LOAd703-induced tumor microenvironment gene engineering in combination with atezolizumab in metastatic malignant melanoma: a phase I/II trial

Domain: chemistry
Published: 2026-02-16
Editorial outcome: minor_revision

Abstract
This phase I/II study evaluated the capacity of tumor microenvironment (TME) gene engineering by intratumoral injection of a viral vector encoding a designed CD40L and 4-1BBL combined with intravenous atezolizumab (anti-PD-L1 antibody) to induce immune activation in 24 patients with stage IV malignant melanoma refractory to PD-1 inhibition. Primary objective was tolerability. Tumor response, pharmacokinetics, and biomarker evaluations were secondary. Treatment was well-tolerated in both dose cohorts (1×1011 and 5×1011 viral particles). Th1 immune biomarkers was increased in the TME (NanoString) as well as in blood (Olink). In long-term survivors, we observed increased markers for T cell fitness and for the immunoproteasome. The overall response rate was 17% accordingly to RECIST 1.1 and disease control was noted in 54%. Forty-six percent of patients were still alive two years post enrollment. The lower dose showed very encouraging results with a median progression-free survival of 9.7 months and median overall survival of 26.3 months (post-hoc analyses). In conclusion, TME gene engineering may have re-sensitized refractory patients to checkpoint treatment or acted alone to control tumor growth. The small sample size and single arm design limits effect interpretation but the data shows promise for continued clinical investigation. Study registration: NCT04123470.

Introduction
Cutaneous malignant melanoma (CMM) is the fifth most common form of cancer in the Western world, with a tripled incidence in the US over the last decades1. Immune checkpoint inhibition (ICI) targeting PD-1, CTLA-4, and LAG-3 has revolutionized advanced CMM treatment, increasing the 5-year survival rate to 50% and is standard-of-care together with protein kinase inhibitors for BRAFV600E-mutated CMM2–4. Despite these breakthroughs, therapy resistance is common5. Resistance depends on low numbers of tumor-infiltrating T cells and their inability to kill tumor cells. An approach for ICI-resistant patients is to reprogram the tumor microenvironment (TME) to sensitize the patients to ICI and to promote in vivo generation of T cells.

The TME consists of multiple cell types, including fibroblasts, vascular endothelium, immune cells, and products released by them that collectively induce immunosuppression6,7,8 and reduce activation of a type 1 T helper cell (Th1) response needed to combat the tumor9.

LOAd703 is a TME gene engineering adenoviral vector with oncolytic capacity, transferring Th1-type immunostimulatory genes to the TME10–12. While it can infect and induce expression of the genes in any infected cell, the replication-induced oncolysis occurs only in tumor cells. LOAd703 encodes trimerized, membrane-bound CD40L (TMZ-CD40L) and 4-1BBL. CD40L is an important initiator of immune responses by inducing maturation of DCs, M1 macrophage differentiation, chemokine and cytokine release from stroma cells, and upregulating attachment receptors on endothelial cells13–15. Importantly, LOAd703-stimulated DCs upregulate CCR716,17, which promotes their translocation to lymph nodes where they can induce systemic immune responses. Additionally, 4-1BBL maintains immune responses by increasing cytokine and chemokine release by DCs and T cells, expanding T and NK cell populations, preventing activation-induced cell death of T cells, and stimulating memory T cell differentiation18. Moreover, infection of tumor cells with LOAd703 enhances tumor cell immunogenicity by upregulation of death receptors and major histocompatibility complex class I (MHC-I)19.

LOAd703 combined with chemotherapy has been evaluated in patients with advanced pancreatic cancer20. Herein, we present the clinical phase I/II trial LOKON003, which was conducted to evaluate safety, activity, and biomarkers of LOAd703 combined with an anti-PD-L1 antibody (atezolizumab) in ICI-resistant CMM patients.

Results
Patient characteristics
Between 2021 and 2023, 26 patients were screened for eligibility, of which 24 were recruited (Supplementary Fig. 1): seven to dose cohort 1×1011 viral particles (VP) and 17 to dose cohort 5×1011 VP. The data cut was in July 2024. Patient demographics and disease characteristics are summarized in Table 1. The median age was 64 years. Fifty-four percent were male, and 46% were female. At baseline, 63% of patients were assessed as ECOG-0 and the remaining 38% ECOG-1. Fifty percent had elevated lactate dehydrogenase at screening. PD-1 inhibitory treatment alone, or in different combinations, was given in an adjuvant setting in 38% of patients and/or against systemic disease in 92%. Ten of the 24 patients (42%) had received ≥2 prior treatments with a PD-1 targeting antibody (range for all patients: 1–4), including both adjuvant and systemic regimes. The patients had received a median of two prior treatments for systemic disease (range 0-6). Seven patients had a BRAF-mutated tumor, of which five had received prior treatment with BRAF inhibitors before enrollment in this study. Other treatments for systemic disease included experimental ICI combinations, oncolytic virus (T-VEC), chemotherapy and other cell cytotoxic agents as shown in Table 1.

Safety
Patients received a median of six LOAd703 injections (Table 2), and the most common injection sites were lymph nodes or subcutis/muscles of a limb. The patients received a median of 6 treatments with atezolizumab. Of note, patients in dose cohort 1×1011 VP received a median of 12 LOAd703 doses and 14 doses of atezolizumab, while the 5×1011 dose cohort received fewer LOAd703 injections (median 5), as well as atezolizumab infusions (median 5).

The primary endpoint was to evaluate tolerability in terms of adverse events (AEs). AEs attributed to LOAd703 are listed in Table 3. The most frequent AEs were pyrexia, which occurred in 14 patients (58%), chills and vomiting in five patients (21%), and four patients (17%) had events of cytokine release syndrome (CRS), headache, infusion-related reaction and/or nausea. A vitiligo-like rash was observed in one patient (4%). Overall, most LOAd703-attributed adverse events were mild and transient. There were only two grade 3 dose-limiting toxicities (DLTs) attributed to LOAd703: one case of pyrexia and one injection site reaction, which resolved to baseline. Despite both patients being treated at the 5×1011 dose cohort, MTD could not be reached as the DLT rate was substantially lower than the target (0.3). No LOAd703 dose reductions were necessary, and no patient experienced LOAd703-attributed AEs resulting in treatment discontinuation. All treatment-emergent adverse events, regardless of an attributed relationship to the study drugs or not, are listed in Supplementary Table 1. There were 17 AEs reported as serious (SAEs,) of which two were attributed to LOAd703 (Supplementary Table 2). These were CRS and pyrexia, both listed as grade 1–2 events.

