Background: Despite significant advancements in the treatment of malignant melanoma, metastatic mucosal melanoma remains a therapeutic challenge due to its complex pathogenesis, distinct pathological characteristics, and limited response to immunotherapy. Combining different immunotherapeutic approaches offers a potential strategy to address these challenges. Tumor-infiltrating lymphocyte (TIL) therapy and oncolytic virus therapy represent promising treatment modalities that may synergize with each other. Patient and methods: We present a case of a 48-year-old woman with metastatic sinonasal mucosal melanoma who achieved a durable complete pathological response following treatment with multiple injections of the oncolytic virus TILT-123 (igrelimogene litadenorepvec) and a single infusion of TILs, without preconditioning chemotherapy or postconditioning interleukin-2. Results: Immunohistochemical analysis and single-cell sequencing revealed interesting alterations in injected and noninjected tumors as well as in peripheral blood, during the treatment course, suggesting that TILT-123 facilitated TIL engraftment into the tumor, ultimately leading to a complete response. Conclusions: This case underscores the potential of combined immunotherapeutic approaches as a promising strategy for patients with metastatic mucosal melanoma.

While the presence of tumor-infiltrating lymphocytes (TILs) associates with improved survival prognosis in ovarian cancer (OvCa) patients, TIL therapy benefit is limited. Here, we evaluated an oncolytic adenovirus coding for a human variant IL-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, as an immunotherapeutic strategy to enhance TIL responsiveness towards advanced stage OvCa tumors. Fragments of resected human OvCa tumors were processed into single-cell suspensions, and autologous TILs were expanded from said samples. OvCa tumor specimens were co-cultured with TILs plus vIL-2 virus, and cell killing was assessed in real time through cell impedance measurement. Combination therapy was further evaluated in vivo through a patient-derived xenograft (PDX) ovarian cancer murine model. The combination of vIL-2 virus plus TILs had best cancer cell killing ex vivo compared to TILs monotherapy. These results were supported by an in vivo experiment, where the best OvCa tumor control was obtained when vIL-2 virus was added to TIL therapy. Furthermore, the proposed therapy induced a highly cytotoxic phenotype demonstrated by increased granzyme B intensity in NK cells, CD4+ T, and CD8+ T cells in treated tumors. Our results demonstrate that Ad5/3-E2F-d24-vIL2 therapy consistently improved TILs therapy cytotoxicity in treated human OvCa tumors.

Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Tumor Infiltrating Lymphocyte (TIL) therapy has recently proved effective in patients with metastatic melanoma. Oncolytic adenovirus TILT-123 (igrelimogene litadenorepvec) is armed with tumor necrosis factor-alpha and interleukin-2 which were selected specifically for their ability to enhance TIL cytotoxicity without the need for preconditioning chemotherapy or postconditioning IL2. In a phase I, open-label, 3+3 dose-escalating multicenter trial, patients with stage IV melanoma were treated with multiple intravenous and intratumoral injections of TILT-123 and a one- or two-time treatment with TILs. TILs were grown from resected tumor tissue and administered without pre- or post-conditioning treatment regimens. The primary endpoint of the study was safety of TILT-123. Secondary endpoints included safety and efficacy of TILT-123 in combination with TILs. Sixteen patients with CPI resistant progressive cutaneous (7), mucosal (5) and uveal (4) metastatic melanoma were treated. Median age was 65.5 years (25-75). The most frequently reported adverse events (AEs) related to TILT-123 were fever (63%) and pain at the injection site (44%) while most frequent AEs related to TIL therapy were fever (50%) and chills (24%). No dose-limiting toxicity was observed and the combination of TILT-123 and TIL therapy did not increase the severity of AEs. 31% of patients experienced treatment related serious adverse events. In d78 imaging, RECIST1.1 responses were observed in two patients while disease control rate was 38 % (6/16). Responders included one patient (cutaneous) with an ongoing partial response and one patient (mucosal) with a durable complete response. Further, two patients (uveal and cutaneous) had long lasting stable disease (> 10 months). PET evaluation on d78 revealed disease control in 6/13 evaluable patients, including 4 partial or minor responses. On d36, after 4 injections of TILT-123 and before TILs, 4 partial or minor responses were seen in PET. The combination of TILT-123 and TIL therapy is safe and feasible in patients with metastatic melanoma. Clinical activity in hard-to-treat melanoma subtypes was observed.

