Syövän T-soluterapioiden tehostus aseistettujen onkolyyttisten adenovirusten avulla

Adoptive T-cell therapies (ACT) are emerging as essential treatments for cancer patients with immunologically active tumours, such as melanoma. Immunologically silent tumours, however, require further stimulation. Oncolytic viruses provide an intriguing option for immune system activation, as they induce danger signalling, immunogenic cell death, and tumour epitope spreading. This study investigated oncolytic adenovirus coding for human Tumour Necrosis Factor alpha (TNFα) and Interleukin 2 (IL2) as an enhancer for ACT. Syrian hamsters permissive for human adenovirus replication provided a model for tumour-infiltrating lymphocyte (TIL) therapy with oncolytic Ad5/3-E2F-d24-hTNFα-IRES-hIL2. Complementary studies were conducted in mice with replication-incompetent viruses, providing data on adenovirally-delivered transgenes with receptor-modified T cells. Both replication-incompetent viruses and oncolytic viruses were able to enhance the antitumour efficacy of ACT. Combined with TIL therapy, Ad5/3-E2F-d24-hTNFα-IRES-hIL2 was able to cure 100% of the animals from tumours. The cured animals resisted tumour rechallenge, indicating formation of immunologic memory. TNFα enhanced chemokine expression in tumours, which attracted the infused cell graft into tumours. The transgenes also induced the presence of T cells, B cells, natural killer cells, and antigen-presenting cells in tumours, yet they lowered the levels of immunosuppressive M2 macrophages. Moreover, local treatment induced systemic antitumour efficacy in non-injected distant tumours. Both tumours showed similar profiles in intratumoural immune cells, indicating systemic changes in the immune system. The animals treated with cytokine-armed viruses showed no signs of systemic toxicity. Furthermore, the local delivery of IL2 was safer and more efficient than systemic IL2 in regard to ACT, suggesting that adenovirally delivered IL2 could replace the toxic, systemic IL2 in ACT protocols. To conclude, oncolytic adenovirus coding for immunostimulatory cytokines is a potential enabler for T-cell therapies.Syövän immunoterapiat pyrkivät aktivoimaan kehon omaa puolustusjärjestelmää tunnistamaan ja tuhoamaan syöpäsolukkoa. Immunoterapioihin kuuluvat muun muassa hoidot immuunijärjestelmää aktivoivilla sytokiineilla ja vasta-aineilla, syöpäsoluihin kohdennetut onkolyyttiset virukset ja T-soluterapian eri muodot. Tässä työssä yhdistettiin toisiaan tukevia immunoterapiamuotoja: onkolyyttiset virukset muokattiin tuottamaan sytokiineja, jotka potentiaalisesti tehostavat T-soluterapiaa. Onkolyyttiset virukset tuhoavat monistuessaan syöpäsoluja ja levittävät elimistöön syövän merkkiaineita, jotka aktivoivat immuunijärjestelmää hyökkäämään kasvainta vastaan. Tämä ei kuitenkaan yleensä riitä tuhoamaan kasvainta kokonaan. Sen vuoksi liitimme viruksen genomiin kaksi sytokiinigeeniä: interleukiini 2:n ja tuumorinekroositekijä alfan, joita infektoituneet syöpäsolut alkavat tuottaa viruksen monistuessa. Molemmat sytokiinit aktivoivat immuunijärjestelmän T-soluja, jotka ovat elimistössä päävastuussa epätavallisten solujen tuhoamisesta. Lisäksi sytokiinit houkuttelevat T-soluja kasvaimeen ja edistävät niiden jakaantumista. Koska viruksen kantamat sytokiinit vaikuttavat pääasiassa T-soluihin, virushoito yhdistettiin T-soluterapiaan. T-soluterapiassa potilaasta eristetään syöpää tunnistavia T-soluja, joita voidaan monistaa ja muokata ennen kuin ne annetaan potilaalle takaisin. T-soluhoidot tehoavat hyvin verisyöpiin, mutta kiinteissä kasvaimissa ne toimivat heikosti. Tässä väitöskirjassa osoitettiin, että valitut virusten kuljettamat sytokiinit parantavat T-soluterapian tehoa kiinteissä kasvaimissa paremmin yhdessä kuin erikseen. Yhdessä T-soluterapia ja sytokiineja tuottavat virukset paransivat kaikki hoitoryhmän hamsterit. Lisäksi parantuneet eläimet saivat immunologisen suojan uusien saman tyyppisten kasvainten muodostumista vastaan. Virushoidon vaikutus ei jäänyt vain paikalliseksi: kun eläin kantoi kahta kasvainta, joista vain toista hoidettiin, myös hoitamattoman kasvaimen kasvu hidastui. Lisäksi sytokiineja tuottavien virusten avulla T-solusiirre kulkeutui tehokkaammin sekä hoidettuun että hoitamattomaan kasvaimeen verrattuna aseistamattomaan virukseen. Sytokiineja tuottavien virusten avulla pystyttiin myös korvaamaan T-soluterapian vaatima perinteinen sytokiinihoito, joka aiheuttaa usein vakavia sivuoireita. Hoito sytokiineja tuottavilla viruksilla ei aiheuttanut haitallisia muutoksia kudoksissa. Tutkimustulokset tukevat kliinistä koetta, jossa virushoitojen ja T-soluterapian turvallisuutta testataan melanoomapotilaissa

