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We developed an autologous hematopoietic stem cell-based platform designed to deliver IFNa, by a transcriptional and post-transcriptional control mechanism mediated by miRNA target sequences, specifically into the tumor microenvironment (TME) via Tie-2 expressing monocytes (Temferon). As of Feb 2022, 3 escalating doses of Temferon (0.5-2.0x106/kg) were tested across 15 newly diagnosed, unmethylated MGMT GBM patients assigned to 5 cohorts. Follow-up from surgery is 6–28mo (2–25mo after Temferon). To date, no DLTs have been identified. As expected, 1mo after the administration of the highest tested dose, the hematopoietic system of Temferon-treated patients was composed of up to 30% of CD14+ modified cells. Temferon-derived progeny persisted, albeit at lower levels, up to 18mo (longest time of analysis). Despite the substantial proportion of engineered cells, very low concentrations of IFNα were detected in the plasma and in the CSF, indicating tight regulation of transgene expression. SAEs were mostly attributed to conditioning chemotherapy (infections) or disease progression (seizures). 1SUSAR (persistent GGT elevation) occurred. Median OS is 15mo from surgery. Homing of transduced cells to the tumor was demonstrated by the presence of gene-marked cells in the 2nd surgery specimens of 3 out 4 pts belonging to low dose cohorts. Single-cell RNA seq of the TME highlighted a Temferon signature associated with the induction IFNa responsive genes and macrophage repolarization. Potential long-term benefit with Temferon was identified in a patient from cohort 3, who had PD at D+120 with two distant enhancing lesions, and increased tumor necrosis. 1y following Temferon, with no 2nd-line therapy added, there was approximately 40% reduction in enhancing tumor volume compared to D+180 with a stable clinical and imaging picture thereafter. The results provide initial evidence of Temferon’s potential to modulate the TME of GBM patients, and anecdotal evidence for long lasting effects of Temferon in prevention of disease progression.

Computational Neurosurgery is a novel field where computational modeling and artificial intelligence (AI) are used to analyze diseases of neurosurgical interest. Our aim is to apply AI models to brain tumor (BT) images to a) automatically segment BTs on pre-operative MRI, b) predict the genetic subtype of glioma on intra- and post-operative histological specimens; and c) predict the extent of resection according to connectomics data. For the segmentation task, we used 510 BT images to train a deep learning (DL) model for automatic segmentation of the tumors’ edges and comparison of the AI-generated masks versus experts’ consensus (quantified by means of the dice score). For the histopathology task, we digitalized 266 hematoxylin/eosin slides of gliomas (including 130 IDH-wildtype and 136 IDH-mutant) and applied a DL architecture to predict the IDH genetic status, then validated by immunohistochemistry and genetic sequencing. The datasets were also augmented by generating synthetic glioma images by means of a Generative Adversarial Network methodology. The resection of 10 BTs was also customized according to connectomics data. In the segmentation experiment, we reached a dice score of ~0.9 (out of 1.0), while further demonstrating that only the T1, T1 after gadolinium, and FLAIR sequences are necessary for accurate automatic segmentation. In the histopathology task, we were able to predict the genetic status with accuracy between 76% and 95% using the DL model. The machine learning-based connectome analysis allowed us to perform safe supramaximal resection. We have shown the robustness of applying AI methodology or the automatic segmentation of BTs in MR imaging. Moreover, we have also shown that AI can be used to predict the genetic status, specifically, IDH, in histopathology images of gliomas. Our results support the use of AI in the clinical scenario for a fast and objective computerized characterization of patients affected by BTs.

Temferon is an ex vivo gene therapy consisting of autologous HSPCs genetically modified to deliver IFN-α2 within the tumor microenvironment (TME) by Tie-2 expressing macrophages. TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed GBM patients with unmethylated MGMT. Autologous HSPCs are transduced with a LVV encoding for IFN-a2 gene. As of 30th April 2021, 18 patients have been enrolled; 13 received Temferon (D+0) with follow-up of 8 – 662 days. After conditioning and Temferon infusion, a rapid engraftment and hematological recovery occurred, with median neutrophil and platelet engraftment at D+13 and D+12, respectively. No dose limiting toxicities were reported. Temferon-derived cells were found within 14 days post treatment and persisted albeit at lower levels in the long-term. Five deaths occurred: one at +478, three at +322, +340 and +402 days due to PD, and the fourth at +60 due to complications following the conditioning regimen. Eight patients had PD (-12 to +239). SAEs include respiratory tract infections, pulmonary embolism, CMV and C.Diff infections, febrile neutropenia, hemiparesis, seizure, brain abscess, worsening of performance status and respiratory failure compatible with ASCT, concomitant medications and PD. Four patients underwent second surgery. Recurrent tumors had gene-marked cells present and increased expression of ISGs compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. Characterization of T-cell immune repertoire suggests the expansion of tumor-associated clones. TME characterization by scRNA and TCR sequencing is ongoing. Interim results show that Temferon is well tolerated, with no dose limiting toxicities identified to date and provide initial evidence of potential immune system activation within the TME.

Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies.

BACKGROUND Most patients with GBM & an unmethylated O-6-methylguanine-DNA methylase (MGMT) gene promoter, have a poor prognosis with approximately 20% of patients surviving to 2 years. Poor prognosis is likely related to factors including a highly immunosuppressive tumor microenvironment (TME). The TME in GBM is mainly composed of tumor associated macrophages (TAMs) & microglia. A subset of tumor-infiltrating macrophages characterized by expression of the angiopoietin receptor Tie2 (TEMs) have features of M2-TAMs, promote tumor angiogenesis & are infrequently found in normal organs. Tie2 is significantly upregulated upon homing to tumors. Gene therapy technology has allowed TEMs to be used as carriers for the local and tumor restricted release of interferon-α (IFN). IFN has antitumor effects, inhibits angiogenesis & modulates the immune system. Cell-based delivery of IFN into the TME by TEMs is expected to provide efficacy, taking advantage of pleiotropic anti-tumor effects & avoiding tolerability issues associated with systemic IFN treatment. We are currently conducting a Phase I/IIa clinical study in Milan (INCB & OSR) to evaluate this therapeutic approach (Temferon) in 21 patients with GBM & unmethylated MGMT promoter (EudraCT Number 2018-001404-11). Eligible patients are identified immediately after first surgical resection. After screening, harvesting of HSPCs occurs followed by 6 weeks of radiotherapy. Non-myeloablative conditioning consists of BCNU & thiotepa followed by administration of non-manipulated HSPCs and Temferon. RESULTS In Part A of the study, 3 cohorts of 3 patients will receive escalating doses of Temferon. After Part A, a single dose of Temferon will be studied in a further 12 patients (Part B). Criteria for study eligibility are the same for both Part A and Part B. Part A is ongoing with the first 2 patients recruited (June 2019) and Temferon is scheduled for administration. An update on the status and progress of the study will be provided.

BACKGROUND Glioblastoma (GBM) remains the most common malignant primary brain tumor in adults. Most patients with GBM & an unmethylated O-6-methylguanine-DNA methylase (MGMT) gene promoter, have a poor prognosis with approximately 20% of patients surviving to 2 years. Poor prognosis is likely related to a number of factors including a highly immunosuppressive tumor microenvironment (TME). The TME in GBM is mainly composed of tumor associated macrophages (TAMs) & microglia. A subset of tumor-infiltrating macrophages characterized by expression of the angiopoietin receptor Tie2 (TEMs) have features of M2-TAMs, promote tumor angiogenesis & are infrequently found in normal organs. Tie2 is significantly upregulated upon homing to tumors. Gene therapy technology has allowed TEMs to be used as carriers for the local and tumor restricted release of interferon-α (IFN). IFN has antitumor effects, inhibits angiogenesis & modulates the immune system. Cell-based delivery of IFN into the TME by TEMs is expected to provide efficacy, taking advantage of pleiotrophic anti-tumor effects & avoiding tolerability issues associated with systemic IFN treatment. MATERIAL AND METHODS We are currently conducting a Phase I/IIa clinical study in Milan to evaluate this therapeutic approach (Temferon) in 21 patients with GBM & unmethylated MGMT promoter (EudraCT Number 2018- 001404-11). The study recruits & follows up patients at a specialist neurosurgical & neuro-oncology unit (INCB); administration of Temferon & hematological follow up takes place at a specialist hematology & bone marrow transplantation unit at OSR. Potentially eligible patients are identified immediately after first surgical resection of GBM once the MGMT promoter methylator status is known. Once screening procedures have been completed, harvesting of HSPCs occurs followed by 6 weeks of radiotherapy. Patients receive a non-myeloablative conditioning regimen consisting of BCNU & thiotepa. This is followed by administration of non-manipulated HSPCs and Temferon. In-patient monitoring occurs until hematological recovery. Thereafter, regular follow-up of patients occurs up to 2 years (+720 days) and patients will be invited to participate in a long term follow-up study lasting an additional 6 years. RESULTS In Part A of the study, 3 cohorts of 3 patients will receive escalating doses of Temferon. On completion of Part A, a single dose of Temferon will be selected to be studied in a further 12 patients in Part B. Criteria for study eligibility are the same for both Part A and Part B. In the event that GBM disease progression occurs, patients will be managed with second line therapies including second surgery, TMZ, BCNU, fotemustine or any other approved therapy for GBM. Patient recruitment for Part A is ongoing with the first dose of Temferon administered in July 2019.