Bispecific T cell engager (BsTe) is a fusion recombinant protein comprised of two single–chain variable fragments with dual specificity for a tumor–associated antigen (TAA) and T cell receptor (usually CD3ε). Immunotherapy with BsTe has shown efficacy in patients with hematologic malignancies and uveal melanoma. However, the antitumor efficacy of BsTe in most solid tumors has been limited due to their short serum half-life and insufficient tumor concentration. We designed a novel serotype 5/3 oncolytic adenovirus encoding for a BsTe cross-linking Mucin1 (MUC1) to CD3, Ad5/3–E2F–d24–aMUC1aCD3.

Tumor-infiltrating lymphocytes (TILs) and natural killer (NK) adoptive cell therapies have shown promising results in advanced-stage melanoma and haematological malignancies, respectively. However, their limited persistence in vivo in absence of an exogenous source of IL-2, and migration to neoplastic sites have impaired their effectiveness in immunosuppressive tumor microenvironments, such as ovarian cancer. Here, we propose the use of an engineered oncolytic adenovirus encoding a vIL-2 cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), to improve said cell therapies. Oncolytic adenoviruses are immunogenic agents that lyse infected cancer cells and recruit immune cells to the neoplastic site. Moreover, the vIL-2 virus continuously expresses a vIL-2 cytokine that preferentially stimulates effector lymphocyte proliferation over T regulatory cells. Fragments of resected human ovarian cancer tumors were received and processed into single-cell suspensions. Autologous TILs were expanded from said sample fragments, while PBMC cells from healthy donors were used as the source of allogeneic NK cells. To evaluate cancer cell killing, ovarian cancer ex vivo tumor cultures were co-cultured either with autologous TILs or with allogeneic NK cells in the presence or absence of the vIL-2 virus. Additionally, in vivo combination therapies efficacy was assessed with a patient-derived xenograft (PDX) ovarian cancer model. Immune cell profiling was performed following patient sample co-cultures and PDX tumor treatments. The addition of vIL-2 virus to the ovarian cancer ex vivo tumor cultures improved both TIL and NK cell therapies efficacy in vitro. Similarly, significantly better tumor control was achieved when the vIL-2 virus was given in conjunction with cell therapies compared to their respective controls. Mechanistically, vIL-2 virus treatment enhanced cell cytotoxicity of adoptively transferred TILs and NK cells in PDX tumors, and in ovarian cancer tumor cultures. Ad5/3-E2F-d24-vIL2 virus treatment seems to be a promising immunotherapeutic candidate to improve the response of adoptive cell therapies in human immunosuppressive solid tumors.

In recent years, cancer therapy has witnessed the successful development and clinical implementation of a wide array of tools to fight cancer, including, for example, monoclonal antibodies and adoptive cell therapies (ACT). Immunotherapies employing such tools to reinvigorate T-cells continue to change patient care by providing durable benefit in patients with advanced solid tumors. The latter is offset, however, by a large share of patients that present little response to immunotherapy, in part due to T-cell dysfunction.

Adoptive T-cell therapy using tumor-infiltrating lymphocytes (TIL) has demonstrated that potent and long-lasting antitumor responses can be achieved with cellular cancer immunotherapy in solid tumor patients. Yet, the therapy yields various toxicities, due to toxic lymphodepleting preconditioning and IL-2 postconditioning. With the intent of decreasing conditioning-related toxicities, while retaining efficacy, we developed TILT-123 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2), a novel oncolytic adenovirus designed to reinvigorate antitumor T-cells.

Treatment of unresectable metastases remains a challenge in oncology. In special because the abscopal effect remains limited in the clinic mainly due to the suppressive microenvironment surrounding cancer cells. The systemic use of immune checkpoint inhibitors, such as anti- anti-PD-1 (anti programmed cell-death protein 1), has shown promise in the treatment of various tumour types, but only a minority of patients respond. Oncolytic virotherapy (OV) is an interesting strategy able to prime the host immune system against tumour epitopes to generate anti-tumour immunity. Then, theoretically complementing anti-PD-1 therapy in an appealing manner. The challenge with OV is that not all metastases can usually be injected. Therefore, it is important to study if local treatment can induce distant responses. While our previous work has shown synergy between oncolytic adenovirus and anti-PD-1, here we sought to establish if local adenovirus injection can impact also non-injected tumours in a pre-clinical model in the context of systemic anti-PD-1 therapy. We engrafted melanoma mouse cells (B16.OVA) in both flanks of immunocompetent mice. Anti-PD-1 treatments were administered systemically, but only one tumour received local virotherapy with the non-replicative adenovirus. Progression was followed in both tumours and compared for possible systemic effects following local treatment, including immune responses. Tumour growth control and overall survival were significantly better in non-injected tumours in the group receiving the anti-PD-1 plus virus, over monotherapy and control groups. In particular, treatment with anti-PD-1 plus virus was the only group that induced complete response of both tumours. Further information, including immunological mechanism-of-action data, will be presented. TNFa and IL-2 armed adenovirus seems a promising approach to improve the efficacy of checkpoint blockade, and the effects can be seen in both injected and non-injected tumours. Here, the systemic anti-tumour effects result from local injection of the virus. A clinical trial is in progress (NCT04217473).

