Onkolyyttisten immunoterapioiden yhdistelmät kasvainresistenssin murtamiseksi

According to latest estimates, cancer is becoming an increasing health risk on a global scale. Consequently, novel cancer treatment modalities are urgently needed, especially for the treatment of metastatic solid tumors that are refractory to standard therapies. One promising approach in the treatment of such advanced cancers is immunotherapy which aims to elicit de novo immune responses and/or to boost pre-existing anti-tumor immunity. Different forms of cancer immunotherapy include oncolytic viruses, which selectively replicate in and destroy cancer cells, and adoptive T-cell therapy, in which the patient is given vastly amplified numbers of tumor-targeting T-cells. Both of these have shown capacity to elicit anti-tumor immunity but efficacy in clinical settings has been suboptimal due to different resistance mechanisms employed by solid tumors. Anti-viral resistance represents a major hurdle in oncolytic virotherapy, as repeated administration of the same virus can lead to induction anti-viral rather than anti-tumor immunity. Moreover, cancer cells in some tumors may intrinsically be resistant to virus infection. In study I, we examined whether this could be circumvented by heterologous prime-boost setting, i.e. by switching between oncolytic adenovirus (Ad) and vaccinia virus (VV) during therapy. The results showed that presence of one virus does not preclude the infection of another and treatment with heterologous Ad-VV therapy can delay the onset of anti-viral resistance. Moreover, we found that restricted replication of the priming (adeno)virus can affect the efficacy of heterologous virotherapy. In study IV, we studied the role of anti-viral signaling in adenovirus replication in cancer cells and whether this could be augmented with Janus Kinase 1/2 inhibitor Ruxolitinib. Interestingly, we found that although exposure to type I interferon does not inhibit progressive Ad replication in vitro, significant improvement in anti-tumor efficacy of the virus was observed in vivo when combined to concomitant Ruxolitinib treatment. These results underline the possible approaches that could be taken to reduce naturally acquired or therapy-induced resistance, which interferes with viral spread and may hinder the therapeutic efficacy. Adoptive T-cell therapy (ACT) can be a potent form of immunotherapy. Despite the large number of anti-tumor T-cells infused during ACT, immunosuppression and immune evasion of advanced tumors can render tumor-infiltrating lymphocytes (TILs) inactive. In study II, we examined whether oncolytic adenovirus could increase anti-tumor efficacy of adoptively transferred T-cell receptor (TCR) transgenic T-cells. Indeed, intratumoral injections of adenovirus were able to counteract immunosuppression by activating antigen-presenting cells (APCs) and anti-tumor T-cells. Moreover, an endogenous T-cell response against other, non-related tumor antigens was detected and this polyclonal response contributed to systemic anti-tumor immunity. In study III, we analyzed whether cellular composition of tumor microenvironment could be modified by local administration of immunostimulatory recombinant cytokines. When combined to adoptive T-cell transfer, intratumoral injections of interleukin 2 (IL-2), interferon α (IFN-α) and interferon γ (IFN-γ) resulted in significant anti-tumor efficacy, increased tumor-levels of stimulatory immune cells and reduced exhaustion of CD8+ TILs. In contrast, administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced tumor growth and recruited immunosuppressive cell types such as monocytic myeloid-derived