De-Chao Yu

Pre-Existent Adenovirus Antibody Inhibits Systemic Toxicity and Antitumor Activity of CN706 in the Nude Mouse LNCaP Xenograft Model: Implications and Proposals for Human Therapy

Pre-existent humoral antibody to adenovirus potentially confounds human clinical trials involving intravascular administration of adenovirus. Using the LNCaP prostate cancer xenograft model in BALB/c nu/nu mice and the prostate-specific attenuated replication-competent adenovirus (ARCATM) CN706, we developed an animal model that systematically controls both the dose of intravascularly administered adenovirus and the titer of the pre-existent anti-Ad5 antibody, and then measures the virus-induced toxicity as well as antitumor activity. We prepared hyperimmune sera to adenovirus in rabbits, passively injected the purified rabbit anti-Ad5 antibody into tumor-bearing mice, and established measurable humoral anti-Ad5 antibody titers. CN706 was intravenously injected into the tail vein of animals 24 hr after passive anti-Ad5 antibody administration. In the absence of pre-existent antibody, the lethal dose (LD100) for BALB/c nu/nu mice was 2.5x10(11) CN706 particles, whereas 1x10(11) CN706 particles was not lethal. However, in the presence of a 1:80 pre-existent titer of Ad5 neutralizing antibody (NAb), intravenous injection of 5x10(11) CN706 particles was no longer lethal. In addition, pre-existent antibody also prevented antitumor activity in a dose-dependent manner: 1x 10(11) CN706 particles prevented LNCaP xenograft tumor progression, but antitumor activity was eliminated by a pre-existent 1:80 NAb titer. These results led us to propose transient removal of pre-existent adenovirus antibody by immunoapheresis. An affinity column of cloned virus capsid proteins was constructed that was able to specifically remove adenovirus antibody from human clinical serum samples. A 5-min disposable immunoassay was also developed to monitor the level of pre-existent antibody in sera before and after immunoapheresis. Clinically, this approach may enable controlled clinical studies of intravenously administered adenovirus in patients with pre-existent anti-adenovirus antibody.

CG0070, a Conditionally Replicating Granulocyte Macrophage Colony-Stimulating Factor–Armed Oncolytic Adenovirus for the Treatment of Bladder Cancer

Purpose: The purpose of this study was to examine the tumor specificity, cytotoxicity, and granulocyte macrophage colony-stimulating factor expression of CG0070, a conditionally replicating oncolytic adenovirus, in human bladder transitional cell carcinoma (TCC) cell lines and determine its antitumor efficacy in bladder TCC tumor models. Experimental Design: Virus yield and cytotoxicity assays were used to determine tumor specificity and virus replication-mediated cytotoxicity of CG0070 in a panel of human bladder TCC cell lines and primary cells in vitro. Two s.c. and one orthotopic bladder TCC xenograft tumor models were used to assess antitumor activity of CG0070. Results: In a matched isogenic pair of cell lines with differing retinoblastoma (Rb) pathway status, CG0070 showed selective E1a and granulocyte macrophage colony-stimulating factor (GM-CSF) expression in Rb pathway–defective cells. CG0070 replicated in Rb-defective bladder TCC cell lines as efficiently as wild-type adenovirus but produced 100-fold less virus in normal human cells. CG0070 was up to 1,000-fold more cytotoxic in Rb pathway–defective bladder TCC cells in comparison with normal human cells. Antitumor activity of CG0070 was shown in two bladder TCC s.c. xenograft tumor models following intratumoral injections and intravesical treatment in an orthotopic xenograft tumor model when compared with PBS treatment. Conclusions:In vitro and in vivo studies showed the selective replication, cytotoxicity, GM-CSF production, and antitumor efficacy of CG0070 in several bladder TCC models, suggesting a potential utility of this oncolytic agent for the treatment of bladder cancer. Further studies are warranted to show the role of human GM-CSF in the antitumor efficacy of CG0070.

Development of transcriptionally regulated oncolytic adenoviruses.