Abstract Background During the last decade, a revived enthusiasm about T-cell related therapies emerged after promising clinical outcomes. Nevertheless, many patients (especially with solid tumours) still do not have adequate therapeutic options. The complexity of the tumour microenvironment is a likely factor acting in detriment of many of those therapies by multiple suppressive mechanisms. To tackle a complex mechanism, an oncolytic adenovirus 2 (Ad5/3-E2F-d24-hTNFa-IRES-hIL2, a.k.a. TILT-123) was engineered to enable T-cell therapies in those circumstances. Methods To study the efficacy of TILT-123 together with different T-cell related therapies (adoptive cell transfer, checkpoint inhibitors and CAR T cell therapy) different models were used including mouse, Syrian Hamster and patient derived in vivo models for different indications were tested. Results Antitumor efficacy analyses showed complete responses in all animals receiving a T-cell therapy or checkpoint inihibitor (aPD1 or aPDL-1) and the T-cell enabling virus. Further, other aspects such as safety, influence on different immune populations, abscopal effect, antitumor memory and ability to replace lympho-depleting chemotherapy and postconditioning with high-dose IL-2 treatment were studied. Conclusion The use of TILT-123 to enable T-cell therapies (including checkpoint-inhibiting antibodies) delivered encouraging preclinical results pointing to it as a valuable approach to increase the number of patients that benefit from T-cell therapies. Those results include not only cell based therapies but also checkpoint inhibitors, a kind of therapy that is making the difference in the field and becoming first-line treatment for an increasing number of indications. Because of the favourable preclinical studies, the first in human clinical trials with TILT-123 will start in the upcoming months. Legal entity responsible for the study The authors. Funding TILT Biotherapeutics. Disclosure V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics. R. Havunen: Full / Part-time employment: TILT Biotherapeutics. J.M. Santos: Full / Part-time employment: TILT Biotherapeutics. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics. All other authors have declared no conflicts of interest.

Background The ovarian tumour microenvironment is an abundant source of tumour-infiltrating lymphocytes (TILs) which can be readily harnessed for infusion in the context of adoptive TIL therapy. This strategy has been proven feasible, but it appears clinically ineffective in ovarian cancer patients due to the absence of tumour-reactive TILs and the presence of immunosuppression. Hence, we hypothesized that an oncolytic adenovirus expressing Tumour Necrosis Factor (TNF)-alpha(a) and Interleukin (IL)-2 (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) could overcome this by generating a proinflammatory microenvironment and reinvigorating TIL anti-tumour activity. Methods Fresh explants from metastatic or primary tumour sites were obtained from stage III-IV ovarian cancer patients and short-term cultures were established in the absence or presence of oncolytic adenovirus. The remainder of the tumour explant was used to generate clinically relevant TILs which were co-cultured with autologous T cell-depleted single cell suspensions pre-treated with or without oncolytic adenoviruses. Cytokine content changes and TIL reactivity was assessed during culture by flow cytometry or interferon (IFN) gamma(g) enzyme-linked immune sorbent assay. Results Treatment of short-term cultures with oncolytic adenovirus coding for TNFa and IL-2 introduced profound changes within the microenvironment, which were characterized by an increase in proinflammatory cytokines and decrease in suppressive cytokines. Further benefits were seen in T-cell depleted ovarian cancer single-cell suspensions infected with cytokine-coding oncolytic adenovirus, which enabled significant production of IFNg by autologous TILs. Such levels were not seen in co-cultures where no virus or the backbone oncolytic adenovirus (no cytokine transgenes) was added. Conclusion These data illustrate the potential of oncolytic adenovirus coding for TNFa and IL-2 to rewire the ovarian tumour microenvironment for effective TIL anti-tumour reactivity. This approach may improve the efficacy of adoptive TIL therapy in ovarian cancer patients, thus warranting further clinical investigation. Legal entity responsible for the study TILT Biotherapeutics Ltd. Funding TILT Biotherapeutics Ltd. Disclosure J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. M. Siurala: Full / Part-time employment: TILT Biotherapeutics Ltd. T. de Gruijl: Advisory / Consultancy: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.

