Targeting the tumor stroma with an oncolytic adenovirus secreting a fibroblast activation protein-targeted bispecific T-cell engager

BackgroundOncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor-associated stroma limits the efficacy of oncolytic viruses by forming a barrier that blocks efficient viral penetration and spread. The stroma also plays a critical role in progression, immunosuppression and invasiveness of cancer. Fibroblast activation protein- (FAP) is highly overexpressed in cancer-associated fibroblasts (CAFs), the main cellular component of tumor stroma, and in this study we assessed whether arming oncolytic adenovirus (OAd) with a FAP-targeting Bispecific T-cell Engager (FBiTE) could retarget infiltrated lymphocytes towards CAFs, enhancing viral spread and T cell-mediated cytotoxicity against the tumor stroma to improve therapeutic activity.MethodsThe bispecific T-cell Engager against FAP was constructed using an anti-human CD3 single-chain variable fragment (scFv) linked to an anti-murine and human FAP scFv. This FBiTE was inserted in the oncolytic adenovirus ICOVIR15K under the control of the major late promoter, generating the ICO15K-FBiTE. ICO15K-FBiTE replication and potency were assessed in HT1080 and A549 tumor cell lines. The expression of the FBiTE and the activation and proliferation of T cells that induced along with the T cell-mediated cytotoxicity of CAFs were evaluated by flow cytometry in vitro.In vivo, T-cell biodistribution and antitumor efficacy studies were conducted in NOD/scid/IL2rg(-)/(-) (NSG) mice.ResultsFBiTE expression did not decrease the infectivity and replication potency of the armed virus. FBiTE-mediated binding of CD3(+) effector T cells and FAP(+) target cells led to T-cell activation, proliferation, and cytotoxicity of FAP-positive cells in vitro. In vivo, FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. The antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus.ConclusionsCombination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP-expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development

Arming Oncolytic Adenoviruses: Effect of Insertion Site and Splice Acceptor on Transgene Expression and Viral Fitness

Oncolytic adenoviruses (OAds) present limited efficacy in clinics. The insertion of therapeutic transgenes into OAds genomes, known as "arming OAds", has been the main strategy to improve their therapeutic potential. Different approaches were published in the decade of the 2000s, but with few comparisons. Most armed OAds have complete or partial E3 deletions, leading to a shorter half-life in vivo. We generated E3+ OAds using two insertion sites, After-fiber and After-E4, and two different splice acceptors linked to the major late promoter, either the Ad5 protein IIIa acceptor (IIIaSA) or the Ad40 long fiber acceptor (40SA). The highest transgene levels were obtained with the After-fiber location and 40SA. However, the set of codons of the transgene affected viral fitness, highlighting the relevance of transgene codon usage when arming OAds using the major late promoter

Enhanced antitumor efficacy of an oncolytic adenovirus armed with an EGFR-targeted BiTE using menstrual blood-derived mesenchymal stem cells as carriers

Poor tumor targeting of oncolytic adenoviruses (OAdv) after systemic administration is considered a major limitation for virotherapy of disseminated cancers. The benefit of using mesenchymal stem cells as cell carriers for OAdv tumor targeting is currently evaluated not only in preclinical models but also in clinical trials. In this context, we have previously demonstrated the enhanced antitumor efficacy of OAdv-loaded menstrual blood-derived mesenchymal stem cells (MenSCs). However, although significant, the antitumor efficacy obtained was modest, and we hypothesized that a greater antitumor efficacy could be obtained arming the OAdv with a therapeutic transgene. Here we show that combining MenSCs with ICOVIR15-cBiTE, an OAdv expressing an epidermal growth factor receptor (EGFR)-targeting bispecific T-cell engager (cBiTE), enhances the antitumor efficacy compared to MenSCs loaded with the unarmed virus ICOVIR15. We found that MenSCs properly produce cBiTE after viral infection leading to a greater antitumor potency both in vitro and in vivo. These findings indicate the mutual benefit of combining MenSCs and armed OAdv and support the combination of ICOVIR15-cBiTE and MenSCs as a cancer treatment.We thank CERCA Program/Generalitat de Catalunya for their institutional support. The authors also thank Dolores Ramos and Silvia Torres for their lab technical support and Vanessa Cervera for samples processing. This work was supported by the Asociación Española Contra el Cáncer, BIO2017-897554-C2-1-Rgrant to R. Alemany from the Ministerio de Economía y Competitividad of Spain, Adenonet BIO2015-68990-REDT to R. Alemany from the Ministerio de Economía y Competitividad of Spain, RedADVANCE(CAT) project COMRDI15-1-0013 to R. Alemany from Ris3CAT and 2017SGR449 research grant to RA from the ‘Generalitat de Catalunya’. Cofunded by the European Regional Development Fund, a way to Build Europe to RA

