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.

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.