Changes initiated at the cellular and systemic levels as a result of viral infection or neoplastic transformation share significant overlap. Therefore, the use of replicating viruses to treat tumors has long been postulated as a promising avenue for oncolytic therapy. Over the last 10 years, transcriptionally regulated adenoviruses have become a popular platform for the development of such oncolytic viruses. Placement of heterologous promoters in front of key adenoviral transcription units to achieve tumor- or tissue-specific viral replication is well documented. Various derivatives of this general strategy have led to considerable insight into its limitations, pitfalls, and potential. Although a general process can be described by which to develop transcriptionally regulated adenoviruses, it is apparent that few set rules can yet be defined as to what constitutes a safe, stable, and therapeutically effective vector. Clinical experiences to date suggest the short-term potential for this class of therapeutics lies in combination therapy regimens. Such lessons from the clinic suggest the next generation of transcriptionally regulated oncolytic adenoviruses take advantage of the ability of the platform to carry transgenes in order to deliver a multimodal therapy from a single agent. Beyond this ‘arming’ of the vectors lies the detargeting, retargeting, and coating of adenoviruses to improve the delivery of the agent to the treatment site(s). As a therapeutic platform, transcriptionally regulated adenoviruses are at an early stage of development with considerable opportunities for advancement.

Oncolytic adenovirus CG7870 in combination with radiation demonstrates synergistic enhancements of antitumor efficacy without loss of specificity.

Conditionally replicating adenoviruses that selectively replicate in tumor cells, but not in normal cells, are being explored as virotherapeutic agents for cancer. A prostate-specific oncolytic adenovirus, CG7870 is currently being evaluated in phase 1/2 clinical trials for the treatment of prostate cancer. To decrease the effective dose and further increase the therapeutic efficacy of CG7870, the combination of virotherapy with radiation therapy was explored in this study. CG7870 is an oncolytic adenovirus in which tumor-specific promoters are driving the expression of E1A and E1B proteins. The effects of combined treatment with CG7870 and radiation on cultured cells were determined in cytotoxicity and virus yield assays. The antitumor efficacy of CG7870 (1 × 107 particles/mm3 of tumor), 10 Gy of local radiation or both was evaluated in established subcutaneous LNCaP xenografts in nude mice. In vitro, the dual agent treatment resulted in synergistically enhanced potency at suboptimal doses of radiation and virus. Virus yield in irradiated cells increased relative to yield in nonirradiated cells without compromising the specificity of the vector for its target cell types. In vivo, CG7870 treatment alone suppressed tumor growth and extended tumor nonprogression time. The average tumor-volume of the groups treated with CG7870 only and radiation only was 121 and 126% of baseline, respectively, 39 days after treatment. The average tumor-volume of the combination group was 34% of baseline 39 days after a single dose of treatment. No significant body weight loss was observed in any treatment group. There was a significant drop in serum level of prostate-specific antigen (PSA) in the combination group compared to the group treated with either agent alone. In mice treated with CG7870 only or radiation only, serum PSA levels changed to 26 and 383% of baseline, respectively, by study day 46. In contrast, PSA levels in mice treated with CG7870 plus radiation decreased to less than 11% of baseline by study day 46. Histological analysis of tumor sections collected from the combination group revealed enhanced necrosis and more apoptotic cells. Combination of CG7870 with radiotherapy significantly increased antitumor efficacy compared to either agent alone. These results suggest that CG7870 in combination with radiation has improved antitumor efficacy at lower doses and with no additional side effects.

Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors.

The use of adenovirus type 5 (Ad5) for cancer therapy is limited by deficiency of its primary cell attachment receptor, coxsackie and adenovirus receptor (CAR), on cancer cells. Ad5 retargeting to alternate receptors through fiber genetic modification can be used to circumvent CAR dependence of its tropism, and thereby achieve infectivity enhancement. Here we propose and test a novel “complex mosaicism” approach for fiber modification, which combines serotype chimerism with peptide ligand(s) incorporation in a single-fiber molecule. We incorporated integrin-binding peptide RGD-4C in the HI-loop, at the carboxy (C)-terminus, or both locales of the Ad3 knob, in the context of Ad5/3 chimera fiber in order to retarget simultaneously the Ad vector to integrins and Ad3 receptors. The infectivity enhancement of the fiber modifications was assessed in various cancer cell lines as cancer-targeting models. Replication-defective complex mosaic Ad-luc vectors bearing chimeric fiber (F.5/3), with or without C-terminal RGD-modification of Ad3 knob, demonstrated up to 55-fold gene transfer increase in bladder cancer cell lines. Although this augmentation was primarily due to Ad3 receptor targeting, some contribution of RGD-mediated integrin-targeting was also observed, suggesting that complex mosaic modification can function in a dual-receptor targeting via a single Ad3 fiber knob.

Enhanced gene transfer and oncolysis of head and neck cancer and melanoma cells by fiber chimeric oncolytic adenoviruses.