Immunterapi vid cancer - ett tillväxtområde

English summar

Cytokine-Coding Oncolytic Adenovirus TILT-123 Is Safe, Selective, and Effective as a Single Agent and in Combination with Immune Checkpoint Inhibitor Anti-PD-1

Oncolytic viruses provide a biologically multi-faceted treatment option for patients who cannot be cured with currently available treatment options. We constructed an oncolytic adenovirus, TILT-123, to support T-cell therapies and immune checkpoint inhibitors in solid tumors. Adenoviruses are immunogenic by nature, are easy to produce in large quantities, and can carry relatively large transgenes. They are the most commonly used gene therapy vectors and are well tolerated in patients. TILT-123 expresses two potent cytokines, tumor necrosis factor alpha and interleukin-2, to stimulate especially the T-cell compartment in the tumor microenvironment. Before entering clinical studies, the safety and biodistribution of TILT-123 was studied in Syrian hamsters and in mice. The results show that TILT-123 is safe in animals as monotherapy and in combination with an immune checkpoint inhibitor anti-PD-1. The virus treatment induces acute changes in circulating immune cell compartments, but the levels return to normal by the middle of the treatment period. The virus is rapidly cleared from healthy tissues, and it does not cause damage to vital organs. The results support the initiation of a phase 1 dose-escalation trial, where melanoma patients receiving a tumor-infiltrating lymphocyte therapy are treated with TILT-123 (NCT04217473).Peer reviewe

TNFa and IL2 Encoding Oncolytic Adenovirus Activates Pathogen and Danger-Associated Immunological Signaling

In order to break tumor resistance towards traditional treatments, we investigate the response of tumor and immune cells to a novel, cytokine-armed oncolytic adenovirus: Ad5/3-d24-E2F-hTNFa-IRES-hIL2 (also known as TILT-123 and OAd.TNFa-IL2). There are several pattern recognition receptors (PRR) that might mediate adenovirus-infection recognition. However, the role and specific effects of each PRR on the tumor microenvironment and treatment outcome remain unclear. Hence, the aim of this study was to investigate the effects of OAd.TNFa-IL2 infection on PRR-mediated danger- and pathogen-associated molecular pattern (DAMP and PAMP, respectively) signaling. In addition, we wanted to see which PRRs mediate an antitumor response and are therefore relevant for optimizing this virotherapy. We determined that OAd.TNFa-IL2 induced DAMP and PAMP release and consequent tumor microenvironment modulation. We show that the AIM2 inflammasome is activated during OAd.TNFa-IL2 virotherapy, thus creating an immunostimulatory antitumor microenvironment.Peer reviewe

