Experimental adenovirus gene therapy for cancer patients with advanced tumors : Translational medicine from basic research to the patient's bedside

The treatment results of many cancers have improved, but there are still challenges, such as metastatic cancers that mostly lack curative treatments. Oncolytic adenoviruses provide a new therapeutic option. Adenoviruses can be modified to replicate specifically in cancer cells and thereby causing the death of cancer cells (oncolysis). Adenovirus treatments are well tolerated and they can be combined with conventional treatments. In the pre-clinical section of this thesis we evaluated the combination of adenoviruses and radiotherapy. We studied the mechanism of radiation-mediated upregulation of adenoviral transgenes. Three cancer cell lines were used: M4A4-LM3 (breast cancer), PC-3MM2 (prostate cancer) and LNM35/eGFP (lung cancer). Cancer cells were exposed to radiation and infected with various replication-deficient adenoviruses. The transgene expression with or without radiation was evaluated. The radiation response was also modulated by DNA protein kinase-, heat shock protein 90- and topoisomerase-I inhibitor. Radiation increased adenovirus transgene expression (luciferase or GFP) regardless of the transgene, promoter, cancer cell line or radiation dose. We showed that enhancement of transgene expression is mediated through genotoxic stress regulation and repair. This synergy can be used for therapeutic benefit. In the clinical section 157 cancer patients with advanced solid tumors were treated with different oncolytic adenoviruses. The safety, efficacy and immunological responses of treatments were evaluated. The safety was monitored by clinical chemistry, cytokine measurements and CTCAE analysis for adverse events. Immunological responses were quantified by neutralizing antibodies, viral genomes in the blood and measuring responses in tumor- and adenovirus-specific T-cells. RECIST analysis and tumor markers were applied to treatment efficacy analysis. We also compared the safety and efficacy of a serial treatment scheme, three rounds of virus, to a single treatment. Adenovirus treatments were well tolerated and commonly detected clinical adverse events were of grade 2 or less including injection or tumor site pain, nausea, fever and fatigue. Six out of 157 treated patients (3.8 %) experienced a grade 4 adverse event. Serious adverse events were seen in 11 patients (7.0 %). No treatment related deaths occurred. According to RECIST analysis disease control (= stable disease or better) was seen in 40.0 - 74.0 % of patients and a stabilization or a decrease in tumor markers for 23.1 - 70.6 % of patients. Serial treatment was as well tolerated as single treatment, but results suggested a better median survival even though statistical significance was not reached. These findings represent a good justification for forthcoming clinical trials.Useiden syöpien hoitotulokset ovat parantuneet viime vuosina, mutta kuitenkaan useimpiin levinneisiin syöpiin ei ole tarjolla parantavia hoitoja. Uusia hoitomenetelmiä tulee siis jatkuvasti kehittää. Onkolyyttiset adenovirukset ovat eräs lupaava uusi hoitomuoto. Adenovirukset ovat yleisiä luonnossa esiintyviä viruksia, jotka aiheuttavat ihmisissä lieviä sairauksia kuten hengitystieinfektioita. Onkolyyttisiä adenoviruksia on muokattu niin, että ne tuhoavat vain syöpäsoluja (onkolyysi) säästäen terveitä soluja. Hoidot tällaisilla viruksilla ovat yleensä potilaille hyvin siedettyjä ja niitä voidaan yhdistää myös muihin hoitoihin. Tässä väitöskirjassa tutkittiin sädehoidon ja adenovirusten yhteisvaikutuksia rinta, eturauhas- ja keuhkosyöpäsoluissa. Adenovirukset kykenevät kuljettamaan mukanaan syöpäsoluihin geenejä joiden tuottamilla proteiineilla on syöpäsoluja tuhoava vaikutus. Tällaisia geenejä kutsutaan siirtogeeneiksi. Tutkimuksessa arvioitiin miten sädehoito vaikuttaa näiden adenovirusten siirtogeenien määrään. Siirtogeenien ja niiden tuottamien proteiinien määrä oli suurempi sellaisissa soluissa joita oli säteilytetty ennen adenovirusten antamista. Näiden tulosten perusteella vaikuttaa olevan hyödyllistä yhdistää adenovirusgeeniterapia sekä sädehoito. Tulokset luovat biologisen perustan yhdistelmähoidolle, joka voi parhaimmillaan tulevaisuudessa parantaa hoidettujen potilaiden ennustetta. Väitöskirjan kliinisessä osassa tutkittiin adenovirushoidon turvallisuutta, tehoa ja immunologista vastetta levinnyttä syöpää sairastavilla potilailla (157 potilasta). Turvallisuutta arvioitiin verikokein ja hoitoihin liittyvät haittavaikutukset luokiteltiin vakavuuden mukaan tasoihin 1 5. Hoidon immunologista vastetta potilaissa arvioitiin mm. mittaamalla viruksen ja T-valkosolujen määrää verenkierrossa. Hoidon tehoa arvioitiin radiologisella kuvantamisella sekä syöpämerkkiaineilla. Lisäksi sarjahoitoa, joka tarkoittaa kolmea peräkkäistä virushoitoa, verrattiin kertahoitoon. Adenovirushoidot olivat hyvin siedettyjä ja yleisesti haitat olivat lieviä tasoa 1 - 2 kuten kiputuntemukset, pahoinvointi, kuume ja väsymys. Kuudella potilaalla (3.8 %) havaittiin tasoa 4 oleva haittavaikutus ja vakavia haittoja 11 potilaalla (7.0 %). Hoitoon liittyviä kuolemia ei ollut. Radiologinen hoitovaste saavutettiin 40.0 - 74.0 %:lle potilaista ja kasvainmerkkiaineilla mitattuna 23.1 - 70.6 %:lle. Sarjahoito oli yhtä turvallinen kuin kertahoito, mutta sillä oli suotuisa vaikutus potilaiden elossaoloaikaan, joskaan ero ei ollut tilastollisesti merkittävä. Tuloksemme ovat lupaavia ja siksi Pohjois-Euroopan ensimmäinen kliininen tutkimus onkolyyttisellä viruksella on aloitettu