Evidence of Anti-tumoral Efficacy in an Immune Competent Setting with an iRGD-Modified Hyaluronidase-Armed Oncolytic Adenovirus

To enhance adenovirus-mediated oncolysis, different approaches that tackle the selectivity, tumor penetration, and spreading potential of oncolytic adenoviruses have been reported. We have previously demonstrated that insertion of the internalizing Arginine-Glycine-Aspartic (iRGD) tumor-penetrating peptide at the C terminus of the fiber or transgenic expression of a secreted hyaluronidase can improve virus tumor targeting and spreading. Here we report a new oncolytic adenovirus ICOVIR17K-iRGD in which both modifications have been incorporated. In xenografted A549 tumors in nude mice, ICOVIR17K-iRGD shows higher efficacy than the non-iRGD counterpart. To gain insights into the role of the immune system in oncolysis, we have studied ICOVIR17K-iRGD in the tumor isograft mouse model CMT64.6, partially permissive to human adenovirus 5 replication, in immunodeficient or immunocompetent mice. Whereas no efficacy was observed in the immunodeficient setting due to insufficient viral replication, partial efficacy and a polymorphonuclear and CD8+ T cell infiltrate were observed in the immunocompetent mice. The results indicate that the elicitation of a virus-induced anti-tumoral immune response is responsible for the observed partial antitumoral effect

Oncolytic adenoviruses expressing transgenes targeting the tumor stroma to enhance the antitumor efficacy

[eng] Oncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor- associated stroma limits the efficacy of oncolytic viruses by forming a barrier that block efficient viral penetration and spread. Another important hurdle for the efficacy of OVs is the antiviral immune responses, where virus-specific infiltrating T cells clear adenovirus-infected cells without compromising tumor burden. In this thesis, these hurdles have been addressed in separate chapters. We first hypothesized that arming an oncolytic adenovirus with a FAP-targeting bispecific T cell engager (FBiTE) could retarget infiltrated lymphocytes towards cancer-associated fibroblasts (CAFs), enhancing viral spread and favoring antitumor rather than anti-viral immune responses. The engineered ICO15K-expressing FBiTE virus showed similar infectivity and replication potency than the non-armed virus. FBiTE-mediated binding of CD3+ effector T cells and FAP+ target cells led to T-cell activation, proliferation, and cytotoxicity against FAP-positive cells in vitro. In vivo, FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. Finally, the antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus. The data presented in this thesis strongly supports that the combination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP- expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development. Aiming to induce stroma disruption, we secondly generated a panel of oncolytic adenoviruses expressing FAP-targeting immunotoxins and a nitroreductase (NfrA)- activatable prodrug. During the development of these projects, we successfully rescued and characterized all the viruses. However, although immunotoxins molecules were properly expressed and secreted from modified-virus infected cells, no promising results were obtained. In contrast, NfrA-armed virus showed replication-dependent enzymatic activity on target cells, leading to increased oncolytic potency in vitro. These preliminary results indicate that this last strategy could be considered to foster viral spread in stroma-abundant tumors, encouraging its validation in an in vivo setting.[cat] Les teràpies basades en virus oncolítics pel tractament de tumors sòlids es consideren molt prometedores degut a la seva capacitat de combinar la lisi directa de cèl·lules canceroses i la mort cel·lular per l’activació del sistema immune. No obstant, l’estroma associat al càncer forma una barrera que bloqueja la penetració i distribució del virus en el tumor, limitant l’eficàcia dels virus oncolítics. Una altra limitació important és la resposta immune contra el virus. Les cèl·lules T citotòxiques específiques contra el virus que infiltren el tumor eliminen, normalment, les cèl·lules infectades per l’adenovirus sense comprometre la massa tumoral. En aquesta tesi, aquestes limitacions han estat abordades en capítols separats. Primer vam hipotetitzar que un adenovirus oncolític armat amb un bispecific T cell engager (BiTE) contra FAP (FBiTE) podria redirigir els limfòcits infiltrats contra els fibroblasts associats al càncer (CAFs), millorant la distribució viral i afavorint la resposta antitumoral vers l’antiviral. El virus ICO15K que expressa el FBiTE va mostrar un patró d’infectivitat i de replicació similars al virus no armat. La unió de les cèl·lules T efectores CD3+ i les cèl·lules diana FAP+ mitjançada pel FBiTE va provocar l’activació, la proliferació i la citotoxicitat de les cèl·lules T contra la cèl·lules FAP positives in vitro. In vivo, l’expressió de FBiTE va induir l’acumulació intratumoral de les cèl·lules T i la disminució dels nivells de FAP, un marcador de CAFs, en els tumors. Finalment, l’activitat antitumoral dels adenovirus armats amb el FBiTE va ser superior que la del virus parental. Els resultats presentats en aquesta tesi aporten fortes evidències que la combinació de l’oncolisi viral de les cèl·lules canceroses i la citotoxicitat dels CAFs FAP+ mitjançada pel FBiTE pot ser una estratègia efectiva per superar les limitacions claus de la viroteràpia. Aquests resultats incentiven el desenvolupament d’aquesta estratègia pel seu ús en la clínica. Amb l’objectiu de destruir l’estroma, vam generar un panell d’adenovirus oncolítics que expressaven diferents immunotoxines específiques contra FAP i una nitroreducatasa (NfrA) activadora de prodroga. Durant el desenvolupament d’aquests projectes, vam obtenir i caracteritzar tots els virus. No obstant, encara que les diferents immunotoxines van ser adequadament expressades i secretades per les cèl·lules infectades pels virus, no vam obtenir capresultat prometedor. El virus armat amb la NfrA, en canvi, va mostrar una activació enzimàtica depenent de la replicació del virus en les cèl·lules diana, incrementant la potència oncolítica del virus in vitro. Aquests resultats preliminars indiquen que aquesta última estratègia podria fomentar la distribució viral en tumors rics en estroma i incentiven la seva validació en models animals