Abstract Background Dendritic Cell (DC) therapy is considered as a promising immunotherapeutic approach for treatment of advanced cancer. However, the tumor microenvironment is highly immunosuppressive that leads to DC dysfunction. Therefore, in clinical trials DC therapy has generally failed to fulfill its expectations. Oncolytic adenoviruses are well tolerated and have shown to preferentially target and kill cancer cells. Therefore, to improve the therapeutic efficacy of DC therapy, we armed oncolytic adenovirus with CD40 ligand (CD40L). CD40L is well known to regulate immune responses through its capacity to stimulate dendritic cells that lead to the activation of cytotoxic T-cells. Methods In this study, we generated a novel virus Ad3-hTERT-CMV-hCD40L (Ad3-hCD40L), which is fully serotype 3 adenovirus. It features a human telomerase reverse transcriptase promoter for tumor specificity and expresses human CD40L (hCD40L) under a cytomegalovirus promoter for induction of antitumor immune responses. Animal experiments were implanted in immunocompetent and in humanized mice model. To further deeply dissect if Ad3-hCD40L can modulate tumor microenvironment, tumor histocultures derived from prostate patients were used. Results In syngeneic studies in animal models, DC therapy with Ad3-hCD40L showed significant antitumor immune response. This enhanced therapeutic effect is associated with increased tumor specific T-cells and induction of T-helper type 1 immune response. Moreover, Ad3-hCD40L and human DCs showed 100 percent survival in conjunction with tumor control. Tumor histocultures treated with Ad3-hCD40L showed that virally expressed hCD40L in the tumor microenvironment leads to significant activation of dendritic cells and induction of Th1 immune response. Conclusion To conclude, CD40L armed oncolytic adenovirus 3 improves DC therapy by favorable alteration of tumor microenvironment. These findings support clinical trials where DC therapy is enhanced with oncolytic adenovirus. Legal entity responsible for the study Cancer Gene Therapy Group, University of Helsinki. Funding University of Helsinki. Disclosure V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.

Releasing the patient's immune system against their own malignancy by the use of checkpoint inhibitors is delivering promising results. However, only a subset of patients currently benefit from them. One major limitation of these therapies relates to the inability of T cells to detect or penetrate into the tumor resulting in unresponsiveness to checkpoint inhibition. Virotherapy is an attractive tool for enabling checkpoint inhibitors as viruses are naturally recognized by innate defense elements which draws the attention of the immune system. Besides their intrinsic immune stimulating properties, the adenoviruses used here are armed to express tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2). These cytokines result in immunological danger signaling and multiple appealing T-cell effects, including trafficking, activation and propagation. When these viruses were injected into B16.OVA melanoma tumors in animals concomitantly receiving programmed cell-death protein 1 (PD-1) blocking antibodies both tumor growth control (p < 0.0001) and overall survival (p < 0.01) were improved. In this set-up, the addition of adoptive cell therapy with OT-I lymphocytes did not increase efficacy further. When virus injections were initiated before antibody treatment in a prime-boost approach, 100% of tumors regressed completely and all mice survived. Viral expression of IL2 and TNFa altered the cytokine balance in the tumor microenvironment towards Th1 and increased the intratumoral proportion of CD8+ and conventional CD4+ T cells. These preclinical studies provide the rationale and schedule for a clinical trial where oncolytic adenovirus coding for TNFa and IL-2 (TILT-123) is used in melanoma patients receiving an anti-PD-1 antibody.