Purpose: The purpose of this study was to evaluate a fiber knob replacement strategy to improve infectivity and efficacy of Ad5 fiber chimeric oncolytic viruses for treatment of melanoma and head and neck cancers (HNC). Experimental Design: Adenoviral receptors and transduction levels were used to determine the level of infectivity of fiber-modified, green fluorescent protein–expressing, replication-deficient viruses in a panel of melanoma and HNC cell lines in vitro. Virus yield and cytotoxicity assays were used to determine the tumor specificity and virus replication-mediated cytotoxicity of the fiber-modified oncolytic viruses in the same panel of melanoma and HNC in vitro. Xenograft tumor models were used to assess the antitumor activity of those fiber-modified chimeric viruses compared with the parental virus. Results: Marker gene expression following gene transfer of the fiber chimeric vectors in melanoma and HNC cell lines was ∼10-fold higher than that obtained with parental Ad5 vector. The fiber chimeric oncolytic variants mediated killing of melanoma and HNC cells that was 2- to 576-fold better than with the parental virus. In addition, fiber chimeric variants produced 2- to 7-fold more progeny virus in tumor cells than the parental virus. Moreover, a high multiplicity of infection was needed for the fiber chimeric viruses to produce cytotoxicity in normal cells. A significantly stronger antitumor response and survival advantage were shown in the tested melanoma and HNC xenograft models following i.t. injections. Conclusions:In vitro and in vivo studies showed the improved transduction, replication, cytotoxicity, antitumor efficacy, and survival advantage in melanoma and HNC tumor models, suggesting a potential use of these oncolytic agents for the treatment of melanoma and HNCs.

Dual promoter-controlled oncolytic adenovirus CG5757 has strong tumor selectivity and significant antitumor efficacy in preclinical models.

Purpose: Transcriptionally controlled oncolytic adenovirus CG5757 is engineered with two tumor-specific promoters from E2F-1 and human telomerase reverse transcriptase genes. This virus has broad anticancer spectrum and higher specificity. The objective of the current study is to show its antitumor selectivity and therapeutic potential. Experimental Design: The antitumor specificity of E2F-1 and human telomerase reverse transcriptase promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and cytotoxicity assays followed by a newly developed ex vivo tumor culture assay. Subsequently, in vivo antitumor efficacy and toxicology studies were carried out to assess the therapeutic potential of this oncolytic agent. Results: In a broad panel of cells, E2F-1 and human telomerase reverse transcriptase promoters were activated in a tumor-selective manner. Under the control of these promoters, expression of E1a and E1b genes appears only in tumor cells. This specificity is extended to viral replication and hence the cytotoxicity in a broad range of cancer cells. Furthermore, CG5757 only replicates in cancer tissues but not in normal tissues that are derived from clinical biopsies. The safety profile was further confirmed in in vivo toxicology studies, and strong efficacy was documented in several tumor xenograft models after CG5757 was given via different routes and regimens. Conclusions: CG5757 has strong antitumor selectivity and potency. It has low toxicity and has great potential as a therapeutic agent for different types of cancers.

Chapter 10 – Development of Attenuated Replication Competent Adenoviruses (ARCAs) for the Treatment of Prostate Cancer

Publisher Summary This chapter discusses the use of attenuated replication competent adenoviruses (ARCAs) for the treatment of prostate cancer. To test the feasibility of the ARCA technology, the prostate-specific enhancer (PSE) fragment was engineered into the adenovirus genome and generated a first generation virus, CV706. Another ARCA, CV787 was created to test the possibility of increasing virus cytotoxicity. The efficacy of the ARCAs was evaluated in many animal models. CV787 is capable of eliminating distant mouse xenograft tumors with a single intravenous injection. Synergy of prostate cancer-specific adenovirus variants has been demonstrated when combined with conventional therapies including radiotherapy and chemotherapy. Synergy of CV787 and docetaxel has opened a single-dose curative therapeutic window. CV787 will resolve the issues of safety and efficacy and hopefully point to a new mode of cancer chemotherapy, one that includes the use of targeted cytolytic adenoviruses. Clinical trials with CV706 and CV787 have proved to be effective against human prostate tumors.