Biomarkers and mechanisms of action
Secondary endpoints included the evaluation of biomarkers for mechanisms of action and tumor response. All analyzed patients (n=17) had pre-existing anti-adenovirus antibodies (ADA) which were significantly increased in blood already at week nine post-treatment initiation and thereafter remained stable during study participation (Fig. 1A). There was no significant correlation of the week nine antibody level with the last known date of survival (Fig. 1B). Only 7 of the 22 patients (32%) had detectable levels of antibodies against atezolizumab at any time point post treatment initiation (Fig. 1C). Presence of virus DNA in blood and shedding via urine, saliva, and stool is being evaluated in a cross trial analysis and will be presented elsewhere.

Biopsies from the injected metastases were collected at baseline (n=16) and at week nine post-treatment initiation (n=15). In Fig. 2A, an mRNA heatmap of all patients pre- and post-treatment initiation indicated an induction of immune biomarkers by LOAd703 and atezolizumab. Figure 2B and Supplementary Data 1: NanoString displayed genes that were significantly altered by the treatment, revealing the presence of activated T and NK cells, chemokines attracting them to the TME, as well as receptors that allow them to transmigrate from blood vessels into the TME. Further, markers for antigen presentation and B-cell activation were upregulated. Another observation was that checkpoints such as CTLA-4, TIGIT, LAG-3 and PD-L2 (PDCD1LG2) were amplified as well. Patients were divided according to above or below median OS at baseline (Fig. 2C, E and Supplementary Table 3) and at nine weeks post-treatment initiation (Fig. 2D, Fig. 2F and Supplementary Table 4). When adjusting for multiple testing, no difference in gene expression between the patients below and above the median OS was identified, but the sample size was small when the cohort was divided. As these tests are not compensating for biological relevance (i.e., a small difference may yield a biological difference of one biomarker while another biomarker will need a substantial change in expression for a biological response to occur), there may be false negatives samples masked in small datasets. Hence, we analyzed the data set without compensating for multiple comparisons to understand if the data can indicate whether certain biomarkers should be further evaluated in upcoming studies. Without adjusting for multiple comparisons in these smaller sub-cohorts, patients who survived longer than the median had expression of genes involved in antigen presentation and protection against a viral response at baseline, and expression of genes involved in antigen presentation, activation of T and NK cells at week nine post treatment initiation. Instead, patients who survived shorter than the median expressed genes associated with immunosuppressive cells. In blood, protein biomarkers were analyzed with an immune oncology (Fig. 2G and Supplementary Table 5) and an oncology panel (Fig. 2H, Supplementary Table 6). When adjusting for multiple comparisons by the false discovery rate (5%) of Benjamini-Hochberg, only PD-L1 remained significantly elevated (p=4.4×10-14), likely due to the previously reported high PD-L1 in blood after treatment with anti-PD-L1. However, when disregarding multiple comparison compensation for the same reasons as discussed above, biomarkers for the presence and activation of DCs, T- and NK cells were present in blood along with ICI targets such as PD-L1 and PD-1.

Efficacy
The overall response rate (ORR) was 17% (n=24) (Table 2), with 14% and 18% in dose cohort 1×1011 VP and 5×1011 VP, respectively. However, 38% (n=9) experienced stable disease for at least eight weeks, resulting in a disease control rate (DCR) of 54% as calculated post-hoc. Of note, in dose cohort 1×1011 VP, 57% of the patients showed long-term disease stability (DCR ≥6 months) versus 29% in cohort 5×1011 VP. Tumor control over time was noted in target lesions (Fig. 3A), including in the LOAd703 injected lesions (Fig. 3B). Nine patients were evaluated with FDG-PET/CT scans, allowing for a post-hoc evaluation of the metabolic ORR. The metabolic ORR was 75% versus 60% in dose cohort 1×1011 (n=4) and 5×1011 (n=5) VP, respectively (Supplementary Table 7). Progression-free survival (PFS) and overall survival (OS) were also evaluated post-hoc. PFS was 3.4 months (n=24). Divided by dose cohort, the median PFS was 9.7 and 3.0 months in the 1×1011 VP and 5×1011 VP dose cohorts, respectively (Table 2, Fig. 3C). Median OS was 19.3 months (n=24). Grouped by dose cohort in a post-hoc analysis, the median OS was 26.3 and 12.4 months in the 1×1011 VP and 5×1011 VP dose cohorts, respectively (Table 2, Fig. 3D). Of note, the 24-month survival was 57% in dose cohort 1×1011 and 41% in cohort 5×1011 VP. In a post-hoc sub-analysis, patients were divided into those who had received one prior treatment (adjuvant or for systemic disease, n=8) prior to enrollment versus those who received >1 prior treatment (n=16). Median PFS was 3.0 versus 3.5 months in these groups (Fig. 3E), while median OS was 30.7 versus 10.9 months (Fig. 3F). Patients who received at least one of their LOAd703 injections in a lymph node (n=10) had poorer median OS compared to patients with other injection sites (10.0 months versus 35.0 months, respectively; Fig. 3G). Patients with M1a status had longer median OS (17.9 months) than M1c patients (12.4 months) as shown in an additional post-hoc analysis, but the curves did not separate (Fig. 3H). There were only two patients with M1b status, and the median OS was not reached. Note that in these post-hoc analyses, the groups were too small to show a statistically significant difference at a 95% confidence interval. In univariate Cox regression analysis, no clinical parameters were associated with OS except for ECOG status at enrollment that was near statistical significance (p=0.075) as well as receiving four or five previous treatments (p=0.065 and p=0.083, respectively; Supplementary Table 8).