Oncolytic Viruses as a Platform for the Treatment of Malignant Brain Tumors

Malignant brain tumors remain incurable diseases. Although much effort has been devoted to improving patient outcome, multiple factors such as the high tumor heterogeneity, the strong tumor-induced immunosuppressive microenvironment, and the low mutational burden make the treatment of these tumors especially challenging. Thus, novel therapeutic strategies are urgent. Oncolytic viruses (OVs) are biotherapeutics that have been selected or engineered to infect and selectively kill cancer cells. Increasingly, preclinical and clinical studies demonstrate the ability of OVs to recruit T cells and induce durable immune responses against both virus and tumor, transforming a “cold” tumor microenvironment into a “hot” environment. Besides promising clinical results as a monotherapy, OVs can be powerfully combined with other cancer therapies, helping to overcome critical barriers through the creation of synergistic effects in the fight against brain cancer. Although many questions remain to be answered to fully exploit the therapeutic potential of OVs, oncolytic virotherapy will clearly be part of future treatments for patients with malignant brain tumors

Oncolytic Viruses as a Platform for the Treatment of Malignant Brain Tumors

Malignant brain tumors remain incurable diseases. Although much effort has been devoted to improving patient outcome, multiple factors such as the high tumor heterogeneity, the strong tumor-induced immunosuppressive microenvironment, and the low mutational burden make the treatment of these tumors especially challenging. Thus, novel therapeutic strategies are urgent. Oncolytic viruses (OVs) are biotherapeutics that have been selected or engineered to infect and selectively kill cancer cells. Increasingly, preclinical and clinical studies demonstrate the ability of OVs to recruit T cells and induce durable immune responses against both virus and tumor, transforming a "cold" tumor microenvironment into a "hot" environment. Besides promising clinical results as a monotherapy, OVs can be powerfully combined with other cancer therapies, helping to overcome critical barriers through the creation of synergistic effects in the fight against brain cancer. Although many questions remain to be answered to fully exploit the therapeutic potential of OVs, oncolytic virotherapy will clearly be part of future treatments for patients with malignant brain tumors

Targeting the tumor stroma with an oncolytic adenovirus secreting a fibroblast activation protein-targeted bispecific T-cell engager

