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In 1996, when the first suicide gene therapy with an adeno­virus vector carrying HSV-tk gene (Ad-HSV-tk) for prostate cancer was conducted at Baylor College of Medicine,24 our research group had started international collaborative studies to develop cancer gene therapy at Okayama University. From March 2001 to July 2005, we completed the first clinical study on suicide gene therapy for prostate cancer in Japan, demonstrating the safety, gene expression, and clinical effects of Ad-HSV-tk gene therapy for locally advanced CRPC.11 Our initial development strategy on in situ gene therapy for prostate cancer was to combine suicide gene therapy with immune gene therapy using interleukin (IL)-12 (Ad-IL-12), in order to augment direct and indirect “bystander” effects.25 As for Ad-IL-12, a Phase I/IIa dose escalation study, start­ing from 1.0 × 1010 vp and reaching 5.0 × 1012 vp for locally advanced CRPC and mCRPC, was conducted at Okayama University from May 2008, following approval from the Japanese Government. In March 2010, the 10th patient was enrolled into this clinical study, as the first patient in dose level 4 of 5 × 1011 vp. This patient showed clear objective responses, with gradual PSA decline and disappearance of a single metastatic internal iliac LN, after three scheduled monthly injections into prostatic lesions. However, adverse events, such as high fever (grade 2–3) and liver dysfunction (grade 2), were also noted. The patient refused to continue treatment after two additional monthly injections, probably due to high fever following each injection. Therefore, this study was put on hold until we could obtain sufficient evi­dence on the safety of Ad-IL-12 from other investigational studies.

Contrary to Ad-IL-12, Ad-REIC showed remark­able safety profiles, although we used the same replication-deficient, the so-called “first-generation” adenovirus type 5, vectors. In fact, the present patient received 15 repeated injec­tions with 1 × 1012 vp for two years and showed only transient fever, treatable with antipyretics. The first-generation adeno­virus vectors are reported to have two major limitations: first, these vectors usually mediate a short-term gene expression, and, second, these vectors tend to elicit relatively strong immune and inflammatory responses.26 In terms of drug development, we already have a huge amount of long-term accumulated data on safety and pharmacology derived from various clinical trials with first-generation adenoviruses. As for type-specific neutralizing antibodies raised against ade­novirus vector particles, it was reported as early as 1999, by Swisher et al, that even with high titers, antibodies in blood would not reduce gene transfer by repeated intratumoral injections, as shown in their initial study on adenovirus-mediated p53 gene transfer (Ad-p53) in advanced non-small-cell lung cancer.27 In addition, many thousands of patients have received Ad-p53 gene therapy in clinical trials, mostly in the USA and China, with some remarkable clinical out­comes. Unfortunately, Ad-p53 (Advexin®) did not receive the U.S. Federal Drug Administration (FDA) approval.28 On the other hand, in 2003, Ad-p53, trademarked as Gen­dicine, was approved by the Chinese State Food and Drug Administration, and Gendicine has been used to treat head and neck squamous cell carcinoma and other cancers, with­out any serious adverse events related to the first-generation adenovirus vectors.29 Therefore, the next challenge for in situ gene therapy using the first-generation adenovirus vectors is to find ways to improve direct and indirect “bystander” effects with these well-characterized vectors.

