is expressed upon viral replication. In this manuscript, we analyze the cytotoxic and oncolytic effects of an IFN-expressing oncolytic adenovirus in EAC and the role of the Cox2 promoter in providing for selective replication in human tissues. An infectivity-enhanced IFN-expressing CRAd (5/3 Cox2 CRAd ΔE3 ADP IFN) and other control viruses were first tested in vitro with cell lines. For the in vivo study, EAC xenografts in nude mice were treated with a single intratumoral dose of virus. An ex vivo analysis with live tissue slices was conducted using surgically resected EAC patient specimens. Expression of IFN significantly enhanced the cytotoxic and oncolytic effect of a Cox2-promoter controlled CRAd. This virus showed significant tumor growth suppression in a xenograft model. Furthermore, in human EAC samples, the promoter-controlled virus demonstrated selective replication in cancerous tissues, leaving normal esophageal tissue unaffected. An IFN-expressing CRAd driven by the Cox2 promoter has strong oncolytic effects as well as cancer-specific replication. Our novel vector possesses critical characteristics that make it a potential candidate for clinical translation to treat EAC.

The genome position of a therapeutic transgene strongly influences the level of expression in an armed oncolytic human adenovirus vector

Efficacy of oncolytic, conditionally-replicating adenovirus (CRAd) vectors can be enhanced by “arming” the vector with therapeutic transgenes. We examined whether inclusion of an intact early region 3 (E3) and the reptilian reovirus fusogenic p14 fusion-associated small transmembrane (FAST) protein enhanced vector efficacy. The p14 FAST transgene was cloned between the fiber gene and E4 region, with an upstream splice acceptor for replication-dependent expression from the major late promoter. In A549 cells, this vector expressed p14 FAST protein at very low levels, and showed a poor ability to mediate cell-cell fusion, relative to a similar vector encoding p14 FAST within the E3 deletion. Although expression of E3 proteins from the CRAd increased plaque size, poor expression of p14 FAST protein compromised the fusogenic capacity of the vector. Thus, location of a therapeutic transgene within a CRAd can significantly impact expression of the transgene and is an important consideration in vector design.

Abstract PO-082: Radiation therapy enhances the potential of the oncolytic virus in the treatment of osteosarcoma

Abstract Background: Combination of neoadjuvant chemotherapy and surgery is the standard treatment approach for osteosarcoma. Despite aggressive treatment protocol, ~40% of patients do not respond and relapse, the survival rate is poor for these patients. Conditionally replicative adenovirus (CRAds) destroying cancer cell while sparing normal cells is a new treatment approach for cancers. We developed a new oncolytic adenovirus which grows using mRNA stabilization system found in many cancer cells. Our recent report showed that this virus replicated in cancer cells where AU-rich element (ARE) containing mRNA is stabilized. ARE-mRNA is stabilized in the majority types of cancers mediate by binding with HuR (Human antigen R), which exports target ARE-mRNA to the cytoplasm. Radiotherapy (RT) is not a first-line treatment option for osteosarcoma as osteosarcoma cells are not killed by radiation easily. But in certain conditions radiotherapy can be used where surgical resection is not technically possible without undue morbidity or when surgery is refused. In response to ionizing radiation and UV radiation DNA damage response (DDR) can influence HuR action either by the increasing cytoplasmic HuR abundance or affecting the binding affinity to target ARE-mRNA. The purpose of the present study was to analyze the efficacy of the combination treatment of RT and oncolytic adenovirus for osteosarcoma. Materials and Methods: E4rof6-deleted adenovirus (dl355) and human osteosarcoma cell lines U2OS and Saos2 were used in this study. Cell viability assays were carried out to evaluate the cytotoxicity and synergism of dl355 and RT combination. Chou and Talalay’s median-effect method was used to calculate the combination index. CPE assay was used to assess the cytopathic effect of the combination. Viral replication with or without radiation combination was investigated by hexon staining. Western blot analysis was performed to examine viral protein expression. Results: We found that RT treatment enhanced the cytoplasmic relocalization of HuR; thus irradiation up-regulated the propagations of dl355. Also, radiation-induced upregulation of viral protein expression was observed by western blotting. The RT and dl355 combination treatment increased the cytolysis of U2OS, and Saos2 cells in a synergistic manner compared to RT or virus only. Conclusion: Radiotherapy up-regulated of oncolytic adenovirus, which grows using the ARE-mRNA stabilization system, and this cytolytic effect provides a rationale for this combination. This combination approach has the possibility of a promising strategy for osteosarcoma. Citation Format: Elora Hossain, Fumihiro Higashino. Radiation therapy enhances the potential of the oncolytic virus in the treatment of osteosarcoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-082.