Patients with a particular benefit from treatment
Although there were no complete responses (CRs) during the study treatment period, we observed two patients with partial response (PR) who became tumor-free after study participation. Firstly, a patient with metastases in the axilla, lung, lung hilum, mediastinum, pleura, liver, lymph nodes and subcutis was enrolled. At study week 5, the patient received a thermal ablation of a single pulmonary lesion. After 9 weeks of treatment with LOAd703 and atezolizumab, PR was observed in the non-ablated target lesions, with further response during the study period and completion of all study-related therapy (12 doses of LOAd703 and 19 treatments of atezolizumab). At the end of the treatment period, a complete metabolic response was observed which is maintained currently, without any further treatment, three years after treatment initiation (Supplementary Fig. 2A). Secondly, a patient with metastases in the pectoral muscle and in the subcutis of the abdominal and dorsal side of the trunk experienced a PR at week 18, which eventually deepened throughout therapy. At 11 months post study initiation, a single pectoral metastasis remained, and it was surgically excised (Supplementary Fig. 2B). The patient still remains tumor-free two years and two months after treatment initiation.

Methods
Study design
The study was a multi-center, single-armed, open-label, dose-escalation Phase I/II clinical trial (LOKON003; NCT04123470) evaluating the safety and effect of LOAd703 in combination with atezolizumab in patients with unresectable or metastatic melanoma at three recruiting centers: Uppsala University Hospital (Uppsala, Sweden), The Angeles Clinic and Research Institute, A Cedars Sinai Affiliate (Los Angeles, CA) and Baylor College of Medicine (BCM, Houston, TX). The study followed a Bayesian Optimal Interval (BOIN) design testing LOAd703 at two dose levels (1×1011 and 5×1011 VP) with a target DLT rate of 0.3 and a maximum of 25 patients enrolled at the maximum tolerated tested dose (MTD). The DLT period encompassed at least two doses of LOAd703, with at least 3 weeks of AE evaluation post-second dose. Sample size was determined based on an assumed response rate of 30% (Study Protocol is found in Supplementary Data 1). The study was reviewed and approved by the ethical review boards Etikprövningsmyndigheten (Uppsala University Hospital), WCG IRB (The Angeles Clinic and Research Institute) and the IRB at Baylor College of Medicine (BCM) (Sweden DNr 2019-05974, 2019-12-18; US IRB: 20193343, 2020-01-03). It was conducted in accordance with the ICH Harmonized Tripartite Guideline for Good Clinical Practice and the principles of the Declaration of Helsinki. All patients provided written informed consent and had the right to withdraw consent to further study participation at any time.

Patient enrollment and eligibility
Enrolled patients had stage IV CMM (n=23) or mucosal melanoma (n=1) with documented disease progression after one or several treatments with a PD-1 inhibitor. Swedish patients with BRAF-mutated melanoma were required to have received previous BRAF and/or MEK inhibitor therapy. Other previous treatments were allowed. Patients were required to have measurable disease according to RECIST 1.1 criteria with at least one visible injectable tumor lesion exceeding 10 mm on CT scan or MRI. If injected tumor lesions had been irradiated, confirmed radiological growth after irradiation was required. Age >18 years, Eastern Cooperative Oncology Group Performance Status (ECOG) performance status 0–1, and acceptable blood laboratory values (hematology, coagulation, liver aminotransferases and serum albumin) were also prerequisites for study participation.

Patients were excluded if they had rapid disease progression at the time of enrollment, clinically active cerebral metastases, a history of leptomeningeal disease, another malignant disease within the past two years, and concurrent or recent medication that could increase toxicity or interfere with the study treatment (including corticosteroids in doses of >10 mg prednisone equivalent per day). Further, patients with active, severe autoimmune disease or a history of grade 4 immune-related adverse events from earlier ICI monotherapy that had required steroid treatment for >12 weeks were also excluded. Patients with uncontrolled intercurrent illness that could compromise compliance with study requirements were ineligible.

Procedures
Enrolled patients were treated with LOAd703 (1×1011 or 5×1011 VP; manufactured by Baylor College of Medicine, TX) administered by image-guided, percutaneous intratumorally injection every third week. Atezolizumab (provided by Hoffmann-La Roche) was administered intravenously at a fixed dose of 1200 mg every third week. Patients could receive up to 12 cycles of combined treatment followed by up to seven infusions of atezolizumab monotherapy, resulting in a total maximal study treatment duration of 57 weeks.

Adverse events were collected throughout study participation and evaluated accordingly to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE v.5.0). Radiological evaluation was performed at baseline and every eight weeks. Patients with disease burden localized to the chest and abdominal area were evaluated with CT scans only. Patients with disease burden localized to the limbs or head and neck area were evaluated with FDG-PET/CT scans, and response was determined accordingly to RECIST 1.1. while the metabolic response was evaluated according to the European Organization for Research and Treatment of Cancer (EORTC). After the end of study participation, disease progression and survival were monitored every four months until death, withdrawal of consent or study end. Samples for ADA and biomarkers were taken at baseline and at multiple time points as indicated in Supplementary Fig. 1.

Objectives and endpoints
The primary objective of the study was to evaluate the tolerability of LOAd703 in combination with atezolizumab, determined by evaluation of adverse events according to CTCAE v5.0.

The secondary objectives were to determine anti-tumor activity, pharmacokinetics, and biological mechanisms-of-action of LOAd703 in combination with atezolizumab, including biomarker, shedding and anti-drug antibodies (PK and shedding are evaluated in a cross-trial evaluation and will be presented elsewhere). The endpoint for anti-tumor activity is ORR. Further, disease control (CR+PR + stable disease (SD)) and ≥6 months disease control were calculated post-hoc. Additionally, the median PFS and median OS were calculated post-hoc. Date of enrollment to date of progression (including clinical progression at the discretion of the investigator) or date of death was used to calculate PFS, while date of enrollment to date of death was used to calculate OS. Patients still alive or lost to follow-up at the cut-off were censored.