In an orthotopic prostate cancer model with pre-established lung metastases using RM-9 mouse prostate cancer cells, in situ Ad-mouse (m)IL-12 gene therapy clearly sup­pressed pre-established lung metastases.25 Although the com­bination of Ad-HSV-tk plus ganciclovir and Ad-mIL-12 gene therapy inhibited local tumor growth more than single treat­ment protocols, combination therapy did not suppress pre-established lung metastases more than Ad-mIL-12 gene therapy alone. Combination therapy also did not achieve significantly better animal survival compared to single treat­ment protocols, probably due to complex cooperative thera­peutic activities and host immunological responses.30 In the same orthotopic mouse model, however, Ad-mREIC as well as Ad-REIC significantly suppressed local tumor growth and pre-established lung metastases, and prolonged mice sur­vival.22,31 These animal studies clearly demonstrate that, in terms of the induction of direct and indirect “bystander” effects, Ad-REIC monotherapy is extremely simple and effec­tive, in comparison to combination with Ad-HSV-tk plus gan­ciclovir and Ad-IL−12. Moreover, the results mentioned above indicate that the powerful “bystander” effects medicated by Ad-REIC might overcome several unresolved problems related to conventional adenovirus vectors, including the inability to infect every cell in the tumor, short-term gene expression, and inability of systemic targeted delivery.

More recently, we have reviewed previous fundamental studies and summarized the anticancer mechanisms of in situ Ad-REIC gene therapy, featuring the characteristic synergis­tic effects between cancer-selective apoptosis and augmenta­tion of antitumor immunity.23 Paul Ehrlich’s “Magic Bullet” concept based on selective toxicity has inspired many scientists to develop innovative therapeutics for more than 100 years.32 As for selective toxicity, Ad-REIC offered a new, sophisti­cated way to induce cancer-selective killing according to the differences in Ad-REIC-induced unfolded protein responses (UPR) between cancer cells and normal cells. Since REIC/Dkk‑3 expression is significantly downregulated in a broad range of human cancer cells18–20,33–38 while being expressed ubiquitously in normal cells,39 these endogenous expression statuses of REIC/Dkkk-3 proteins appear to define the sensitivity to Ad-REIC-induced UPR. As a new “Magic Bullet” for cancer gene therapy, ER-stress-induced apopto­sis due to sustained overactivation of UPR was mediated in Ad-REIC-infected cancer cells, while ER-stress-induced overproduction of IL-7 was mediated in co-infected normal cells including cancer-associated fibroblasts.21 In addition to the fact that IL-7 excessively produced by Ad-REIC activates innate immunity involving NK cells, the secreted REIC pro­teins with potent immunomodulatory functions augment antitumor immunity. As demonstrated in Figure 7, dendritic cells, induced by secreted REIC proteins, acquire cancer anti­gens from apoptotic cancer cells and induce class 1-restricted CD8+ cytotoxic T lymphocytes (CTLs).22,23 These CTLs play a major role in systemic antitumor immunity of in situ Ad-REIC as a personalized therapeutic cancer vaccine.

In the current patient, two initial injections of Ad-REIC, of as little as 1.2 mL of vector solution, induced almost com­plete degeneration of the injected metastatic obturator lymph node of more than 30 mL in volume. This suggests that, in an optimum environment, the direct effects of Ad-REIC are strong enough to overcome the inability of the adenovirus to infect all targeted cancer cells. Furthermore, similar degenera­tion developed in distant, noninjected metastatic common and external iliac LNs, indicating the induction of robust systemic antitumor immunity by in situ Ad-REIC. Although complete control of metastatic pelvic LNs was achieved following several boosting injections into pelvic LNs, newly developed multiple para-aortic LN metastases progressed in spite of the presence of Ad-REIC-mediated systemic antitumor immunity. For this progression, there are two possible interpretations: first, heterogenic clones of cancer cells developed with tumor-associated antigens significantly different from those of primary metastatic cancer cells, and, second, same clones of primary metastatic cancer cells developed under a different immuno­suppressive tumor microenvironment. The latter is supported by the recent concept of “cancer immunoediting” and find­ings related to immunosuppressive networks and checkpoints controlling antitumor immunity.40,41 In the treatment of para-aortic LNs, since each injection showed a remarkable effect only on the injected LN, injections into every metastatic LNs including re-enlarged LNs were necessary for sufficient con­trol of metastatic LNs. Probably, the induced systemic effects were compromised by an immunosuppressive microenviron­ment established in each metastatic LN.