Evaluation of tumor immunity after administration of conditionally replicative adenoviral vector in canine osteosarcoma patients

Osteosarcoma is one among the most common neoplasms in dogs. Current treatments show limited efficacy and fail to prevent metastasis. Conditionally replicative adenoviruses (CRAd) replicate exclusively in targeted tumor cells and release new virus particles to infect additional cells. We proposed that OC-CAVE1 (CAV2 with the E1A promoter replaced with the osteocalcin promotor) may also enhance existing immunity against tumors by overcoming immune tolerance via exposure of new epitopes and cytokine signaling. Eleven client-owned dogs with spontaneously occurring osteosarcomas were enrolled in a pilot study. All dogs were injected with OC-CAVE1 following amputation of the affected limb or limb-sparing surgery. Dogs were monitored for viremia and viral shedding. There was minimal virus shedding in urine and feces by the 6th day and no virus was present in blood after 4 weeks. CAV-2 antibody-titers increased in all of the patients, post-CRAd injection. Immunological assays were performed to monitor 1) humoral response against tumors, 2) levels of circulatory CD11c + cells, 3) levels of regulatory T cells, and 4) cytotoxic activity of tumor specific T cells against autologous tumor cells between pre-CRAd administration and 4 weeks post-CRAd administration samples. Administration of the CRAd OC-CAVE1 resulted in alteration of some immune response parameters but did not appear to result in increased survival duration. However, 2 dogs in the study achieved survival times in excess of 1 year. Weak replication of OC-CAVE1 in metastatic cells and delay of chemotherapy following CRAd treatment may contribute to the lack of immune response and improvement in survival time of the clinical patients.

Enhanced tumor suppression by conditionally replicating adenovirus in multidrug-resistant human ovarian carcinoma

Background: Breast and ovarian cancers are among the most common and deadly gynecologic malignancies. Today, interest in the non-cytotoxic drug development is at an all-time high, albeit platinum-based combination chemotherapy is still the standard of care for ovarian cancer. However, the treatment of advanced malignancies is restricted due to chemotherapy resistance, which in most cases is acquired. Therefore, identifying novel anti-cancer strategies for gynecological cancers is imperative. We have developed a highly tumor-targeting oncolytic adenovirus by controlling its replication via tumor-specific promotor, survivin. Hitherto, we have tested the anti-tumor potency of this genetically engineered oncolytic virus against breast and lung cancers and found it very toxic and tumor-targeting. In the present study, we continued our investigation of using survivin responsive conditionally replicating adenovirus (CRAd) to chemotherapy-resistant cancers of different origins.

Absent in melanoma 2 enhances anti-tumour effects of CAIX promotor controlled conditionally replicative adenovirus in renal cancer.

Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIXpromotor)‐controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIXpromotor‐AIM2 adenoviruses (Ad‐CAIXpromotor‐AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad‐CAIXpromotor, Ad‐CAIXpromotor‐AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786‐O cells or OSRC‐2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC‐2‐xenograft mice. The tumour growth was remarkably inhibited in Ad‐CAIXpromotor‐AIM2‐treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD‐CMK resulted in the reduction of anti‐tumour activity by Ad‐CAIXpromotor‐AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad‐CAIXpromotor‐AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour‐specific Ad‐CAIXpromotor‐AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2‐based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.

Survivin-responsive conditionally replicating adenovirus for patients with advanced sarcoma demonstrated potent and long-term efficacy and high safety in a phase I clinical trial.

11512 Background: Whereas one of oncolytic viruses (OVs), inducing selective tumor killing and systemic anti-tumor immunity, was approved by FDA in 2015, the best OV that more safely and efficiently treats intractable cancers has not been successfully developed. By our platform technology to efficiently construct next-generation OVs, i.e., “conditionally replicating adenoviruses (CRAs) that target and/or treat tumor cells with multiple factors” (m-CRAs), we identified that among candidates, survivin-responsive m-CRAs (Surv.m-CRAs) exhibited the most potent antitumor efficacy and cancer selectivity ( i.e., safety) in preclinical studies ( Cancer Res, 2005 et al.). Here we present the data of First-In-Human phase I clinical trial of Surv.m-CRA-1 for musculoskeletal tumors (MST). Methods: This single-arm, open label study included 9 patients with unresectable and advanced MST. Patients underwent a single intratumoral injection of either 1×10^10 viral particle (vp) (low), 1×10^11 vp (mid) or 1×10^12 vp (high). The primary endpoints were safety and tolerability. The secondary endpoints included the local control of treated tumor at one month, defined by RECIST and Choi criteria, analysis of dissemination of Surv.m-CRA-1, serum cytokine and adenoviral antibody. Long-term follow-up was done in some patients. Results: Four patients (44.4%) had grade 3 or higher adverse events, including lymphopenia, leukocytopenia and mildly elevated liver transaminase in 2, 1 and 1 patient, respectively. Virus excretions, including second peak of viremia from viral replication in tumor, were observed in 1, 2 and 3 patients of low, mid and high dose, respectively. Out of 9 patients, 5 PR, 3 SD and 1 PD by Choi, and 8 SD and 1 PD by RECIST were observed. During follow-up, another 1 and 2 patients became PR by Choi and RECIST, respectively. Of note, long-term PR (over 2 years) after a single injection of Surv.m-CRA-1 was achieved in two chordoma cases in low dose. Conclusions: Surv.m-CRA-1 was well tolerated and showed antitumor activity for prolonged periods against advanced MST. We about to start Phase I/II study of multiple injections of Surv.m-CRA-1 for advanced solid tumors in two-arms for musculoskeletal tumors and pancreatic cancer. Clinical trial information: R000026464 .