Anti-drug antibody analyses
Serum from patients collected at baseline and at several time points during study participation was analyzed by an anti-human adenovirus (Hxn) IgG ELISA kit (Alpha Diagnostic, San Antonio, TX, USA) accordingly to the company instructions. Samples were analyzed with a 3-tiered approach (screening, confirmation and titer) to detect antibodies against atezolizumab using a service provided by ICON plc (Farmingdale, NY, USA). Significant changes were evaluated by mixed-effects analysis for repeated analyses, alpha 0.05, and antibody units at week nine were correlated to the last known survival date (10 patients censored from 17 months onwards) using a Simple linear regression analysis at a significance level of 0.05. Both analyses were done using GraphPad Prism 10.4.1 (La Jolla, CA, USA).

Immune biomarkers
Tumor biopsies were collected at baseline and at week nine post-treatment initiation. From tumor biopsies, mRNA was purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). The mRNA from each patient was analyzed with NanoString technology (NanoString Technologies, Seattle, WA, USA) using the nCounter® PanCancer Immune Profiling Panel and the assay was performed by the Clinical Genomics Unit at the Uppsala University Hospital, Uppsala, Sweden. Data was normalized with RUV-4 in RStudio for the fold change between baseline and week 9, and then the approximate individual value was calculated by removing unwanted variation from the observed log2 value. For evaluation of alterations in gene expression post-treatment, multiple paired t tests were conducted with correction for multiple comparisons by the false discovery rate (5%) of Benjamini-Hochberg. For examination of differences in gene expression pre and post-treatment based on OS, unpaired t-tests were conducted. Because of the small sample size of the compared groups, no adjustment for multiple comparisons was applied.

Plasma from patients collected at baseline and at week nine post-treatment initiation was analyzed with the Olink Target 96 Immuno-Oncology panel and the Olink Target 96 Oncology II (Olink Proteomics AB, Uppsala, Sweden). The assay was performed by the Olink Proteomics Service Facility, Uppsala, Sweden. The difference in plasma protein levels pre and post-treatment was assessed by a paired t-test. Statistical testing was done using RStudio or GraphPad Prism version 10.4.1.

Statistical analyses for clinical assessments
Descriptive statistics were presented with median, range and interquartile range (IQR) for continuous variables. Categorical variables were enumerated by numbers and percentages when appropriate. Efficacy prediction (sample size) was evaluated using a one-sided binomial test with a significance level of 0.025. At a maximum enrollment of 25 patients at the maximum tolerated dose (MTD), the null hypothesis could be rejected with a probability of 0.9 given an assumed true response rate of ≥30% (Study protocol: https://clinicaltrials.gov/study/NCT04123470?term=LOKON003&rank=1). PFS and OS were assessed using the Kaplan-Meier estimator for survival analysis. Cox regression analysis was conducted in RStudio IDE, AGPL v3.