311. Oncolytic Adenovirus CG5757 Preferentially Infects Primary Human Tumor Tissues and Shows Strong Anti-Tumor Activity in Tumor Models Following Systemic Administration

Top of pageAbstract A human adenovirus serotype 5 based oncolytic virus CG5757 was designed to preferentially replicate in and kill cancer cells that have a defective retinoblastoma (Rb) pathway and overexpress telomerase. Tumor selectivity of CG5757 was examined using a newly developed ex vivo primary tumor tissue culture model. Primary human colorectal tumors were obtained after surgical resection and placed in Millicell insert with media containing hormones, which can keep tissue viable for about 5 days. The paired tumor or normal tissues from same tissue sample were used in a viral infection experiment to determine the tumor selectivity of CG5757. Cultured tissues were infected with CG5757 or wild type (wt) Ad5 for five days, and progeny viral production was determined by TCID50 assay. Results of the paired tissue cultures from five colon cancer patients indicate that wt Ad5 has similar viral productivity in tumor and normal tissues, whereas CG5757 has higher productivity in tumor tissues. In general, CG5757 produces 100- to 1,000-fold more virus in tumor tissue compared to normal tissue. Immunohistochemical staining for adenoviral E1A shows that CG5757 has positive infection only in the cancer tissue (colon), whereas no signal is detected in normal tissues of colon, pancreas or spleen. Similarly in vitro viral specificity characterization using a panel of tumor cell lines also suggest a strong tumor selectivity of CG5757. Anti-tumor efficacy of CG5757 following intravenous injection was examined in a subcutaneous tumor model. In this study, pre-established prostate cancer LNCaP xenografts in nude mice were treated with two intravenous injections of 4|[times]|1010 viral particles of CG5757 given at a 3-day interval. Significant anti-tumor efficacy was observed in CG5757-treated animals. Eight weeks after treatment, CG5757-treated animals exhibited approximately 72% inhibition of tumor growth. These studies demonstrate the potential therapeutic efficacy of such dual promoter-controlled oncolytic adenoviruses in cancers that are Rb-defective and telomerase-positive.

445. CG5757, an Oncolytic Adenovirus for the Treatment of Retinoblstoma (Rb) Pathway-Defective and Telomerase-Positive Cancers

Replication-selective oncolytic viruses hold promise for the treatment of cancer. Among this novel group of therapeutics are oncolytic adenoviruses engineered with tumor-specific transcriptional response elements (TRE) controlling essential genes. These vectors replicate selectively in cancer cells, leading to expression of toxic viral products and oncolysis mediated by viral replication. We and others have compared different transcriptional control strategies by placing one or more tumor-specific TREs upstream of different viral genes including E1A, E1B and E4. Relocation of the viral packaging signal was also investigated. One such oncolytic virus, CG5757, was generated by replacing the E1A and E1B endogenous promoters with promoters derived from the human E2F-1 and telomerase reverse transcriptase (hTERT) genes, respectively. The E2F-1 promoter is activated in Rb-defective tumor types, a pathway mutated in approximately 85% of all cancers. Likewise, telomerase is aberrantly expressed in over 90% of tumors. CG5757 also has a deletion in the coding region of the E1B 19k gene, a Bcl2-like viral antiapoptotic protein, to increase vector cytotoxicity. CG5757 shows strong tumor selectivity. In vitro, expression of E1A and E1B genes was highly restricted to Rb-defective and hTERT-positive cancer cells, including Hep3B (hepatocellular carcinoma), LoVo (colorectal carcinoma), A549 (lung cancer), Panc-1 (pancreatic cancer), 253J B-V (bladder cancer), and Hela (cervical cancer). In normal cells, including a lung fibroblast cell line (WI-38) and several other human primary cell lines (HRE, BSMC, PrEC, HMEC and HMVEC-L), no E1 expression could be detected from infection with CG5757. The transcriptional control of E1 gene expression also correlated with selective viral replication in target cells. CG5757 replicates similarly to wild-type virus in tumor cells, but its replication is, on average, 1,000-times less efficient in normal cells. In a viral cytotoxicity assay, CG5757 destroys tumor cells 100- to 10,000-times more efficiently than normal cells. Comparisons of the cytoxicity of CG5757 in tumor cells versus normal cells (normalized for transduction efficiency with wild-type adenovirus 5) yielded high selectivity indices, some of which were greater than 1000. In vivo, strong antitumor activity was seen using CG5757 in NCR nude mice with subcutaneous lung cancer (A549) and bladder transitional cell carcinoma (253J B-V) xenografts. With respect to the 253J B-V model, four weeks after treatment the average tumor volume in animals treated with four consecutive daily intratumoral injections of CG5757 (4×108 particles/mm3 of tumor) decreased to 72% of baseline while the control group had an increase to 944% of baseline. Furthermore, 50% of treated animals had complete regression of the 253J B-V tumor xenografts. The potential therapeutic efficacy of such dual promoter controlled oncolytic adenoviruses in cancers that are Rb-defective and hTERT-positive has been demonstrated.