Adenovirus Coding for Interleukin-2 and Tumor Necrosis Factor Alpha Replaces Lymphodepleting Chemotherapy in Adoptive T Cell Therapy

Lymphodepleting preconditioning with high-dose chemotherapy is commonly used to increase the clinical efficacy of adoptive T cell therapy (ACT) strategies, however, with severe toxicity for patients. Conversely, oncolytic adenoviruses are safe and, when engineered to express interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-alpha), they can achieve antitumor immunomodulatory effects similar to lymphodepletion. Therefore, we compare the safety and efficacy of such adenoviruses with a cyclophosphamide-and fludarabine- containing lymphodepleting regimen in the setting of ACT. Human adenovirus (Ad5/3-E2F-D24-hTNF-alpha-IRES-hIL-2; TILT-123) replication was studied using a Syrian hamster pancreatic tumor model (HapT1) infused with tumor- infiltrating lymphocytes (TILs). Using the oncolytic virus instead of lymphodepletion resulted in superior efficacy and survival. Immune cells responsive to TNF-alpha IL-2 were studied using an immunocompetent mouse melanoma model (B16. OVA) infused with ovalbumin-specific T (OT-I) cells. Here, the adenovirus approach improved tumor control together with increased intratumoral Th1 cytokine levels and infiltration of CD8+ T cells and CD86+ dendritic cells. Similar to humans, lymphodepleting preconditioning caused severe cytopenias, systemic inflammation, and damage to vital organs. Toxicity was minimal in adenovirus- and OT-Itreated mice. These findings demonstrate that ACT can be effectively facilitated by cytokine-coding adenovirus without requiring lymphodepletion, a rationale being clinically investigated.Peer reviewe

Systemic Delivery of Oncolytic Adenovirus to Tumors Using Tumor-Infiltrating Lymphocytes as Carriers

Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.Peer reviewe

Interleukin 8 activity influences the efficacy of adenoviral oncolytic immunotherapy in cancer patients

After the landmark approval of T-VEC, oncolytic viruses are finding their way to the clinics. However, response rates have still room for improvement, and unfortunately there are currently no available markers to predict responses for oncolytic immunotherapy. Interleukin 8 (IL-8) production is upregulated in many cancers and it also connects to several pathways that have been shown to impair the efficacy of adenoviral immunotherapy. We studied the role of IL-8 in 103 cancer patients treated with oncolytic adenoviruses. We found high baseline serum IL-8 concentration to be independently associated with poor prognosis (p <0.001). Further, normal baseline IL-8 was associated with improved prognostic potential of calculation of the neutrophil-to-lymphocyte ratio (p <0.001). Interestingly, a decrease in IL-8 concentration after treatment with oncolytic adenovirus predicted better overall survival (p <0.001) and higher response rate, although this difference was not significant (p=0.066). We studied the combination of adenovirus and IL-8 neutralizing antibody ex vivo in single cell suspensions and in co-cultures of tumor-associated CD15+ neutrophils and CD3+ tumor-infiltrating lymphocytes derived from fresh patient tumor samples. These results indicate a role for IL-8 as a biomarker in oncolytic virotherapy, but additionally provide a rationale for targeting IL-8 to improve treatment efficacy. In conclusion, curtailing the activity of IL-8 systemically or locally in the tumor microenvironment could improve anti-tumor immune responses resulting in enhanced efficacy of adenoviral immunotherapy of cancer. © Taipale et al.Peer reviewe

Abscopal Effect in Non-injected Tumors Achieved with Cytokine-Armed Oncolytic Adenovirus

