Rompiendo la inmunotolerancia en el entorno tumoral mediante el uso de vacunas oncolíticas

La inmunoterapia contra el cáncer está mejorando día a día y cobrando mayor importancia gracias a los avances que han mejorado el conocimiento del sistema inmune en esta enfermedad y el proceso de inmunoedición. Uno de los objetivos principales de estos tratamientos es revertir la inmunosupresión inherente al entorno tumoral para así facilitar la creación de una respuesta antitumoral específica por parte del sistema inmune. Una estrategia incluida en este grupo terapéutico es la de las vacunas oncolíticas, una herramienta que consiste en el uso de virus modificados genéticamente para destruir selectivamente las células cancerosas. La destrucción de células cancerosas se consigue de dos formas: a corto plazo (oncolisis por acción directa del virus) y a largo plazo (reversión de la inmunosupresión y generación de la respuesta inmune). A la hora de diseñar los virus no sólo se fomenta la especificidad por células tumorales sino que también se han probado con éxito otras modificaciones que les permitan conseguir una mayor respuesta del sistema inmunitario. El uso de virus oncolíticos ofrece una gran variedad de estrategias de tratamiento diferentes debido a la relativa facilidad con la que se puede modificar un virus para armarlo con genes que supongan un tratamiento más efectivo

Enhanced Stability and Bioactivity of Natural Anticancer Topoisomerase I Inhibitors through Cyclodextrin Complexation

The use of cyclodextrins as drug nano-carrier systems for drug delivery is gaining importance in the pharmaceutical industry due to the interesting pharmacokinetic properties of the resulting inclusion complexes. In the present work, complexes of the anti-cancer alkaloids camptothecin and luotonin A have been prepared with β-cyclodextrin and hydroxypropyl-β-cyclodextrin. These cyclodextrin complexes were characterized by nuclear magnetic resonance spectroscopy (NMR). The variations in the 1H-NMR and 13C-NMR chemical shifts allowed to establish the inclusion modes of the compounds into the cyclodextrin cavities, which were supported by docking and molecular dynamics studies. The efficiency of the complexation was quantified by UV-Vis spectrophotometry and spectrofluorimetry, which showed that the protonation equilibria of camptothecin and luotonin A were drastically hampered upon formation of the inclusion complexes. The stabilization of camptothecin towards hydrolysis inside the cyclodextrin cavity was verified by the quantitation of the active lactone form by reverse phase liquid chromatography fluorimetric detection, both in basic conditions and in the presence of serum albumin. The antitumor activity of luotonin A and camptothecin complexes were studied in several cancer cell lines (breast, lung, hepatic carcinoma, ovarian carcinoma and human neuroblastoma) and an enhanced activity was found compared to the free alkaloids, particularly in the case of hydroxypropyl-β-cyclodextrin derivatives. This result shows that the cyclodextrin inclusion strategy has much potential towards reaching the goal of employing luotonin A or its analogues as stable analogues of camptothecin

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

Enabling PD-1 axis inhibitors against solid tumours with engineered adenoviruses armed with TNFa and IL-2