Data availability
Data sharing requests will be considered on a case-by-case basis to comply with the protection of personal data regulations. Requests by academic study groups for deidentified patient data with the intent to achieve aims of the original proposal can be forwarded to the corresponding author, who will evaluate if the request is in line with approved use by Ethical Review Boards and Regulatory Bodies, and if so, share a data file to be used by the academic study groups under a signed agreement. The Study Protocol is available in Supplementary Information). Source data are provided (SourceData), other data is provided in the Article and/or Supplementary Information. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have to some extend addressed my comments including adding information on M1 stages which points towards marked differences in subgroups among the two patient cohorts. Thus, the low dose cohort includes only 14 % M1c patients while the high dose has 41% M1c. The authors have added text and a figure on survival according to M1 stage in the result section. However, the total number of patients as well as the numbers of patients in the different M1 stages do not harmonize between table and figure/figure legend. Table: M1b: n=11, M1b: n=5, M1c n=:8, with a total of 24 patients. Figure 3H: M1b: n=12, M1b: n=2, M1c: n=16 with a total of 30 patients. Figure 3H legend: M1c, n=12; M1c, n=2; M1c, n=10 with a total of 24 patients. If the numbers given in the table is to be trusted then new calculations of survival in Figure 3H needs to be carried out and associated text revised.\nFurther, the authors have now included a cox regression analysis claiming that “ no clinical parameters impacted on survival”. However, from the related suppl tabl S9 it appears that M1 stage is not included. M1 staging is one of the strongest prognostic indicators for survival and should be included in the cox analysis.\nThe impact of M1 stage in regard to interpretation of results between VP dose cohorts in the trial, and in comparison with other trials, have not been integrated in the Discussion. It is recommended to incorporate a paragraph on trial limitations highlighting this together with the potential impact of post PD treatment on OS as previously mentioned.", "reviewer_2": "I thank the authors for their comprehensive replies.\nIn the discussion, I still miss some future perspectives with this treatment strategy. Should this be investigated in earlier treatment lines or in combination with other drugs? Could this be a biomarker-driven drug and if so which one? The authors should scientifically comment on this regardless on strategic decisions of the biotech company producing the drug.\nFurthermore, I have some small spelling suggestions:\nINTRODUCTION:\nImmune checkpoint inhibition (ICI) targeting PD-1, CTLA-4, and LAG-3, HAS revolutionized … and IS standard-of-care\ntogether with protein kinase inhibitors for BRAFV600E-mutated CMM\nDISCUSSION:\nadditional ICI than atezolizumab", "reviewer_3": "The paper reports results from a Phase I/II trial that employed a BOIN design for dose escalation across two dose levels. The\ntrial planned to enroll 25 evaluable subjects at the MTD (up to 50 total), but was terminated early due to lack of efficacy\nsignal. Dose escalation occurred after seven patients were enrolled at the initial dose level. Given the small sample size (N\n= 24), all analyses are exploratory and should be interpreted with caution, as noted by the authors. Nevertheless, the\nstatistical reporting requires greater rigor and improvement. Specific recommendations include:\n1. The rationale for N = 25 appears to be based on efficacy assumptions. However, the statement lacks key details, such as\nthe assumed true response rate under the alternative hypothesis. The study protocol includes this information; authors\nshould reference the protocol as supplementary material or cite the published protocol if available.\n2. All point estimates (e.g., ORR, PR, SD rates, median PFS/OS) should be accompanied by 95% confidence intervals, as\nspecified in the protocol.\n3. Statistical methods used for all analyses should be described in the main text. For example, methods mentioned in figure\ntitles (Figures 1–2) should be expanded with sufficient detail.\n4. AE table (Table 3) should present adverse events by dose level, consistent with the study SAP.\n5. Figure 1A compares anti-adenoviral antibody levels over time by dose level. The use of two-way ANOVA for longitudinal\nmeasurements is not appropriate; a repeated-measures or mixed-effects model would be more suitable.\n6. Figure 1B correlates antibody units at week nine with overall survival using simple linear regression, which is\ninappropriate given the presence of censored survival data. A Cox proportional hazards model or similar approach should\nbe considered.\n7. It is unclear why some analyses in Figure 2 include multiple comparison adjustments while others do not. Adjustment\nshould be applied consistently across marker comparisons.\n8. Information on study drug compliance is missing and should be included.\nMinor Comments\n1. Phrases such as “significant difference at a 95% confidence interval” are unconventional. Recommend revising to “at a\nsignificance level of 0.05.”\n2. In the Results section (page 4, lines 85–89), some numbers cited in the text do not appear in Table 1.\n3. In the Methods section titled “Statistical analyses for clinical assessment” (page 19, line 522), the term “one-sided\nbinominal t-test” is unclear. For a binary endpoint like ORR, a one-sided binomial test would be appropriate, but not a t-test.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to the previous comments from Nature Medicine’s referees\nReviewer #1 (Remarks to the Author):\nComment 1\nLOAd703-Induced Tumor Microenvironment Gene Engineering in Combination with Atezolizumab in\nMetastatic Malignant Melanoma – A Phase I/II Trial. Hamid et al.\nThe authors report from a phase I/II trial where 24 patients were treated with a CD40L and 4-1BBL\ngene modified oncolytic virus at two dose levels in combination with atezolizumab. The rational for\nthis strategy is substantiated and represents and interesting approach. The manuscript, however,\nsuffer from some weaknesses. Thus, important data on prognostic patient characteristics is lacking\nand further the authors are prone to unsubstantiated interpretation of efficacy results despite the\nlow patient numbers and the insufficient prognostic information.\nAccording to the protocol ‘At least 25 response evaluable patients will be enrolled at the MTD for\nevaluation of their response’. Please explain the reason for closing the trial after 17 patients at the\nhighest dose level? And add information of this deviation in the manuscript including how this affect\nthe validity of the reported ORR according to the statistical considerations in the protocol.\nResponse: The study was the first trial treating melanoma patients with LOAd703 and also the first\nstudy combining LOAd703 with atezolizumab. Hence, the early stage character of the study called for\na cautious design with an appropriate number of patients. The statistical design built on overall\nresponse rate which in turn consider only CRs and PRs. After enrolling a total of 24 response\nevaluable patients (7 at dose level 1 and 17 at dose level 2) we realized that the ORR was not as high\nas we stipulated in the statistics and enrolling one last patient at dose level 2 would not make us\nreach the primary endpoint independently of response. Hence, the study was stopped. However, we\nalso learnt that the dose level 1 may be better than dose level 2 in terms of survival. The mode-of-\naction of our treatment called for another type of study design based on tumor control and overall\nsurvival as the next step. We believe this was the correct decision for this study and our result show\nthe importance that consider survival also in earlier studies, perhaps more important for\nimmunotherapy studies that can induce long term tumor control beyond treatment termination due\nto the immunological memory achieved.