Background: Adoptive cell transfer (ACT) has shown promising yet suboptimal results in clinical trials for melanoma. We aimed to enhance the efficacy of ACT by coupling it with adenoviruses coding for tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2). Methods: Non-replicating adenoviruses (Ad5-CMV-mIL2 and Ad5-CMV-mTNFa) were constructed and confirmed to produce biologically active murine cytokines in vitro and in vivo, and subsequently used in combination with ovalbumin-specific (OT-I) T-cell transfer. Oncolytic adenoviruses expressing human TNFa and/or human IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2 also known as TILT-123) were also constructed and used in combination with tumor-infiltrating lymphocytes (TIL) transfer in adenovirus-permissive Syrian hamsters. Results: The non-replicating virus vectors used in combination with OT-I T-cells effectively controlled tumor growth of established murine melanoma (B16.OVA) tumors. In further experiments, a triple combination of Ad5-CMV-mIL2 + Ad5-CMV-mTNFa (1:1 ratio) and OT-I T-cells improved antitumor efficacy over double combinations. Mechanistic studies revealed that intratumoral virus injections induce trafficking of adoptively transferred T-cells to tumors. Further, the cytokine-coding adenoviruses caused favorable alterations in the tumor microenvironment. In hamsters, established pancreatic cancer tumors were eradicated with TILT-123 injections coupled with TIL transfer. The cured animals were protected from tumor rechallenge. In addition, we have demonstrated that adenovirally delivered IL-2 is superior to systemically administered recombinant IL-2 with regard to safety and efficacy in the preclinical setting. Conclusions: The combinatorial approach studied here highlights the potential of adenovirotherapy to enable T-cell therapies. TILT Biotherapeutics is in the process of confirming the results in clinical trials. Legal entity responsible for the study: TILT Biotherapeutics Ltd Funding: TILT Biotherapeutics Ltd, Finland; Jane and Aatos Erkko Foundation, Finland; European Commission Marie Curie Innovative Training Network (ITN) grant VIRION (H2020-MSCA-ITN-2014 project number 643130); Doctoral Program in Clinical Research, University of Helsinki, Finland; Helsinki University Central Hospital (HUCH) Research Funds, Finland; Sigrid Juselius Foundation, Finland; Biocentrum Helsinki, Finland; Biocenter Finland, Finland; Finnish Cancer Organizations, Finland. Disclosure: J.M. Santos, M. Siurala, R. Havunen, V. Cervera-Carrascon, S. Sorsa: Employee in TILT Biotherapeutics Ltd. A. Hemminki: Shareholder of Targovax SA and employee in TILT Biotherapeutics Ltd.

Background: Immune checkpoint inhibitors have been delivering promising results in an increasing number of indications. This approach proved the potential benefits of the use of the immune system as a tool against malignant cells. However, there is still room from improvement as there is a considerable number of patients not benefiting from these therapies. One of the biggest obstacles to overcome for those unresponsive patients is the lack of immune presence in the tumor. To overcome that limitation, we hypothesize that viruses are an appealing tool, as they are inherently able to attract the attention of the immune system and to express different transgenes. In our case, the viruses were armed with tumor necrosis factor alpha (TNFα) and interleukin-2 (IL-2), two cytokines to improve the trafficking to the tumor and enable T-cell mediated responses in the tumor. Methods: To study the previously described scenario, an in vivo model of subcutaneous melanoma (B16.OVA) was used. Three different experiments were carried out (n = 48, 75 and 94) in order to assess the benefits and to understand the effect in the tumor microenvironment after the treatments. Results: When both therapies where given together, they delivered significantly better results than single treatment groups in term of overall survival and tumor growth control. From all the tested conditions, the treatments delivered better results when virotherapy was initiated before checkpoint blockade, in a "prime and boost" approach. In this set up, a 100% complete response was achieved (HR = 0.026 [0,005; 0,139] when compared with checkpoint blockade alone and HR = 0.069 [0,015; 0,327] when compared with virotherapy alone). The biological samples obtained through the study revealed a shift towards a Th1 cytokine status in the tumor while profile of the immune cell population also showed a significant change towards to the antitumor subsets. Conclusions: The purpose of this study was to try to overcome poor immune infiltration in order to enable checkpoint blockade therapies, based on the results, the objectives were achieved after the use of a viral platform delivering immunostimulatory cytokines in the tumor that helped to redesign the tumor microenvironment. Legal entity responsible for the study: Cancer Gene Therapy Group Funding: TILT Biotherapeutics Ltd. Disclosure: V. Cervera-Carrascon, J.M. Santos, M. Siurala, R. Havunen and S. Sorsa: Employee of TILT Biotherapeutics. A. Hemminki: Shareholder in Targovax ASA (Oslo, Norway) and in TILT Biotherapeutics Ltd. (Helsinki, Finland). Employee of TILT Biotherapeutics.