Cisplatin Synergistically Enhances Antitumor Potency of Conditionally Replicating Adenovirus via p53 Dependent or Independent Pathways in Human Lung Carcinoma.

Cisplatin is ranked as one of the most powerful and commonly prescribed anti-tumor chemotherapeutic agents which improve survival in many solid tumors including non-small cell lung cancer. However, the treatment of advanced lung cancer is restricted due to chemotherapy resistance. Here, we developed and investigated survivin promoter regulating conditionally replicating adenovirus (CRAd) for its anti-tumor potential alone or in combination with cisplatin in two lung cancer cells, H23, H2126, and their resistant cells, H23/CPR, H2126/CPR. To measure the expression of genes which regulate resistance, adenoviral transduction, metastasis, and apoptosis in cancer cells, RT-PCR and Western blotting were performed. The anti-tumor efficacy of the treatments was evaluated through flow cytometry, MTT and transwell assays. This study demonstrated that co-treatment with cisplatin and CRAd exerts synergistic anti-tumor effects on chemotherapy sensitive lung cancer cells and monotherapy of CRAd could be a practical approach to deal with chemotherapy resistance. Combined treatment induced stronger apoptosis by suppressing the anti-apoptotic molecule Bcl-2, and reversed epithelial to mesenchymal transition. In conclusion, cisplatin synergistically increased the tumor-killing of CRAd by (1) increasing CRAd transduction via enhanced CAR expression and (2) increasing p53 dependent or independent apoptosis of lung cancer cell lines. Also, CRAd alone proved to be a very efficient anti-tumor agent in cancer cells resistant to cisplatin owing to upregulated CAR levels. In an exciting outcome, we have revealed novel therapeutic opportunities to exploit intrinsic and acquired resistance to enhance the therapeutic index of anti-tumor treatment in lung cancer.

Combination therapy with F5/35 fiber chimeric conditionally replicative adenoviruses expressing IL-24 enhances the antitumor effect of temozolomide against melanoma.

BackgroundTemozolomide (TMZ) is widely used to treat melanoma; however, response rates to TMZ are low because of rapid and frequent resistance. Conditionally, replicative adenoviruses (CRAds) are an effective and promising approach. The receptor for adenovirus is coxsackie‐adenovirus receptor (CAR), which is poorly expressed in most cells. However, CD46, which is the receptor of species B adenoviruses (Ads), is highly expressed in many cells.MethodsWe constructed CRAd F5/35‐ZD55‐IL‐24, which uses the viral receptors CAR and CD46 for entry into cells. We investigated the antitumor effect of F5/35‐ZD55‐IL‐24 in combination with TMZ to treat melanoma in vitro and in vivo.ResultsThe \\results indicated that F5/35‐ZD55‐IL‐24 in combination with TMZ produced additive or synergistic antitumor and pro‐apoptotic effects in melanoma cells. The combination of F5/35‐ZD55‐IL‐24 and TMZ significantly inhibited the growth of melanoma in vivo. In addition, the antitumor effect of F5/35‐ZD55‐IL‐24 was superior to that of ZD55‐IL‐24 and ZD55‐IL‐24 combined with TMZ.ConclusionsThe use of F5/35‐ZD55‐IL‐24 in conjunction with TMZ is a promising approach for anti‐melanoma therapy.Our results indicated that F5/35‐ZD55‐IL‐24 in combination with TMZ produced additive or synergistic antitumor effect and pro‐apoptotic effect in melanoma cells highly expressed CD46. The combination of F5/35‐ZD55‐IL‐24 and TMZ significantly inhibited the growth of melanoma in vivo. We also found the antitumor effect of F5/35‐ZD55‐IL‐24 was superior to ZD55‐IL‐24, the combination of F5/35‐ZD55‐IL‐24 and TMZ had a more significant antitumor effect than ZD55‐IL‐24 combining with TMZ.

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.