Over the last ten years, the use of immunotherapeutic treatments for cancer has exponentially increased due to the promising responses across different tumour types. One of the particularities of the approach is that patients can develop long lasting curative responses. Unfortunately, those encouraging complete responses happen only in a minority of patients (< 10%). Immune checkpoint inhibitors, like anti-PD-1 or anti-PD-L1, are embodiments of that kind of therapy, whose concept revolves around the concept of stopping inhibitory signals that limit the development of antitumour responses. Results obtained from clinical experiences showed that patients responding better to immune checkpoint inhibitors tend to have “hot tumours”, meaning that there are effector components (such as T cells) and the microenvironment supports their function. To make immune checkpoint inhibitors work in those patients with cold tumours, the use of oncolytic adenoviruses could provide the spark to warm them. Viruses are able to trigger the immune alarm and thus, make tumours hot when their replication is restricted to the tumour. To study the potential of oncolytic viruses to increase the efficacy of immune checkpoint inhibitors, an array of preclinical models that allowed to study the matter from different angles were used. While in vivo murine and hamster models provided the basis for direct antitumour effect and survival assessments, other studies including human tumour sample histocultures and other in vitro experiments were performed to understand the subjacent cellular and molecular mechanisms involved. Due to the critical role of the T-cell compartment surrounding the activity of immune checkpoint inhibitors, different virus families were studied in terms of their ability to trigger T-cell responses. The best T-cell engagement was provided by the adenovirus candidate. An adenovirus engineered to support further antitumour immune responses was used to enable anti-PD-1 and anti-PD-L1 therapies. In both cases, the proposed strategy was able to cure all the animals. Additionally, tumours that were already refractory to anti-PD-1 still respond to virotherapy and the virus resets the tumour microenvironment sensitizing those tumours respond to anti-PD-1. Overall, this work revealed the potential of using engineered adenoviruses to repolarize the tumour microenvironment in general and the T-cell compartment in particular to a point that complement the mechanism of action of PD-1 and PD-L1 inhibitory antibodies. That synergistic combination rendered an increased percentage of responses even in tumours previously resistant or refractory to immune checkpoint inhibitors.Viimeisen kymmenen vuoden aikana immunoterapian käyttö syövän hoidossa on kasvanut eksponentiaalisesti johtuen useissa eri syöpätyypeissä esiintyneistä lupaavista hoitovasteista. Yksi immunoterapian erityispiirre on potilaiden mahdollisuus kehittää pitkäaikaisia parantavia hoitovasteita. Valitettavasti näitä täydellisiä hoitovasteita esiintyy vain pienessä osassa potilaista (< 10%). Immuunivasteen vapauttajat, kuten PD-1- ja PD-L1-vasta-aineet, ovat immunoterapiaa, jonka vaikutusmekanismi perustuu immuunivastetta inhiboivien signaalien estoon. Normaalisti nämä signaalit estäisivät immuunipuolustuksen hyökkäyksen syöpäkudosta vastaan. Kliininen kokemus on osoittanut immuunivasteen vapauttajista hyötyvien potilaiden omaavan useammin ”kuumia kasvaimia”, joille on tyypillistä sekä immuunisolujen esiintyminen, että niiden toimintaa tukeva kasvaimen mikroympäristö. Immuunivasteen vapauttajien tehon parantamiseen myös ”kylmiä kasvaimia” omaavilla potilailla voidaan hyödyntää onkolyyttisiä adenoviruksia. Virukset kykenevät herättämään immuunivasteen tehden kasvaimista ”kuumia” erityisesti silloin, kun niiden lisääntyminen on rajoitettu kasvainkudokseen. Tutkiaksemme onkolyyttisten virusten mahdollisuutta tehostaa immuunivasteen vapauttajien toimintaa, käytimme useita prekliinisillä malleja, jotka mahdollistavat ilmiön tutkimisen useista lähestymiskulmista. In vivo hiiri- ja hamsterimallit muodostivat pohjan suorille kasvainvaikutuksille ja selviytymistutkimuksille, kun taas ihmiskudosten viljelykokeet ja muut in vitro -tutkimukset antoivat lisätietoa solunsisäisistä ja molekylaarisista mekanismeista. T-solut ovat tärkeässä roolissa immuunivasteen vapauttajien toiminnan kannalta, minkä vuoksi tutkimme useiden eri virusperheiden kykyä herättää T-soluvaste. Tutkimuksissa parhaan T-soluvasteen herätti adenoviruksiin kuuluva virus. Modifioitu adenovirus kykeni tehostamaan syöpäkudokseen kohdistuvaa immuunivastetta ja täten parantamaan PD-1- ja PD-L1-vasta-ainehoitojen tehoa. Kummankin vasta-aineen kohdalla yhdistäminen adenovirusterapiaan paransi kaikki koe-eläimet. Lisäksi jo vastustuskyvyn PD-1-vasta-aineelle kehittäneet kasvaimet vastasivat adenovirusterapiaan. Adenovirusterapia myös palautti kasvaimen herkkyyden PD-1-vasta-ainehoidolle. Kokonaisuudessaan tämä väitöstyö osoittaa modifioitujen adenovirusten kyvyn muokata kasvaimen mikroympäristöä tavalla, joka tukee PD-1- ja PD-L1-vasta-ainehoitojen vaikutusmekanismeja erityisesti T-soluihin liittyen. Synergistinen yhdistelmä paransi hoitovastetta jopa kasvaimissa, jotka olivat aiemmin olleet vastustuskykyisiä immuunivasteen vapauttajille