\nComment 2\nAll patients are reported to have stage IV disease, however, it appears from table 1 that for 71% of\nthe patients the M1 stage is not specified. This information is crucial for the interpretation of the\nresults and should be included in the manuscript. Especially as the remaining 29 % of the patients are\nreported to be the good prognosis stage M1a and b and a potential unbalance of stage M1c between\nthe two dose levels might affect the efficacy measures and considerations.\nResponse: We agree that the M stages should be mentioned to make comparisons easier and have\nincluded that in Table 1.\nComment 3\nThe authors spend quite some efforts in Results and Discussion interpreting the efficacy data\nespecially PFS and OS including comparison between the two dose levels as well as comparison with\nefficacy data from published trials on e.g. TIL therapy. The authors should be more cautious in their\ninterpretation especially when claiming comparability in patient characteristics when e.g. a major\nprognostic parameter like M stage is unspecified in the majority of the trial patients.\nFurther, post-trial therapy might significantly impact OS e.g. BRAF/MEKi and TIL therapy or ipi/nivo\nfor patients primary treated with either mono anti-PD1 or Opdivo. In order to speculate on the OS\nobserved data on post-trial therapy should be included in the paper and taken into consideration.\nResponse: We disagree that we discuss the data in the Results section but we do report ORR, PFS\nand OS, as most trials do even if numbers are small in early studies. Further, the readers are\nappropriately notified that this is an early study and the number of patients enrolled. As this is an\nearly study, it is not randomized. Therefore, in the discussion we are putting our data into context by\ndescribing effect data in similar patient populations. This is also commonly done in the discussion\nsection which is supposed to be a discussion of your data and other researchers findings in the same\ntopic.\nComment 4\nPage 3; ‘The TME consists of multiple cell types including fibroblasts, vascular endothelium, immune\ncells, and products released by them such as chemokines, extracellular matrix and vesicles, that\ncollectively induce immunosuppression’, recommend adding ‘cytokines’.\nResponse: as it is many types of factors released, not only cytokines, that affect the suppressive\nnature, we stated “products released by them” instead in sake of keeping the introduction a bit\nshorter.\nComment 5\nPage 4, Patient Characteristics: ‘All patients had stage IV CMM’, recommend to add ‘ progressive’\nResponse: We have shortened the results and the same sentence is not included anymore, however,\nin the methods the word documented disease progression is mentioned.\nComment 6\nPage 5, Safety: ‘the most common injection sites were lymph nodes or subcutis/muscles of a limb’,\nrecommend being explicit about the intratumoral nature of the treatment. Further, it would be\ninformative for the reader to add treatment interval in this section and modify the heading of the\nsection to ‘Treatment and Safety’.\nResponse: the number of treatments are in the results section as requested. We have clearly\nspecified the intratumoral injection procedure in the Methods “Procedures” and it is also mentioned\nin the abstract.\nComment 7\nPage 5, ‘eight AEs due to other attributions led to LOAd703 treatment discontinuation’,\nplease add more details.\nResponse: This sentence was removed in sake of shortening the manuscript but we refer to the AE\nand SAE tables in which adverse reactions due to any cause are shown as in the paper, the AEs\nattributed to LOAd703 are shown.\nComment 8\nPage 6, Immune biomarker analyses, ‘To evaluate the capacity of LOAd703 to stimulate a Th1 type of\ninflammation in the TME’. Atezolizumab should be added to the sentence as the separate impact of\nLOAd703 cannot be assessed in this trial.\nPage 6, the authors report several TME changes based on expression analyses. ‘ The data confirm an\nupregulated gene expression of Th1 biomarkers including markers indicating the presence of T and\nNK cells, their activation and cytotoxic function, chemokines attracting T and NK cells to the TME as\nwell as receptors that allow them to transmigrate from blood vessels into the TME. Further, markers\nfor antigen presentation and B cell activation were upregulated.’ More details with indication of\nspecific markers should be added here in Results instead of in the Discussion.\nPage 6, Significant differences in gene expression level between the two groups are displayed for\nthese groups in heatmaps and volcano plots at baseline. Please revise the sentence and remove ‘for\nthese groups’\nResponse: In sake of space, the biomarker description was needed to be shortened, however, in the\nsupplementary tables full lists of all significant makers are shown. It also made more sense to discuss\nthe markers in the discussion but if the Editors of Nature Communication agree, we would be happy\nto provide a longer biomarker section in the results?\nComment 9\nPage 7, please add information on PR and CR rate in addition to ORR.\nResponse: as the reviewer wanted us to not describe the results too much, this was a somewhat\ncontradictory comment (see Comment 3). However, it is mentioned in the section of patients with\nparticular benefit from treatment.\nComment 10\nPage 8, Patients with particular benefit from treatment: ‘we observed two patients with partial\nresponse (PR) who became tumor-free after study participation’. This is a tricky formulation as one\npatient had a residual lesion removed and apparently the other was in complete metabolic response.\nWas a lesion still visible on CT ?\nPage 8, Discussion. It is recommended to remove this unsubstantiated sentence; ‘The survival\nappeared clearly superior to what is expected without treatment.’\nPage 8, ‘ICI targets were simultaneously upregulated in injected metastases, including PD-L2\n[PDCD1LG2], LAG-3, and TIGIT which indicates a benefit from combining LOAd703 with other\ncheckpoints than atezolizumab’. Recommend revising to ‘potential benefit’\nPage 9, top sentence on Opdivo please insert reference on phase III trial.\nPage 9, ‘Patients who survived longer had high levels of genes related to….’. Please take into\nconsideration in the discussion potential differences in prognostic characteristics of the patients.\nPage 9-10, ‘The results seemed clearly superior compared to chemotherapy or best supportive care,\nwhich would have been the alternative options for the vast majority of the study participants…’ Some\nof the BRAF mutated patients did not receive BRAF/MEK inhibitor and ipi/nivo therapy before\ninclusion. Please take this into consideration.\nResponse: The discussion is overall shorter and removed sections that troubled the referee.\nComment 11\nPage 20, ‘Patients with mucosal, acral or uveal melanoma were not included in this study due to the\ndifferences in biology and therapeutic response from CMM.’ According to the protocol in sept 2020\n‘Exclusion criteria no 1 was modified so that patients with mucosal melanoma will no longer be\nexcluded’\nPage 21, procedures: A definition of metabolic response is lacking.\nTable 1, important information is lacking on PDL1 expression, M1 stage, and grading of LDH level\nTable 2, Injection site nomenclature is unclear,´ Abdomen, Anterior chest, Axilla, Leg (gluteus, thigh,\nknee, lower)’, please add info on whether it is sc, LN or muscular lesions.\nResponse: The methods section has been modified to correctly describe the exclusion criteria\naccordingly to the last protocol and actual enrolled patients. The metabolic response is described in\nthe Supplementary Table 8 footnotes. Patients with elevated LDH at baseline and M1 stages are\nmentioned in Table 1 but unfortunately, information about PDL1 expression by histochemistry is not\navailable. Likewise, the collected data on injected tumor site does not provide additional\ninformation.\nReviewer #2 (Remarks to the Author):\nIn this article, the authors report the results of a phase I/II trial of atezolizumab IV plus IT\nadministration of a viral vector encoding trimerized, membrane-bound CD40L and 4-1BBL.\nTherapy\nwas generally well tolerated, with adverse events expected from IT immunotherapy and PD-L1\nimmune checkpoint inhibition (ICI). Biomarker data suggest clear activation of an immune response.\nWhile the ORR is relatively low, OS data seem more promising, especially in this heavily pretreated\npopulation.\nLOAd703 is novel intratumoral viral therapy which encodes and transfers Th1 type\nimmunostimulatory genes to the TME. While combinations of ICI with oncolytic viruses have been\ninvestigated before in phase 1-3 studies (e.g. T-VEC + pembrolizumab, MASTERKEY-265/KEYNOTE-\n034 study, Ribas et al. ESMO 2021), this is - to the best of my knowledge - the first early-phase trial\ninvestigating this specific virotherapy in combination with PD-L1 ICI in PD-1 ICI-refractory melanoma.\nI don't have any remarks on the study design and statistical design. The trial is well executed. The\nmanuscript is well written. Data are qualitative, well presented and informative. The conducted\nexploratory analyses on blood and biopsies are relevant, informative and provide a biological\nsurrogate for drug activity. Furthermore, they seem to identify a population who derives more\nbenefit from this therapeutic approach.\nOn a side note, it is not clear why not all patients underwent biopsies at baseline and during\ntreatment.\nI have some remarks:\nComment 1\n- The authors present data showing that the lower dose of LOAd703 seems associated with better\nOS. I can acknowledge that the number of patients is very low, but I miss some critical insight into\nthis observation. Could this for example be due to more immune exhaustion after repetitive\nexposure to viral antigens? Or because the median number of injections was lower in the high dose\ngroup?\nResponse: In this study we cannot say why the lower dose patients seem to survive longer than the\nhigher dose. As the referee comment, and we discuss, the number of patients are low. However,\ntaken all LOAd703 studies together (LOKON001, 002 and 003) we always see the same trend and we\nagree that repetitive treatment with a higher viral antigen load may be responsible but thus far there\nis no proof to discuss. The number of injections will be lower if the patient progress and go out of\nstudy but that is likely more circumstantial as that patient population progress for another reason\nearlier than those that survive longer.\nComment 2\n- It is unclear whether atezolizumab has an additive/synergistic effect in this combination therapy in\nthis PD-(L)1 ICI refractory population or whether the observed effect is due to LOAd703 alone (see\nalso effect of T-VEC monotherapy for example OPTIm trial). Could the authors comment on this?\nResponse: Of course, in a pivotal study the suitable control would be to have each agent alone and in\ncombination. However, as the patients are refractory to one, or several rounds of a PD-1 antibody\nalone or in combination with other antibodies or even Tvec, continuing to treat these patients with a\nPD-1/PD-L1 targeting antibody without any other additions was not ethically appealing. However, the\nvirus alone can be used in a control group moving forward. Nonetheless, Tvec has not gain a lot of\ninterest to be used as a monotherapy and the combination is likely a better option for the patients.\nOur in vitro experiments and animal data supports the combination to a greater immune activation\nand in animals the combination supports the systemic response post intratumoral injection of\nLOAd703 which is needed in this patient group with multiple metastases.\nComment 3\n- I miss some insights on future perspectives with this novel therapy. The authors state that\ninvestigating nivolumab-relatlimab +/- LOAd703 in the first-line setting would be attractive,\npresumably due to upregulation of LAG-3 shown in this study. However, one could also combine\nCTLA-4 ICI based on these data (also based on the fact that CTLA-4 may have an important\ninvolvement in the TME).\nResponse: It is still debated how to move forward in melanoma with LOAd703 and there are no\nformal decisions made yet. Any or several ICIs may be of interest. The study PIs advice the company\ncurrently sponsoring the clinical development of possibilities.\nComment 4\n- Multiple immune resistance mechanisms have been described, especially in the later-line setting.\nThe exploratory data shown in this study indicate that baseline expression of genes involved in\nantigen presentation and protection against a viral response at baseline appears to be associated\nwith better OS. This could serve as a basis for future biomarker research to select patients for whom\nthis therapy could be of particular benefit. Could the authors comment on this?\nResponse: This is correct, we are currently analyzing all biomarker data with focus on the patients\nwith progressive disease and short survival to determine if they have a joint biomarker signature.\nPreliminary, we have defined a patient population within our study based on biomarkers in which\nnone of the patients survived more than 6 months (median OS 4 months). Analyses are ongoing and\na paper will be prepared within the next few months.\nComment 5\nMinor improvements:\n- Minor spell check required, for example:\n* Introduction:\n** \"has revolutionized...and is standard-of-care\" instead of \"have revolutionized...and are\"\n** \"may depend\" instead of \"may depends\"\n- Table 3:\n* Please review pyrexia AE: 14 G1-2 and 1 G3, but 14 any grade AE.\nResponse: We have completed a correction of the manuscript and in regards to the last comment,\nthe same patient can have more than one pyrexia and it may be of different grades separated in\ntime. Hence, a single patient can be in both G1-2 and G3 group so end number of affected patients is\nstill 14.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have to some extend addressed my comments including adding information\non M1 stages which points towards marked differences in subgroups among the two\npatient cohorts. Thus, the low dose cohort includes only 14 % M1c patients while the\nhigh dose has 41% M1c. The authors have added text and a figure on survival according\nto M1 stage in the result section. However, the total number of patients as well as the\nnumbers of patients in the different M1 stages do not harmonize between table and\nfigure/figure legend. Table: M1b: n=11, M1b: n=5, M1c n=:8, with a total of 24 patients.\nFigure 3H: M1b: n=12, M1b: n=2, M1c: n=16 with a total of 30 patients. Figure 3H\nlegend: M1c, n=12; M1c, n=2; M1c, n=10 with a total of 24 patients. If the numbers given\nin the table is to be trusted then new calculations of survival in Figure 3H needs to be\ncarried out and associated text revised.\nFurther, the authors have now included a cox regression analysis claiming that “ no\nclinical parameters impacted on survival”. However, from the related suppl tabl S9 it\nappears that M1 stage is not included. M1 staging is one of the strongest\nprognostic indicators for survival and should be included in the cox analysis.\nThe impact of M1 stage in regard to interpretation of results between VP dose cohorts in\nthe trial, and in comparison, with other trials, have not been integrated in the Discussion.\nIt is recommended to incorporate a paragraph on trial limitations highlighting this\ntogether with the potential impact of post PD treatment on OS as previously mentioned.\nResponse: Thank for observing the mistake in Table 1. We have revised and performed\nQC checks for the M1 data which is now revised. We also added M1 staging in the Cox\nanalysis in Supplementary Table S9 and revised the manuscript accordingly on pages 6\n(results) and 8 (discussion).\nReviewer #2 (Remarks to the Author):\nI thank the authors for their comprehensive replies.\nIn the discussion, I still miss some future perspectives with this treatment strategy. Should\nthis be investigated in earlier treatment lines or in combination with other drugs? Could\nthis be a biomarker-driven drug and if so which one? The authors should scientifically\ncomment on this regardless on strategic decisions of the biotech company producing the\ndrug.\nFurthermore, I have some small spelling suggestions:\nINTRODUCTION:\nImmune checkpoint inhibition (ICI) targeting PD-1, CTLA-4, and LAG-3, HAS\nrevolutionized … and IS standard-of-care together with protein kinase inhibitors for\nBRAFV600E-mutated CMM\nDISCUSSION:\nadditional ICI than atezolizumab\nResponse: Thank you for noticing the spelling errors, we have modified them accordingly\nto the suggestions. We also agree that it may be of interest for the readers to have a\nmore elaborate future perspective discussion, which we have added on pages 7 and 8.\nReviewer #3 - Biostatistics, clinical trials (Remarks to the Author):\nThe paper reports results from a Phase I/II trial that employed a BOIN design for dose\nescalation across two dose levels. The trial planned to enroll 25 evaluable subjects at the\nMTD (up to 50 total), but was terminated early due to lack of efficacy signal. Dose\nescalation occurred after seven patients were enrolled at the initial dose level. Given the\nsmall sample size (N = 24), all analyses are exploratory and should be interpreted with\ncaution, as noted by the authors. Nevertheless, the statistical reporting requires greater\nrigor and improvement. Specific recommendations include:\n1. The rationale for N = 25 appears to be based on efficacy assumptions. However, the\nstatement lacks key details, such as the assumed true response rate under the alternative\nhypothesis. The study protocol includes this information; authors should reference the\nprotocol as supplementary material or cite the published protocol if available.\nResponse: We thank the reviewer for the statistical review. We agree that a reference to\nthe protocol is suitable for this purpose and have added that in the manuscript study\ndesign on page 18.\n2. All point estimates (e.g., ORR, PR, SD rates, median PFS/OS) should be accompanied by\n95% confidence intervals, as specified in the protocol.\nResponse: We have now included the confidence interval for all point estimates in Table\n2.\n3. Statistical methods used for all analyses should be described in the main text. For\nexample, methods mentioned in figure titles (Figures 1–2) should be expanded with\nsufficient detail.\nResponse: We have now added the statistical methods not only in the Figure Legends\nbut also in the method section at page 20.\n4. AE table (Table 3) should present adverse events by dose level, consistent with the\nstudy SAP.\nResponse: Table 3 is modified to contain AEs by dose level.\n5. Figure 1A compares anti-adenoviral antibody levels over time by dose level. The use of\ntwo-way ANOVA for longitudinal measurements is not appropriate; a repeated-measures\nor mixed-effects model would be more suitable.\nResponse: We agree with the reviewer and has changed the statistics to a mixed-effects\nanalysis.\n6. Figure 1B correlates antibody units at week nine with overall survival using simple\nlinear regression, which is inappropriate given the presence of censored survival data. A\nCox proportional hazards model or similar approach should be considered.\nResponse: We agree that under normal circumstances, censored patients will disable the\nlinear regression analysis. However, the study had an unusually long follow-up for\nsurvival, and the censored patients were all surviving >17 months (n=1), >28 months\n(n=2) or >30 months (n=7), and none of them were among the three patients that had\nhigher anti-adenoviral antibodies. Hence, if the actual death date were later for the\ncensored patients, the week 9 level of anti-adenovirus antibody would still be the same,\nand in the same range of most patients dying early. The regression plot visualizes the\nthree patients with higher level of anti-adenovirus versus the remaining patients that all\nhad a median level of antibody independently of short or long survival in a manner that\nmakes the figure relevant. However, to avoid any confusion or misunderstandings, we\nhave explained the censored patients in the figure legend, in the results (page 5) and\ndiscussion (page 8), and renamed the x-axis from overall survival to last known survival\ndate.\n7. It is unclear why some analyses in Figure 2 include multiple comparison adjustments\nwhile others do not. Adjustment should be applied consistently across marker\ncomparisons.\nResponse: This has now been clarified in the results section page 5 and 6 and discussed\non page 8 to make sure the reader can see that when dividing the data sets they become\ntoo small for multi comparison compensation which increases the risk of false negative\ndata. Hence, these sub-cohorts were also analyzed without multi comparison to evaluate\nif there are possible masked biomarkers that are still relevant to evaluate in upcoming\nstudies with larger cohorts or with single-plex methods. It is a balance to have too harsh\nor too poor statistics as false negatives as well as false positives can lead to wrong\nconclusions. We agree with the reviewer that it is important that the reader understands\nwhat data is presented and the results section is improved in this perspective.\n8. Information on study drug compliance is missing and should be included.\nResponse: The study drugs LOAd703 and Atezolizumab are administered at the hospital\nas an image-guided intratumoral injection versus intravenous infusion, respectively.\nHence, the exact number of doses per patient is registered in the study records and\nsummarized in Table 2.\nMinor Comments\n1. Phrases such as “significant difference at a 95% confidence interval” are\nunconventional. Recommend revising to “at a significance level of 0.05.”\nResponse: We have changed it accordingly to the suggestion from the reviewer.\n2. In the Results section (page 4, lines 85–89), some numbers cited in the text do not\nappear in Table 1.\nResponse: We have now added the missing information in Table 1.\n3. In the Methods section titled “Statistical analyses for clinical assessment” (page 19, line\n522), the term “one-sided binominal t-test” is unclear. For a binary endpoint like ORR, a\none-sided binomial test would be appropriate, but not a t-test.\nResponse: That is correct, and we have corrected the error in the Method section.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We acknowledge that Reviewer 4 co-reviewed this manuscript and did not\nhave further separate questions or comments to us."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have no further comments", "reviewer_3": "We thank authors for fully addressing almost all of our prior comments. The only revision needed is about the reported confidence intervals. By convention, the confidence interval is written as (lower boundary, upper boundary) or lower boundary-upper boundary. But some CIs currently have upper boundary reported first followed by lower boundaries. These should be corrected.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: This has now been corrected in Table 2"}]

Decision letter