Oncolytic Adenovirus With Temozolomide Induces Autophagy and Antitumor Immune Responses in Cancer Patients

Peer reviewe

Immunological Effects of a Tumor Necrosis Factor Alpha-Armed Oncolytic Adenovirus

For long it has been recognized that tumor necrosis factor alpha (TNFa) has anticancer characteristics, and its use as a cancer therapeutic was proposed already in the 1980s. However, its systemic toxicity has limited its usability. Oncolytic viruses, selectively cancer-killing viruses, have shown great potency, and one of their most useful aspects is their ability to produce high amounts of transgene products locally, resulting in high local versus systemic concentrations. Therefore, the overall magnitude of tumor cell killing results from the combination of oncolysis, transgene-mediated direct effect such as TNFa-mediated apoptosis, and, perhaps most significantly, from activation of the host immune system against the tumor. We generated a novel chimeric oncolytic adenovirus expressing human TNFa, Ad5/3-D24-hTNFa, whose efficacy and immunogenicity were tested in vitro and in vivo. The hTNFa-expressing adenovirus showed increased cancer-eradicating potency, which was shown to be because of elevated apoptosis and necrosis rates and induction of various immune responses. Interestingly, we saw increase in immunogenic cell death markers in Ad5/3-d24-hTNFa-treated cells. Moreover, tumors treated with Ad5/3-D24-hTNFa displayed enhanced presence of OVA-specific cytotoxic T cells. We thus can conclude that tumor eradication and antitumor immune responses mediated by Ad5/3-d24-hTNFa offer a new potential drug candidate for cancer therapy

Integrin targeted oncolytic adenoviruses Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of patients with advanced chemotherapy refractory solid tumors

The safety of oncolytic viruses for treatment of cancer has been shown in clinical trials while antitumor efficacy has often remained modest. As expression of the coxsackie-adenovirus receptor may be variable in advanced tumors, we developed Ad5-D24-RGD, a p16/Rb pathway selective oncolytic adenovirus featuring RGD-4C modification of the fiber. This allows viral entry through alpha-v-beta integrins frequently highly expressed in advanced tumors. Advanced tumors are often immunosuppressive which results in lack of tumor eradication despite abnormal epitopes being present. Granulocyte-macrophage colony stimulating factor (GMCSF) is a potent activator of immune system with established antitumor properties. To stimulate antitumor immunity and break tumor associated immunotolerance, we constructed Ad5-RGD-D24-GMCSF, featuring GMCSF controlled by the adenoviral E3 promoter. Preliminary safety of Ad5-D24-RGD and Ad5-RGD-D24-GMCSF for treatment of human cancer was established. Treatments with Ad5-D24-RGD (N = 9) and Ad5-RGD-D24-GMCSF (N = 7) were well tolerated. Typical side effects were grade 1-2 fatigue, fever and injection site pain. 77% (10/13) of evaluable patients showed virus in circulation for at least 2 weeks. In 3 out of 6 evaluable patients, disease previously progressing stabilized after a single treatment with Ad5-RGD-D24-GMCSF. In addition, 2/3 patients had stabilization or reduction in tumor marker levels. All patients treated with Ad5-D24-RGD showed disease progression in radiological analysis, although 3/6 had temporary reduction or stabilization of marker levels. Induction of tumor and adenovirus specific immunity was demonstrated with ELISPOT in Ad5-RGD-D24-GMCSF treated patients. RGD modified oncolytic adenoviruses with or without GMCSF seem safe for further clinical development