Abstract Background Oncolytic virus (OV)-based therapies have an emerging role in the treatment of solid tumors, involving both direct cell lysis and immunogenic cell death. Nonetheless, tumor-associated stroma limits the efficacy of oncolytic viruses by forming a barrier that blocks efficient viral penetration and spread. The stroma also plays a critical role in progression, immunosuppression and invasiveness of cancer. Fibroblast activation protein-α (FAP) is highly overexpressed in cancer-associated fibroblasts (CAFs), the main cellular component of tumor stroma, and in this study we assessed whether arming oncolytic adenovirus (OAd) with a FAP-targeting Bispecific T-cell Engager (FBiTE) could retarget infiltrated lymphocytes towards CAFs, enhancing viral spread and T cell-mediated cytotoxicity against the tumor stroma to improve therapeutic activity. Methods The bispecific T-cell Engager against FAP was constructed using an anti-human CD3 single-chain variable fragment (scFv) linked to an anti-murine and human FAP scFv. This FBiTE was inserted in the oncolytic adenovirus ICOVIR15K under the control of the major late promoter, generating the ICO15K-FBiTE. ICO15K-FBiTE replication and potency were assessed in HT1080 and A549 tumor cell lines. The expression of the FBiTE and the activation and proliferation of T cells that induced along with the T cell-mediated cytotoxicity of CAFs were evaluated by flow cytometry in vitro . In vivo, T-cell biodistribution and antitumor efficacy studies were conducted in NOD/scid/IL2rg − / − (NSG) mice. Results FBiTE expression did not decrease the infectivity and replication potency of the armed virus. FBiTE-mediated binding of CD3+ effector T cells and FAP+ target cells led to T-cell activation, proliferation, and cytotoxicity of FAP-positive cells in vitro. In vivo , FBiTE expression increased intratumoral accumulation of T cells and decreased the level of FAP, a marker of CAFs, in tumors. The antitumor activity of the FBiTE-armed adenovirus was superior to the parental virus. Conclusions Combination of viral oncolysis of cancer cells and FBiTE-mediated cytotoxicity of FAP-expressing CAFs might be an effective strategy to overcome a key limitation of oncolytic virotherapy, encouraging its further clinical development

Enhanced antitumor efficacy of an oncolytic adenovirus armed with an EGFR-targeted BiTE using menstrual blood-derived mesenchymal stem cells as carriers

Poor tumor targeting of oncolytic adenoviruses (OAdv) after systemic administration is considered a major limitation for virotherapy of disseminated cancers. The benefit of using mesenchymal stem cells as cell carriers for OAdv tumor targeting is currently evaluated not only in preclinical models but also in clinical trials. In this context, we have previously demonstrated the enhanced antitumor efficacy of OAdv-loaded menstrual blood-derived mesenchymal stem cells (MenSCs). However, although significant, the antitumor efficacy obtained was modest, and we hypothesized that a greater antitumor efficacy could be obtained arming the OAdv with a therapeutic transgene. Here we show that combining MenSCs with ICOVIR15-cBiTE, an OAdv expressing an epidermal growth factor receptor (EGFR)-targeting bispecific T-cell engager (cBiTE), enhances the antitumor efficacy compared to MenSCs loaded with the unarmed virus ICOVIR15. We found that MenSCs properly produce cBiTE after viral infection leading to a greater antitumor potency both in vitro and in vivo. These findings indicate the mutual benefit of combining MenSCs and armed OAdv and support the combination of ICOVIR15-cBiTE and MenSCs as a cancer treatment.We thank CERCA Program/Generalitat de Catalunya for their institutional support. The authors also thank Dolores Ramos and Silvia Torres for their lab technical support and Vanessa Cervera for samples processing. This work was supported by the Asociación Española Contra el Cáncer, BIO2017-897554-C2-1-Rgrant to R. Alemany from the Ministerio de Economía y Competitividad of Spain, Adenonet BIO2015-68990-REDT to R. Alemany from the Ministerio de Economía y Competitividad of Spain, RedADVANCE(CAT) project COMRDI15-1-0013 to R. Alemany from Ris3CAT and 2017SGR449 research grant to RA from the ‘Generalitat de Catalunya’. Cofunded by the European Regional Development Fund, a way to Build Europe to RA

Arming Oncolytic Adenoviruses: Effect of Insertion Site and Splice Acceptor on Transgene Expression and Viral Fitness

Oncolytic adenoviruses (OAds) present limited efficacy in clinics. The insertion of therapeutic transgenes into OAds genomes, known as “arming OAds”, has been the main strategy to improve their therapeutic potential. Different approaches were published in the decade of the 2000s, but with few comparisons. Most armed OAds have complete or partial E3 deletions, leading to a shorter half-life in vivo. We generated E3+ OAds using two insertion sites, After-fiber and After-E4, and two different splice acceptors linked to the major late promoter, either the Ad5 protein IIIa acceptor (IIIaSA) or the Ad40 long fiber acceptor (40SA). The highest transgene levels were obtained with the After-fiber location and 40SA. However, the set of codons of the transgene affected viral fitness, highlighting the relevance of transgene codon usage when arming OAds using the major late promoter