Cancer treatment with local administration of armed oncolytic viruses could potentially induce systemic antitumor effects, or the abscopal effect, as they self-amplify in tumors, induce danger signaling, and promote tumor-associated antigen presentation. In this study, oncolytic adenovirus coding for human tumor necrosis factor alpha (TNF-alpha) and interleukin-2 (IL-2) Ad5/3-E2F-d24-hTNF-alpha-IRES-hIL-2 (also known as [a.k.a.] TILT-123) provoked antitumor efficacy in tumors that were injected with Ad5/3-E2F-d24-hTNF-alpha-IRES-hIL-2 and those that were left non-injected in the same animal. Importantly, the virus was able to travel to distant tumors. To dissect the effects of oncolysis and cytokines, we studied replication-incompetent viruses in mice. Systemic antitumor effects were similar in both models, highlighting the importance of the arming device. The cytokines induced positive changes in immune cell infiltrates and induced the expression of several immune-reaction-related genes in tumors. In addition, Ad5/3-E2F-d24-hTNF-alpha-IRES-hIL-2 was able to increase homing of adoptively transferred tumor-infiltrating lymphocytes into both injected and non-injected tumors, possibly mediated through chemokine expression. In summary, local treatment with Ad5/3-E2F-d24-hTNF-alpha-IRES-hIL-2 resulted in systemic antitumor efficacy by inducing immune cell infiltration and trafficking into both treated and untreated tumors. Moreover, the oncolytic adenovirus platform had superior systemic effects over replication-deficient vector through spreading into distant tumors.Peer reviewe

Oncolytic Adenovirus Type 3 Coding for CD40L Facilitates Dendritic Cell Therapy of Prostate Cancer in Humanized Mice and Patient Samples

Dendritic cell (DC)-based vaccines have shown some degree of success for the treatment of prostate cancer (PC). However, the highly immunosuppressive tumor microenvironment leads to DC dysfunction, which has limited the effectiveness of these vaccines. We hypothesized that use of a fully serotype 3 oncolytic adenovirus (Ad3-hTERT-CMV-hCD40L; TILT-234) could stimulate DCs in the prostate tumor microenvironment by expressing CD40L. Activated DCs would then activate cytotoxic T cells against the tumor, resulting in therapeutic immune responses. Oncolytic cell killing due to cancer cell-specific virus replication adds to antitumor effects but also enhances the immunological effect by releasing tumor epitopes for sampling by DC, in the presence of danger signals. In this study, we evaluated the companion effect of Ad3-hTERT-CMV-hCD40L and DC-therapy in a humanized mouse model and PC histocultures. Treatment with Ad3-hTERT-CMV-hCD40L and DC resulted in enhanced antitumor responses in vivo. Treatment of established histocultures with Ad3-hTERT-CMV-hCD40L induced DC maturation and notable increase in proinflammatory cytokines. In conclusion, Ad3-hTERT-CMV-hCD40L is able to modulate an immunosuppressive prostate tumor microenvironment and improve the effectiveness of DC vaccination in PC models and patient histocultures, setting the stage for clinical translation.Peer reviewe

Effective Combination Immunotherapy with Oncolytic Adenovirus and Anti-PD-1 for Treatment of Human and Murine Ovarian Cancers

Ovarian cancer (OvCa) is one of the most common gynecological cancers and has the highest mortality in this category. Tumors are often detected late, and unfortunately over 70% of OvCa patients experience relapse after first-line treatments. OvCa has shown low response rates to immune checkpoint inhibitor (ICI) treatments, thus leaving room for improvement. We have shown that oncolytic adenoviral therapy with Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (aka. TILT-123) is promising for single-agent treatment of cancer, but also for sensitizing tumors for T-cell dependent immunotherapy approaches, such as ICI treatments. Therefore, this study set out to determine the effect of inhibition of the immune checkpoint inhibitors (ICI), in the context of TILT-123 therapy of OvCa. We show that simultaneous treatment of patient derived samples with TILT-123 and ICIs anti-PD-1 or anti-PD-L1 efficiently reduced overall viability. The combinations induced T cell activation, T cells expressed activation markers more often, and the treatment caused positive microenvironment changes, measured by flow cytometric assays. Furthermore, in an immunocompetent in vivo C57BL/6NHsda mouse model, tumor growth was hindered, when treated with TILT-123, ICI or both. Taken together, this study provides a rationale for using TILT-123 virotherapy in combination with TILT-123 and immune checkpoint inhibitors together in an ovarian cancer OvCa clinical trial.Peer reviewe