suppressor cells (MDSCs) and M2 macrophages to the tumor bed. These results indicate that immunomodulation by carefully selected cytokines and/or oncolytic adenovirus can sensitize the tumor in favor of adoptively transferred anti-tumor T-cells. In conclusion, different combinatorial approaches can be employed to overcome intrinsic, naturally acquired or therapy-induced resistance to anti-tumor T-cells or oncolytic adenovirus. These advances enable significant improvement in the treatment of solid cancers and can potentially lead to development of curative cancer immunotherapies.Syövän uusiutuminen ja etäpesäkkeiden muodostuminen ovat suurimmat syyt syöpiin liittyvään kuolleisuuteen, minkä johdosta uusille hoitotavoille on kasvava tarve. Yksi tällaisistä seuraavan sukupolven syöpähoidoista on immunoterapia, joka perustuu potilaan oman immuunijärjestelmän aktivointiin syöpäkasvainta vastaan. Eri immunoterapian muotojen kuten onkolyyttisten virusten sekä adoptiivisen T-solusiirron on osoitettu aiheuttavan luontaisia ja hankittuja immuunivasteita syöpäkasvainta vastaan. Silti näiden hoitomuotojen teho on usein jäänyt valitettavan heikoksi, sillä kiinteät syöpäkasvaimet ovat kasvaessaan kehittäneet verkoston erilaisia resistenssimekanismeja, joiden avulla ne pystyvät torjumaan kasvainta vastaan kohdistetut immuunivälitteiset hyökkäykset. Onkolyyttiset adenovirukset ovat syöpäspesifisiksi muokattuja viruksia, jotka lisääntyessään tuhoavat syöpäsoluja. Tämän onkolyysiksi nimetyn tapahtuman ohella onkolyyttiset adenovirukset myös stimuloivat immuunijärjestelmää ja tekevät kasvaimesta immunogeenisemmän, sillä adenovirus sisältää ns. vaarasignaaleja, jotka immuunijärjestelmä tunnistaa vieraiksi. Kuitenkin toistuva annostelu saattaa johtaa adenovirusta estävän resistenssin muodostumiseen. Väitöskirjatutkimuksen ensimmäisessä ja neljännessä osajulkaisussa etsittiin keinoja tämän adenovirus-resistenssin vähentämiseen yhdistämällä onkolyyttinen adenovirushoito joko toisen, täysin erilaisen onkolyyttisen viruksen (vaccinia virus) tai interferoni-signalointia inhiboivan Ruxolitinib-lääkeaineen kanssa. Kummankin lähestymistavan havaittiin hidastavan adenovirus-resistenssin kehittymistä ja pienentävän kasvaimia hiirimalleissa. Näin ollen kyseiset yhdistelmähoidot edustavat uusia mahdollisia tapoja lisätä onkolyyttisten adenovirusten hoitotehoa myös potilaissa. Adoptiivinen T-solusiirto on parhaimmillaan tehokas lähestymistapa syövän hoitoon, sillä kasvainspesifisiksi muokattuja sytotoksisia T-soluja voidaan ensin laboratoriossa monistaa suuriin määriin ja lopulta siirtää ne takaisin syöpäpotilaaseen. Tästä huolimatta siirretyt T-solut menettävät tehonsa nopeasti, sillä kiinteiden kasvainten mikroympäristö on vahvasti immunosuppressiivinen ja syöpäsolut helposti muokkautuvat valintapaineessa, jolloin T-solut eivät enää tunnista syöpäsoluja ja eivät siten kykene tappamaan niitä. Väitöskirjatutkimuksen toisessa ja kolmannessa osajulkaisussa tutkittiin mahdollisuutta parantaa T-solusiirron hoitotehoa yhdistämällä se onkolyyttisen adenoviruksen tai sytokiini-injektioiden kanssa. Molemmissa tapauksissa havaittiin, että paikallinen immunostimulaatio (adenoviruksen tai sytokiinien muodossa) vähensi kasvaimen immunosuppressiota ja aktivoi kasvainspesifisiä T-soluja, johtaen siten merkittävästi parantuneeseen hoitotehoon melanooman hiirimalleissa. Tämän väitöskirjatutkimuksen prekliiniset tulokset edistävät kasvain-immunologian perustietämystä sekä mahdollistavat vastaavien immunoterapiayhdistelmien käytön ihmispotilailla vahvan kasvainspesifisen immuunivasteen, pitkäaikaisen remission ja jopa täydellisen hoitovasteen saavuttamiseksi

Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

<p>Abstract</p> <p>Background</p> <p>T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds.</p> <p>Methods</p> <p>We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays.</p> <p>Results</p> <p>From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells.</p> <p>Conclusions</p> <p>In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening to identify small molecule PTP activators that could function as RTK antagonists in cells.</p

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 decreases the proportion of TIM-3(+) subset of tumor-infiltrating CD8(+) T cells with correlation to improved survival in patients with cancer

Background Oncolytic viruses are a potent form of active immunotherapy, capable of invoking antitumor T-cell responses. Meanwhile, less is known about their effects on immune checkpoints, the main targets for passive immunotherapy of cancer. T-cell immunoglobulin and mucin domain-3 (TIM-3) is a coinhibitory checkpoint driving T-cell exhaustion in cancer. Here we investigated the effects of oncolytic adenovirus on the TIM-3 checkpoint on tumor-infiltrating immune cells and clinical impact in patients with cancer receiving oncolytic immunotherapy. Methods Modulation of TIM-3 expression on tumor-infiltrating immune cells was studied preclinically in B16 melanoma following intratumoral treatment with Ad5/3 increment 24-granulocyte-macrophage colony-stimulating factor oncolytic adenovirus. We conducted a retrospective longitudinal analysis of 15 patients with advanced-stage cancer with tumor-site biopsies before and after oncolytic immunotherapy, treated in the Advanced Therapy Access Program (ISRCTN10141600, April 5, 2011). Following patient stratification with regard to TIM-3 (increase vs decrease in tumors), overall survival and imaging/marker responses were evaluated by log-rank and Fisher's test, while coinhibitory receptors/ligands, transcriptomic changes and tumor-reactive and tumor-infltrating immune cells in biopsies and blood samples were studied by microarray rank-based statistics and immunoassays. Results Preclinically, TIM-3(+) tumor-infiltrating lymphocytes (TILs) in B16 melanoma showed an exhausted phenotype, whereas oncolytic adenovirus treatment significantly reduced the proportion of TIM-3(+) TIL subset through recruitment of less-exhausted CD8(+) TIL. Decrease of TIM-3 was observed in 60% of patients, which was associated with improved overall survival over TIM-3 increase patients (p=0.004), together with evidence of clinical benefit by imaging and blood analyses. Coinhibitory T-cell receptors and ligands were consistently associated with TIM-3 changes in gene expression data, while core transcriptional exhaustion programs and T-cell dysfunction were enriched in patients with TIM-3 increase, thus identifying patients potentially benefiting from checkpoint blockade. In striking contrast, patients with TIM-3 decrease displayed an acute inflammatory signature, redistribution of tumor-reactive CD8(+) lymphocytes and higher influx of CD8(+) TIL into tumors, which were associated with the longest overall survival, suggesting benefit from active immunotherapy. Conclusions Our results indicate a key role for the TIM-3 immune checkpoint in oncolytic adenoviral immunotherapy. Moreover, our results identify TIM-3 as a potential biomarker for oncolytic adenoviruses and create rationale for combination with passive immunotherapy for a subset of patients.Peer reviewe

Oncolytic Adenoviruses Armed with Tumor Necrosis Factor Alpha and Interleukin-2 Enable Successful Adoptive Cell Therapy

Adoptive cell therapy holds much promise in the treatment of cancer but results in solid tumors have been modest. The notable exception is tumor-infiltrating lymphocyte (TIL) therapy of melanoma, but this approach only works with high-dose preconditioning chemotherapy and systemic interleukin (IL)-2 postconditioning, both of which are associated with toxicities. To improve and broaden the applicability of adoptive cell transfer, we constructed oncolytic adenoviruses coding for human IL-2 (hIL2), tumor necrosis factor alpha (TNF-alpha), or both. The viruses showed potent antitumor efficacy against human tumors in immunocompromised severe combined immunodeficiency (SCID) mice. In immunocompetent Syrian hamsters, we combined the viruses with TIL transfer and were able to cure 100% of the animals. Cured animals were protected against tumor re-challenge, indicating a memory response. Arming with IL-2 and TNF-alpha increased the frequency of both CD4(+) and CD8(+) TILs in vivo and augmented splenocyte proliferation ex vivo, suggesting that the cytokines were important for T cell persistence and proliferation. Cytokine expression was limited to tumors and treatment-related signs of systemic toxicity were absent, suggesting safety. To conclude, cytokine-armed oncolytic adenoviruses enhanced adoptive cell therapy by favorable alteration of the tumor microenvironment. A clinical trial is in progress to study the utility of Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (TILT-123) in human patients with cancer.Peer reviewe

Artificially Cloaked Viral Nanovaccine for Cancer Immunotherapy

Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.Peer reviewe

Favorable Alteration of Tumor Microenvironment by Immunomodulatory Cytokines for Efficient T-Cell Therapy in Solid Tumors

Unfavorable ratios between the number and activation status of effector and suppressor immune cells infiltrating the tumor contribute to resistance of solid tumors to T-cell based therapies. Here, we studied the capacity of FDA and EMA approved recombinant cytokines to manipulate this balance in favor of efficient anti-tumor responses in B16. OVA melanoma bearing C57BL/6 mice. Intratumoral administration of IFN-alpha 2, IFN-gamma, TNF-alpha, and IL-2 significantly enhanced the anti-tumor effect of ovalbumin-specific CD8+ T-cell (OT-I) therapy, whereas GM-CSF increased tumor growth in association with an increase in immunosuppressive cell populations. None of the cytokines augmented tumor trafficking of OT-I cells significantly, but injections of IFN-alpha 2, IFN-gamma and IL-2 increased intratumoral cytokine secretion and recruitment of endogenous immune cells capable of stimulating T-cells, such as natural killer and maturated CD11c+ antigen-presenting cells. Moreover, IFN-alpha 2 and IL-2 increased the levels of activated tumor-infiltrating CD8+ T-cells concomitant with reduction in the CD8+ T-cell expression of anergy markers CTLA-4 and PD-1. In conclusion, intratumoral administration of IFN-alpha 2, IFN-gamma and IL-2 can lead to immune sensitization of the established tumor, whereas GM-CSF may contribute to tumor-associated immunosuppression. The results described here provide rationale for including local administration of immunostimulatory cytokines into T-cell therapy regimens. One appealing embodiment of this would be vectored delivery which could be advantageous over direct injection of recombinant molecules with regard to efficacy, cost, persistence and convenience.Peer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival