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The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines. Implications: Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.

PURPOSEThis phase I study aimed to define the recommended phase II dose (RP2D) of tebentafusp, a first-in-class T-cell receptor/anti-CD3 bispecific protein, using a three-week step-up dosing regimen, and to assess its safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity in patients with metastatic uveal melanoma (mUM).METHODSIn this open-label, international, phase I/II study, HLA-A*02 or HLA-A*02:01+ patients with mUM received tebentafusp 20 μg once in week 1 and 30 μg once in week 2. Dose escalation (starting at 54 μg) began at week 3 in a standard 3 + 3 design to define RP2D. Expansion-phase patients were treated at the RP2D (20-30-68 μg). Blood and tumor samples were collected for pharmacokinetics/pharmacodynamics assessment, and treatment efficacy was evaluated for all patients with baseline efficacy data as of December 2017.RESULTSBetween March 2016 and December 2017, 42 eligible patients who failed a median of two previous treatments were enrolled: 19 in the dose escalation cohort and 23 in an initial dose expansion cohort. Of the dose levels investigated, 68 μg was identified as the RP2D. Most frequent treatment-emergent adverse events regardless of attribution were pyrexia (91%), rash (83%), pruritus (83%), nausea (74%), fatigue (71%), and chills (69%). Toxicity attenuated following the first three doses. The overall response rate was 11.9% (95% CI, 4.0 to 25.6). With a median follow-up of 32.4 months, median overall survival was 25.5 months (range, 0.89-31.1 months) and 1-year overall survival rate was 67%. Treatment was associated with increased tumor T-cell infiltration and transient increases in serum inflammatory mediators.CONCLUSIONUsing a step-up dosing regimen of tebentafusp allowed a 36% increase in the RP2D compared with weekly fixed dosing, with a manageable side-effect profile and a signal of efficacy in mUM.

The nuclear deubiquitylase BRCA1-associated protein 1 (BAP1) is frequently inactivated in malignant pleural mesothelioma (MPM) and germline BAP1 mutation predisposes to cancers including MPM. To explore the influence on cell physiology and drug sensitivity, we sequentially edited a predisposition mutation (w-) and a promoter trap (KO) into human mesothelial cells. BAP1w-/KO MeT5A cells express less BAP1 protein and phenocopy key aspects of BAP1 loss in MPM. Stable isotope labeling with amino acids in cell culture–mass spectrometry revealed evidence of metabolic adaptation, with concomitant alteration of cellular metabolites. In MeT5A, BAP1 deficiency reduces glycolytic enzyme levels but increases enzymes involved in the tricarboxylic acid cycle and anaplerotic pathways. Notably both argininosuccinate synthase 1 (ASS1), essential for cellular synthesis of arginine, and its substrate aspartate, are elevated in BAP1w-/KO MeT5A cells. Likewise, ASS1 expression is higher in BAP1-altered MPM cell lines, and inversely correlates with BAP1 in The Cancer Genome Atlas MESO dataset. Elevated ASS1 is also evident by IHC staining in epithelioid MPM lacking nuclear BAP1 expression, with improved survival among patients with BAP1-negative/ASS1-expressing tumors. Alterations in arginine metabolism may sensitize cells to metabolic drugs and we find that BAP1-negative/ASS1-expressing MPM cell lines are more sensitive to ASS1 inhibition, although not to inhibition of purine synthesis by mizoribine. Importantly, BAP1w-/KO MeT5A become desensitized to arginine deprivation by pegylated arginine deiminase (ADI-PEG20), phenocopying BAP1-negative/ASS1-expressing MPM cell lines.Implications:Our data reveal an interrelationship between BAP1 and arginine metabolism, providing a potential means of identifying patients with epithelioid MPM likely to benefit from ADI-PEG20.

Metabolomics MeT5A isogenics

Supplementary Methods, References and List of Figures and Tables

Fig. S1. SNP6.0 analysis of parental MeT5A-BAP1+/+ cells.Fig. S2. Expression of catalytically active BAP1 in parental MeT5A.Fig. S3. Characterisation of BAP1 expression in gene-edited MeT5A.Fig. S4. Whole genome sequencing of gene-edited MeT5A.Fig. S5. Proliferative profiles for gene-edited MeT5A.Fig. S6. Enriched GO terms in gene-edited MeT5A SILAC-MS.Fig. S7. Enriched KEGG pathways in gene-edited MeT5A SILAC-MS include EMT.Fig. S8. Metabolite responses to BAP1 mutation in isogenic MeT5A.Fig. S9. Immunoblotting for differentially expressed metabolic enzymes identified by SILAC-MS in isogenic MeT5A cell lines.Fig. S10. Characterisation of BAP1-status for MPM cell panel.Fig. S11. Evaluating correlation between BAP1 and selected metabolic enzymes in a panel of MPM cell lines and the TCGA MESO pan-cancer dataset.Fig. S12. Validation of ASS1 response to BAP1 alteration.Fig. S13. Improved prognosis for epithelioid MPM patients with loss of nBAP1 and increased expression of ASS1.Fig. S14. Relationship between BAP1 and ASS1 transcripts in the TCGA Pan-Cancer datasets for other cancer types.Fig. S15. The influence of BAP1-status on response to inhibition of purine metabolism.Fig. S16. The influence of BAP1-status on response to ASS1 inhibition.

Background/aim Utilising radiotherapy in the management of head and neck cancer (HNC) often results in long term toxicities. Mandibular osteoradionecrosis (ORN) represents a late toxicity associated with significant morbidity. We aim to identify a panel of common genetic variants which can predict ORN to aid development ofpersonalised radiotherapy protocols. Method Single nucleotide polymorphism (SNP) arrays were applied to DNA samples from patients who had prior HNC radiotherapy and minimum two years follow-up. A case cohort of mandibular ORN was compared to a control group of participants recruited to CRUK HOPON clinical trial. Relevant clinical parameters influencing ORN risk (e.g. smoking/alcohol) were collected. Significant associations from array data were internally validated using polymerase chain reaction (PCR) and pyrosequencing. Results Following inclusion of 141 patients in the analysis (52 cases, 89 controls), a model predictive for ORN was developed; after controlling for alcohol consumption, smoking, and age 4053 SNPs were identified as significant. This was reduced to a representative model of 18 SNPs achieving 92% accuracy. Following internal technical validation, a six SNP model (rs34798038, rs6011731, rs2348569, rs530752, rs7477958, rs1415848) was retained within multivariate regression analysis (ROC AUC 0.859). Of these, four SNPs (rs34798038 (A/G)(p 0.006), rs6011731 (C/T)(p 0.018), rs530752 (A/G)(p 0.046) and rs2348569 (G/G)(p 0.005)) were significantly associated with the absence of ORN. Conclusion This is the first genome wide association study in HNC using ORN as the endpoint and offers new insight into ORN pathogenesis. Subject to validation, these variants may guide patient selection for personalised radiotherapy strategies.

Melanomas arising in the uveal tract of the eye are a rare form of the disease with a biology and clinical phenotype distinct from their more common cutaneous counterparts. Treatment of primary uveal melanoma with radiotherapy, enucleation or other modalities achieves local control in more than 90% of patients, although 40% or more ultimately develop distant metastases, most commonly in the liver. Until January 2022, no systemic therapy had received regulatory approval for patients with metastatic uveal melanoma, and these patients have historically had a dismal prognosis owing to the limited efficacy of the available treatments. A series of seminal studies over the past two decades have identified highly prevalent early, tumour-initiating oncogenic genomic aberrations, later recurring prognostic alterations and immunological features that characterize uveal melanoma. These advances have driven the development of a number of novel emerging treatments, including tebentafusp, the first systemic therapy to achieve regulatory approval for this disease. In this Review, our multidisciplinary and international group of authors summarize the biology of uveal melanoma, management of primary disease and surveillance strategies to detect recurrent disease, and then focus on the current standard and emerging regional and systemic treatment approaches for metastatic uveal melanoma.

Background Sodium valproate (VPA) has been associated with a reduced risk of head and neck cancer development. The potential protective mechanism of action is believed to be via inhibition of histone deacetylase and subsequent epigenetic reprogramming. SAVER is a phase IIb open-label, randomised control trial of VPA as a chemopreventive agent in patients with high-risk oral epithelial dysplasia (OED). The aim of the trial is to gather preliminary evidence of the clinical and biological effects of VPA upon OED and assess the feasibility and acceptability of such a trial, with a view to inform a future definitive phase III study. Methods One hundred and ten patients with high-risk OED will be recruited from up to 10 secondary care sites in the UK and randomised into either VPA or observation only for 4 months. Women of childbearing potential will be excluded due to the teratogenic properties of VPA. Tissue and blood samples will be collected prior to randomisation and on the last day of the intervention/observation-only period (end of 4 months). Clinical measurement and additional safety bloods will be taken at multiple time points during the trial. The primary outcome will be a composite, surrogate endpoint of change in lesion size, change in grade of dysplasia and change in LOH profile at 8 key microsatellite regions. Feasibility outcomes will include recruitment targets, compliance with the study protocol and adverse effects. A qualitative sub-study will explore patient experience and perception of the trial. Discussion The current management options for patients with high-risk OED are limited and mostly include surgical resection and clinical surveillance. However, there remains little evidence whether surgery can effectively lead to a notable reduction in the risk of oral cancer development. Similarly, surveillance is associated with concerns regarding delayed diagnosis of OED progressing to malignancy. The SAVER trial provides an opportunity to investigate the effects of a repurposed, inexpensive and well-tolerated medication as a potential chemopreventive strategy for patients with high-risk OED. The clinical and biological findings of SAVER will inform the appropriateness, design and feasibility of a definitive phase III trial. Trial registration The trial is registered with the European Clinical Trials Database (Eudra-CT 2018-000197-30). (http://www.isrctn.com/ISRCTN12448611). The trial was prospectively registered on 24/04/2018.

Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess therapies using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.

Background Immune checkpoint blockers (ICBs) activate CD8+ T cells, eliciting both anti-cancer activity and immune-related adverse events (irAEs). The relationship of irAEs with baseline parameters and clinical outcome is unclear. Methods Retrospective evaluation of irAEs on survival was performed across primary (N = 144) and secondary (N = 211) independent cohorts of patients with metastatic melanoma receiving single agent (pembrolizumab/nivolumab—sICB) or combination (nivolumab and ipilimumab—cICB) checkpoint blockade. RNA from pre-treatment and post-treatment CD8+ T cells was sequenced and differential gene expression according to irAE development assessed. Results 58.3% of patients developed early irAEs and this was associated with longer progression-free (PFS) and overall survival (OS) across both cohorts (log-rank test, OS: P P = 2.8 × 10−6). Pre-treatment monocyte and neutrophil counts, but not BMI, were additional predictors of clinical outcome. Differential expression of numerous gene pathway members was observed in CD8+ T cells according to irAE development, and patients not developing irAEs demonstrating upregulated CXCR1 pre- and post-treatment. Conclusions Early irAE development post-ICB is associated with favourable survival in MM. Development of irAEs is coupled to expression of numerous gene pathways, suggesting irAE development in-part reflects baseline immune activation.

Introduction Immune checkpoint inhibitors can lead to thyroid dysfunction. However, the understanding of the clinical phenotype of ICI-induced thyroid dysfunction in the real-world population is limited. The purpose of this study was to characterise the clinical patterns of dysfunction and evaluate the demographic, biochemical and immunological features associated with this patient cohort. Materials and methods To characterise the longitudinal clinical course of thyroid dysfunction in patients from a single, UK regional cancer centre, a retrospective review of patients was conducted. Inclusion criteria included all patients treated with antiPD-1 checkpoint inhibitors (ICI), either as monotherapy (pembrolizumab/nivolumab) or in combination with a CTLA-4 inhibitor (ipilimumab). Patterns of toxicity were evaluated together with assessment of antibody titres. Results Over 16 months, thyroid dysfunction was seen in 13/90 and 3/13 patients treated with anti-PD1 monotherapy and in combination with ipilimumab, respectively. Patients either developed hyperthyroidism followed by hypothyroidism (12/16) or de novo hypothyroidism (4/16). Most patients were female (n = 11). All patients required thyroid replacement therapy. There was no relationship between clinical pattern of dysfunction and the presence of thyroid autoantibodies. Conclusions There are two distinct patterns of thyroid dysfunction in ICI-treated patients. Patients with thyroiditis develop subsequent hypothyroidism in the vast majority of cases. The potential benefit from steroids or other therapy to manage the hyperthyroid phase remains unclear. Early detection of these patients through appropriate monitoring will improve clinical management and early hormone replacement, reducing the symptomatic burden of hypothyroidism.

In patients with previously treated metastatic uveal melanoma, the historical 1 year overall survival rate is 37% with a median overall survival of 7.8 months. We conducted a multicenter, single-arm, open-label phase 2 study of tebentafusp, a soluble T cell receptor bispecific (gp100×CD3), in 127 patients with treatment-refractory metastatic uveal melanoma (NCT02570308). The primary endpoint was the estimation of objective response rate based on RECIST (Response Evaluation Criteria in Solid Tumours) v1.1. Secondary objectives included safety, overall survival, progression-free survival and disease control rate. All patients had at least one treatment-related adverse event, with rash (87%), pyrexia (80%) and pruritus (67%) being the most common. Toxicity was mostly mild to moderate in severity but was greatly reduced in incidence and intensity after the initial three doses. Despite a low overall response rate of 5% (95% CI: 2–10%), the 1 year overall survival rate was 62% (95% CI: 53–70%) with a median overall survival of 16.8 months (95% CI: 12.9–21.3), suggesting benefit beyond traditional radiographic-based response criteria. In an exploratory analysis, early on-treatment reduction in circulating tumour DNA was strongly associated with overall survival, even in patients with radiographic progression. Our findings indicate that tebentafusp has promising clinical activity with an acceptable safety profile in patients with previously treated metastatic uveal melanoma, and data suggesting ctDNA as an early indicator of clinical benefit from tebentafusp need confirmation in a randomized trial.

Uveal melanoma (UM) metastasises in ~50% of patients, most frequently to the liver. Surveillance imaging can provide early detection of hepatic metastases; however, guidance regarding UM patient risk stratification for surveillance is unclear. This study compared sensitivity and specificity of four current prognostic systems, when used for risk stratification for surveillance, on patients treated at the Liverpool Ocular Oncology Centre (LOOC) between 2007–2016 (n = 1047). It found that the Liverpool Uveal Melanoma Prognosticator Online III (LUMPOIII) or Liverpool Parsimonious Model (LPM) offered greater specificity at equal levels of sensitivity than the American Joint Committee on Cancer (AJCC) system or monosomy 3 alone, and suggests guidance to achieve 95% sensitivity and 51% specificity (i.e., how to detect the same number of patients with metastases, while reducing the number of negative scans). For example, 180 scans could be safely avoided over 5 years in 200 patients using the most specific approach. LUMPOIII also offered high sensitivity and improved specificity over the AJCC in the absence of genetic information, making the result relevant to centres that do not perform genetic testing, or where such testing is inappropriate or fails. This study provides valuable information for clinical guidelines for risk stratification for surveillance in UM.

Background: Tebentafusp, a bispecific (gp100 x CD3) ImmTAC, is approved for adult HLA-A*02:01+ patients with unresectable or metastatic uveal melanoma (mUM). In the Ph2 IMCgp100-102 study of pts with previously treated mUM (NCT02570308), tebentafusp demonstrated a median OS of 16.8 months and 1-year OS of 61% after a minimum 2 yrs of follow-up, nearly double the historical observed rates.12, Here we present the final analysis of OS of the Ph2 IMCgp100-102 study. Methods: 127 HLA-A*02:01+ 2L+ pts with mUM were dosed weekly with intravenous tebentafusp following intra-patient dose escalation of 20mcg dose 1, 30mcg dose 2 and 68mcg dose 3+. Primary objective was overall response rate and secondary objectives included safety and OS. OS was estimated by Kaplan-Meier method. Survival for ctDNA evaluable patients was assessed. Analyses are based on a 17 Oct 2022 database lock. Results: With a median follow-up of 46 months, median OS was 16.8 (95% CI: 12.3-21.3) months, consistent with previous reports. Estimated 1-yr, 2-yr, 3-yr and 4-yr OS rates were 61%, 36%, 21% and 11%, respectively. ctDNA clearance by week 9 (n=12) was associated with 100% 1-yr, 73% 2-yr, 45% 3-yr and 23% 4-yr survival. OS for patients with poor prognostic factors compared favorably relative to historical benchmarks (Table 1). Mean and median duration of treatment was 9.9 and 5.6 months. Majority of pts who received a subsequent line of therapy received immunotherapy (48/72; 67%) followed by liver-directed therapy (14/72; 19%). No new safety signals were identified. Conclusions: This study provides the longest follow-up of OS for a soluble TCR therapeutic to date. Tebentafusp continued to show unprecedented survival benefit for 2L+ mUM pts, including the poorest prognostic groups, with estimated OS rates that remain nearly double the historical rates observed in this population at a median follow-up of ~4 years. Early ctDNA clearance was associated with indication of survival benefit. 1. Carvajal et al. Nat Med 2022 2. Sacco et al. J Immunother Canc 2021 Table 1. OS in Ph2 IMCgp100-102 versus historical benchmarks Ph2 IMCgp100-102 Historical benchmarks*,† Population 1-yr 2-yr 3-yr 1-yr 2-yr 3-yr Overall 2L+ 61% 36% 21% 37% 15% 9% Subgroups Age ≥ 65 yrs 51% 31% 25% 38% 12% 6% LDH > ULN 44% 22% 11% 23% 8% 3% Baseline largest liver lesion ≥ 3cm 52% 26% 14% 32% 11% 4% *OS benchmark for 2L+ patients calculated from Rantala et al 2019 †OS benchmarks for pt subgroups (all lines of therapy) estimated from digitized OS curves presented in Khoja et al. 2019 Citation Format: Joseph J. Sacco, Richard D. Carvajal, Marcus O. Butler, Alexander N. Shoushtari, Jessica C. Hassel, Alexandra Ikeguchi, Leonel Hernandez-Aya, Paul Nathan, Omid Hamid, Josep M. Piulats, Matthew Rioth, Douglas B. Johnson, Jason J. Luke, Erique Espinosa, Serge Leyvraz, Laura Collins, Chris Holland, Michelle L. McCully, Takami Sato. Long-term survival follow-up of tebentafusp in previously treated metastatic uveal melanoma (mUM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT223.

BackgroundRP1 is an enhanced potency oncolytic HSV-1 which expresses a fusogenic glycoprotein (GALV-GP R-) and granulocyte macrophage colony stimulating factor (GM-CSF).1 In pre-clinical studies, RP1 demonstrated potent GALV-GP R-enhanced anti-tumor activity and immunogenic cell death. This Phase 1/2 (Ph 1/2) study was designed to evaluate the safety and efficacy of RP1 ± nivolumab (nivo) in patients (pts) with advanced solid tumors, including pts whose disease failed prior anti-PD-1/PD-L1 therapy and has reported promising interim data in a number of tumor types including cutaneous squamous cell carcinoma (CSCC) and anti-PD1 failed melanoma to date.2MethodsThis is a multi-center, first-in-human, open label, multi-cohort, non-randomized Ph1 study of RP1 alone and combined with nivo followed by Ph2 in combination with nivo in pts with recurrent advanced solid tumors including those that progressed after prior anti-PD-1/PD-L1 therapy. The Ph 1 monotherapy dose escalation (n=14) and RP-1 combination expansion (n=22) cohorts are fully enrolled. Approximately 260 pts are expected to be enrolled in the ongoing Ph 2 portion across five cohorts; melanoma (n=30, enrollment complete), non-melanoma skin cancer (n=45, to include 15 pts with anti-PD-1/PD-L1 failed disease), anti-PD-1 failed MSI-H/dMMR tumors (n=30), anti-PD-1/PD-L1-failed non-small-cell lung cancer (n=30) and a registration-directed cohort in anti-PD-1 failed cutaneous melanoma (n=125). Pts in the Ph 2 portion receive up to 10 mL of RP1 intratumorally into one or more superficial or deep seated/visceral lesions at the recommended Ph 2 dose (1x10^6 PFU/mL × 1 followed by 1x10^7 PFU/mL × 7, Q2W). Following the first dose of RP1, nivo (240 mg IV Q2W for 4 months then 480 mg IV Q4W for up to 2 years) is subsequently administered in combination. Pts may receive up to 8 additional doses of RP1 if they meet protocol-specified criteria. Tumor assessments are performed Q8W. The primary objectives of the Ph 2 part of the study are to assess the safety, tolerability, and overall response rate (ORR) of RP1 in combination with nivo, by independent review for the anti-PD1 failed melanoma cohort. Secondary objectives include duration of response, complete response rate, disease control rate, PFS, 1-year and 2-year survival rates. Exploratory objectives include biodistribution and shedding analysis of RP1 and biomarker studies, including analyses of tumor biopsies and blood samples. Enrollment is currently ongoing in the UK and US, with additional sites in the EU (including France and Spain) are expected to open in 2021.Trial RegistrationNCT03767348ReferencesThomas S, Kuncheria L, Roulstone V, Kyula JN, Mansfield D, Bommareddy PK, Smith H, Kaufman HL, Harrington KJ, Coffin RS. Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1. J Immunother Cancer 2019;7(1):214.Coffin R, Astley-Sparke P, and Middleton M (2021, June 3rd). Retrieved from https://ir.replimune.com/static-files/f4fe3349-e082-4d41-94a1-106ce7e78a23Ethics ApprovalThe study was approved by institutional review board or the local ethics committee at each site. Informed consent was obtained from patients prior to enrollment.

Immunotherapy using immune checkpoint inhibitors (ICIs) induces durable responses in many metastatic cancers. Metastatic uveal melanoma (mUM), typically occurring in the liver, is one of the most refractory tumours to ICIs and has dismal outcomes. Monosomy 3 (M3), polysomy 8q, and BAP1 loss in primary uveal melanoma (pUM) are associated with poor prognoses. The presence of tumour‐infiltrating lymphocytes (TILs) within pUM and surrounding mUM – and some evidence of clinical responses to adoptive TIL transfer – strongly suggests that UMs are indeed immunogenic despite their low mutational burden. The mechanisms that suppress TILs in pUM and mUM are unknown. We show that BAP1 loss is correlated with upregulation of several genes associated with suppressive immune responses, some of which build an immune suppressive axis, including HLA‐DR, CD38, and CD74. Further, single‐cell analysis of pUM by mass cytometry confirmed the expression of these and other markers revealing important functions of infiltrating immune cells in UM, most being regulatory CD8+ T lymphocytes and tumour‐associated macrophages (TAMs). Transcriptomic analysis of hepatic mUM revealed similar immune profiles to pUM with BAP1 loss, including the expression of IDO1. At the protein level, we observed TAMs and TILs entrapped within peritumoural fibrotic areas surrounding mUM, with increased expression of IDO1, PD‐L1, and β‐catenin (CTNNB1), suggesting tumour‐driven immune exclusion and hence the immunotherapy resistance. These findings aid the understanding of how the immune response is organised in BAP1 − mUM, which will further enable functional validation of detected biomarkers and the development of focused immunotherapeutic approaches. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Malignant pleural mesothelioma (MPM) has limited treatment options and poor prognosis. Frequent inactivation of the tumour suppressors BAP1, NF2 and P16 may differentially sensitise tumours to treatments. We have established chick chorioallantoic membrane (CAM) xenograft models of low-passage MPM cell lines and protocols for evaluating drug responses. Ten cell lines, representing the spectrum of histological subtypes and tumour suppressor status, were dual labelled for fluorescence/bioluminescence imaging and implanted on the CAM at E7. Bioluminescence was used to assess viability of primary tumours, which were excised at E14 for immunohistological staining or real-time PCR. All MPM cell lines engrafted efficiently forming vascularised nodules, however their size, morphology and interaction with chick cells varied. MPM phenotypes including local invasion, fibroblast recruitment, tumour angiogenesis and vascular remodelling were evident. Bioluminescence imaging could be used to reliably estimate tumour burden pre- and post-treatment, correlating with tumour weight and Ki-67 staining. In conclusion, MPM-CAM models recapitulate important features of the disease and are suitable to assess drug targets using a broad range of MPM cell lines that allow histological or genetic stratification. They are amenable to multi-modal imaging, potentially offering a time and cost-efficient, 3Rs-compliant alternative to rodent xenograft models to prioritise candidate compounds from in vitro studies.

Phosphoinositide 3-kinase δ (PI3Kδ) plays a key role in lymphocytes and inhibitors targeting this PI3K have been approved for hematological malignancies. While studies in hematological and solid tumor models in mice have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumor immunity, the impact of PI3Kδi on solid tumors in humans remains unclear. Here, we assessed the effects of the PI3Kδi AMG319 in patients with resectable head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomised phase-II trial. We find that PI3Kδ inhibition decreases tumor-infiltrating immunosuppressive TREG cells and causes heightened cytotoxic potential of tumor-infiltrating CD8+ and CD4+ T cells. Loss of intratumoral TREG cells and an increase in the frequency of activated TREG cells in the blood post-treatment are indicative of systemic effects on TREG tissue retention and maintenance. At the tested AMG319 doses, immune-related adverse events caused treatment discontinuation in 12/21 of AMG319-treated patients, further suggestive of systemic effects on TREG cells. Consistent with this notion, in a murine syngeneic tumor model, PI3Kδi decreased TREG cells in both tumor and non-malignant tissues and affected TREG subtype composition, maintenance and functionality. Our data demonstrate the cancer-immunotherapy potential of PI3Kδ inhibition in humans, but its modulation will need to be carefully balanced to harness its anti-tumor capacity while minimizing immune related toxicity.

Aims Immune checkpoint blockade (ICB) is the mainstay of treatment for metastatic melanoma and lung adenocarcinoma, and their use is growing in cancer. Immune checkpoint blockade induced colitis (ICB colitis) presents a management challenge and its mechanisms remain poorly elucidated. Methods We performed next generation single-cell RNA sequencing of immune cells from patients given ICB. We validated findings using confocal microscopy, drawing comparisons bioinformatically with ulcerative colitis. In parallel, we conducted a review of 1,074 patients given checkpoint inhibitors across two tertiary centres between 2011–2018 to discover patterns in incidence and predictors of clinical outcome. Results Using single-cell RNA sequencing, we discovered excessive local CD8 T cell proliferation was a key feature of ICB colitis, and we were able to visualise higher numbers of replicating CD8 T cells in gut tissue sections of patients with colitis than those without ICB colitis. The degree of replication was greater than seen in ulcerative colitis by bioinformatic analysis. From our clinical review, age, gender and smoking status did not alter the risk of developing colitis, whereas type of immunotherapy did (incidence 9% in PD-1 Monotherapy vs 32% Combination Therapy). Having prior IBD did not guarantee the development of ICB Colitis. Systemic markers of inflammation (C-Reactive Protein, Albumin) did not predict outcome, whereas local markers of inflammation (endoscopic UCEIS scoring, histological Nancy Index) did. Conclusions We putatively link novel insights from bench science to clinical trends. ICB colitis may be driven more by a gut localised inflammation response in comparison to ulcerative colitis. As we also demonstrate, this may explain why endoscopic and histological scoring may have better prognostic value than systemic measures of inflammation.

Background RP2 is an enhanced potency oncolytic HSV-1 expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), a fusogenic protein (GALV-GP R-), and an anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibody-like molecule which is being tested in an open-label, multicenter, phase 1 study alone and combined with PD-1 blockade (NCT04336241). Methods The objectives were to assess initial safety and efficacy and determine the recommended phase 2 dose (RP2D) of RP2 alone and combined with nivolumab. Patients were to be treated using a 3+3 dose escalation at two dose levels of up to 10 mL of RP2 Q2W up to 5 times (dose level 1: 105 PFU/mL then 4 doses of 106 PFU/mL; dose level 2: 106 PFU/mL then 4 doses of 107 PFU/mL). Following determination of the RP2D, additional HSV-1 seronegative patients were to be enrolled such that ≥3 had been dosed with RP2 at the RP2D, and a combination cohort of up to 30 patients dosed up to 8 times with RP2 at the RP2D combined with nivolumab (240 mg Q2W for 4 months from the second RP2 dose, then 480 mg Q4W for 20 months) opened. Lesions were injected directly or under imaging guidance used for visceral lesions. Tumor biopsies were obtained for biomarker analysis. Viral shedding and anti-HSV antibody titers were also monitored. Results Six HSV seropositive patients were enrolled in the dose-escalation phase with primarily Grade 1–2 adverse events, including febrile and other constitutional symptoms, local inflammation, and erythema observed. There were no DLTs requiring dose level expansion. The RP2D was selected as up to 10 mL of 106 PFU/mL followed Q2W by multiple doses of 107 PFU/mL. Of the six patients treated with single agent RP2, three (50%) have ongoing partial responses. Objective responses (including in uninjected tumors) were observed in patients with uveal melanoma (prior ipilumumab/nivolumab; extensive liver metastases), mucoepidermoid carcinoma (prior carboplatin/paclitaxel, bicalutamide, ceralasertib), and esophageal cancer (prior durvalumab, M6620, capecitabine, oxaliplatin, cisplatin, chemoradiation; liver and abdominal node metastases). Enrollment is underway in HSV seronegative patients and in combination with nivolumab. Updated data including biomarker and biodistribution data will be presented. Conclusions The Phase 1 clinical data supports the safety and efficacy of single agent RP2, including demonstration of uninjected tumor response in patients with difficult to treat advanced cancers. This data supports the hypothesis that anti-CTLA-4 delivered intra-tumorally through oncolytic virus replication, with accompanying antigen release and presentation, can provide potent anti-tumor effects.

Background RP1 is an enhanced potency oncolytic HSV encoding a fusogenic protein (GALV-GP R-) and GM-CSF which has previously demonstrated tolerable safety and tumor regression alone and with nivolumab in patients with a number of tumor types. Updated data from the phase 1 expansion with nivolumab, melanoma phase 2 (enrollment complete) and non-melanoma skin cancer (NMSC; enrollment ongoing) cohorts will be presented (NCT03767348). Enrollment of a further 125 patient anti-PD1 refractory cutaneous melanoma cohort; and activation of a cohort of anti-PD1 refractory NSCLC is underway. Methods Stage IIIb-IV melanoma patients for whom anti-PD-1 was indicated or who were refractory to prior anti-PD-1 alone or in combination with anti-CTLA-4, were enrolled. NMSC patients were anti-PD1 naive. Patients received ≤8 doses of RP1 (≤10 mL/visit Q2W; first dose 106 PFU/mL then 107 PFU/mL) with nivolumab (240 mg IV Q2W for 4 months then 480 mg IV Q4W up to 2 years) from the second RP1 dose. Results As of 24th June 2020, 36 melanoma and 16 NMSC patients had been enrolled with follow up of 8 weeks follow up, one of two angiosarcoma patients and seven of eight CSCC patients (5 CR) have achieved response (CSCC ORR 87.5%; CR rate 62.5%, including of uninjected visceral disease). Tumor biopsies in patients continue to routinely show immune activation, including robust recruitment of CD8+ T cells, reversal of T cell exclusion, and increased PD-L1 expression. Treatment remains ongoing, and current data will be presented. Conclusions RP1 and nivolumab have continued to be well tolerated, with continued promising anti-tumor activity in patients with skin cancers, including those with anti-PD1 refractory and other difficult to treat melanomas, and in patients with CSCC.

Patients with locally advanced oral squamous cell cancer (LAOSCC) are treated with adjuvant radiotherapy (RT) or chemoradiotherapy (CRT) following surgical ablation. This depends on the pathological risk factors and aims to reduce the risk of local recurrence and improve survival. Delivery of these aggressive treatments is, however, challenging particularly following major surgery. To inform the adaptations necessary to deliver gold-standard therapy, we aimed to describe real-world delivery of multimodality treatment in LAOSCC, in a UK population with high levels of disease incidence and low socioeconomic status. Patients with LAOSCC (T1-4 N1-3/T3-4 N0) who were treated between October 2014 and October 2016 and had a minimum follow up of 24 months were included. They were identified using the Somerset Cancer Register and data were collected through retrospective case note review. Approval was obtained from the audit departments at the relevant NHS institutions, and data were analysed using IBM SPSS Statistics for Windows version 24 (IBM Corp). The analysis included 129 patients with 82% having an initial performance status (PS) of 0-1. The most frequent change in PS was a one point drop (46%). Twenty of the 93 eligible patients (22%) underwent adjuvant CRT. A total of 37 (40%) began adjuvant CRT/RT within 42 days, and 79 (85%) within 56 days. A delay in initiating adjuvant therapy was associated with higher rates of complications and a longer postoperative hospital stay. Concordance between imaging and pathological nodal staging was poor (cK 0.223). PS frequently declines after complex surgical procedures and long postoperative recovery periods, leading to difficulties providing adjuvant treatments within the national guidance of 42 days. Frequent deviation from planned adjuvant therapies highlights the need for improved treatment strategies.

1Abstract1.1BackgroundImmune checkpoint blockers (ICBs) activate CD8+ T cells to elicit anti-cancer activity but frequently lead to immune-related adverse events (irAEs). The relationship of irAE with baseline parameters and clinical outcome is unclear. We investigated associations between irAE development, CD8+ T cell receptor diversity and expression and clinical outcome in a non-trial setting.1.2MethodsPatients ≥18 years old with metastatic melanoma (MM) receiving combination ICB (ipilimumab plus nivolumab – cICB, n=60) or single-agent ICB (nivolumab/pembrolizumab – sICB, n=78) were prospectively recruited. We retrospectively evaluated the impact of irAEs on survival. This analysis was repeated in an independent cohort of MM patients treated at a separate institution (n=210, cICB:74, sICB:136). We performed RNA sequencing of CD8+ T cells isolated from patients prior to treatment, analysing T cell receptor clonality differential transcript expression according to irAE development.1.3Results48.6% of patients experienced treatment-related irAEs within the first 5 cycles of treatment. Development of irAE prior to the 5th cycle of ICB was associated with longer progression-free and overall survival (PFS, OS) in the primary cohort (log-rank test, PFS: P=0.00034; OS: PP=0.00064). Across cohorts median survival for those patients not experiencing irAE was 14.4 (95% CI:9.6-19.5) months vs not-reached (95% CI:28.9 - Inf), P=3.0×10−7. Pre-treatment performance status and neutrophil count, but not BMI, were additional predictors of clinical outcome. Analysis of CD8+ T cells from 128 patients demonstrated irAE development was associated with increased T cell receptor diversity post-treatment (P=4.3×10−5). Development of irAE in sICB recipients was additionally associated with baseline differential expression of 224 transcripts (FDRCYP4F3 and PTGS2.1.4ConclusionsEarly irAE development post-ICB is strongly associated with favourable survival in MM and increased diversity of peripheral CD8+ T cell receptors after treatment. irAE post-sICB is associated with pre-treatment upregulation of inflammatory pathways, indicating irAE development may reflect baseline immune activation states.Key messageImmune-related adverse events (irAEs) commonly occur in patients with metastatic melanoma treated with immune checkpoint blockade (ICB) therapy. In real world setting we find development of early irAEs post-ICB treatment is associated with survival benefit, indicative of a shared mechanism with anti-tumour efficacy. CD8+ T cells from patients who develop irAE show increased receptor diversity, and pre-treatment samples from patients who develop irAE post single-agent anti-PD1 show over-expression of inflammatory pathways, indicating baseline immune state can determine irAE development.

Outcomes remain poor for patients presenting with locally-advanced oral cancers and it remains imperative to re-evaluate adjuvant therapies to provide improved outcomes, ideally without compromising on long-term quality of life. We present current available evidence that supports the use of immune checkpoint inhibitors (ICI) in squamous cell carcinoma (SCC) of the head and neck and discuss trials examining the integration of ICI into the locoregional management of such lesions that are resectable. We focus particularly on the Neoadjuvant and adjuvant nivolumab as Immune Checkpoint inhibition in Oral cavity cancer (NICO) trial which is investigating the integration of neoadjuvant and adjuvant ICI into the treatment of resectable locally-advanced oral cavity cancers.

Background Immune checkpoint inhibitors (ICI) have revolutionised oncological care but cause immune-related adverse events (irAE). Inflammation of the gastrointestinal tract (enterocolitis) is the most frequent cause of significant morbidity and cessation of ICI. We sought to determine whether Common Terminology Criteria for Adverse Events (CTCAE) classification of colitis, inflammatory bowel disease (IBD) biomarkers, endoscopic scores and histology scores correlate with disease outcome. Methods A retrospective study was performed on patients receiving ICI for metastatic melanoma, non-small cell lung cancer and urothelial cancer from 2012 to 2018 at two UK centres by reviewing the electronic patient records and oncology prescribing databases to identify patients who had irAE colitis. irAE colitis was defined as diarrhoea requiring steroid/infliximab therapy for resolution and/or with endoscopic/histological confirmation. Demographics, clinical data, endoscopic findings, histopathology reports and treatment outcomes were obtained. Patients were divided into three categories: (1) Mild-moderate disease where diarrhoea settled rapidly following a course of steroids ≤60 days; (2) Refractory or moderate–severe colitis requiring steroids >60 days; and (3) Severe colitis requiring infliximab ‘rescue therapy’. Endoscopies were re-analysed with image scoring using Mayo and ulcerative colitis Endoscopic Index of Severity (UCEIS) scores by two independent endoscopists. Histology was scored using the Nancy Index by two expert GI histopathologists. Results 1074 patients were analysed. Twelve per cent (134) developed irAE colitis. Median patient age was 66, 59% were male. Age, sex, smoking status and prior IBD or autoimmune disease were not associated with risk of developing irAE colitis. CTCAE grading did not correlate with steroid duration (p = 0.92) or infliximab requirement (p = 0.18) (Figure 1). CRP, albumin and haemoglobin didn’t correlate with severity of irAE colitis (Figure 2). 22% of patients received rescue therapy with infliximab either as single or multiple doses, with 3 undergoing a colectomy (2.3%; two had ipilimumab and one had combination). The UCEIS (p = 0.008) and Mayo (p = 0.016) scores correlated with disease severity and infliximab requirement. Patients with higher Nancy scores (3/4) were more likely to require infliximab (p = 0.03) (Figure 3). Conclusion Clinical assessment with CTCAE does not accurately reflect immunotherapy-colitis severity thus negatively impacting timely treatment decisions. Our data support endoscopic scoring of colitis severity, and demonstrate the potential prognostic utility of objective histologic scoring. we suggest that clinical guidelines are changed to incorporate new algorithms for the investigation and management of irAE colitis.

Introduction: RP2 is a novel, enhanced potency oncolytic HSV1 which expresses GM-CSF, an anti-CTLA-4 antibody-like molecule and the fusogenic gibbon ape leukemia virus membrane R- glycoprotein (GALV-GP R-). RP2 is in clinical development in a range of solid tumors alone and with nivolumab (nivo). RP2 + nivo has resulted in deep and durable responses in patients who failed prior anti-PD1 therapy (SITC 2021). Here we present biomarker data in patients treated with RP2 alone or combined with nivo from an ongoing clinical trial (NCT04336241). Methods: Tumor biopsies and peripheral blood mononuclear cells (PBMCs) were collected pre-treatment and at D43. The tumor immune microenvironment (TIME) was analyzed by multi-plex (7 color 6-plex - CD8, PD-L1, PD-1, foxp3, CD68 and S100B) immunohistochemistry (IHC) of tumor biopsies using the Opal Human Panel (OHP) 6043 and by gene expression analysis using the NanoString IO360 panel. The tumor inflammation signature score (TIS) was also calculated using an 18 gene signature (Ayers JCI 2017) . Systemic anti-tumor immunity was assessed using PBMCs by sequencing the CDR3 regions of TCRβ chains using immunoSEQ Assay. Correlation analysis of baseline tumor PD-L1 and CD8 status versus clinical responses was also performed. Results: IHC indicated robust increases in CD8 T cell influx and PD-L1 expression post-RP2 alone and with RP2 + nivo. An increase in the CD8/foxp3+ cell ratio was observed by multi-plex IHC. A consistent increase in CD8 and PD-L1 was observed in most of the tested biopsies (~70%), which generally appeared to be co-located (n=20). These increases in CD8 and PD-L1 expression levels were observed in both superficial and visceral tumors. A particularly striking change was observed in a biopsy obtained from a liver lesion from a tebantafusp and ipi/pembro-failed uveal melanoma patient. Clinical responses were independent of baseline CD8 T cell infiltration, PD-L1 expression levels, and prior anti-PD-1 therapy status. Gene expression analysis of tumor biopsies (n=12) indicated increases in the expression of key genes associated with immune activation, particularly those associated with dendritic cell function, major histocompatibility complex-II and interferon-gamma signature. Increases in expression of genes associated with ARG1, cytotoxicity, IDO1, NK cell and Th1 cell abundance were observed, particularly in responding patients. TCR sequencing of PBMCs revealed expansion of pre-existing T cell clones and the appearance of new clones post-RP2 monotherapy and RP2 + nivo, with ~50% of these changes being newly detected clones. Expansion of pre-existing clones and generation of new T cell clones specific for MART-1 was also observed with RP2 monotherapy and in combination. Conclusion: The biomarker data presented indicates broad immune activation by RP2 and demonstrates that clinical response does not correlate with baseline PD-L1 and CD8 expression status. Clinical responses were often associated with increases in gene signatures associated with cytotoxic T, NK and Th1 cells. These data indicate the potential for broad utility of RP2 in a range of tumor types, including in patients with primary or acquired resistance to immune checkpoint blockade. Citation Format: Kevin J. Harrington, Praveen K. Bommareddy, Mark R. Middleton, Joseph J. Sacco, Anna Olsson-Brown, Tze Y. Chan, Pablo Nenclares, Isla Leslie, Francesca Aroldi, Imran Saleem, Christoph M. Ahlers, Henry Castro, Robert S. Coffin. Clinical biomarker studies with an enhanced potency oncolytic HSV expressing an anti-CTLA-4 antibody, as a single agent and combined with nivolumab in patients with advanced solid tumors indicates potent immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT155.

9553 Background: RP1 is an enhanced potency oncolytic version of HSV1 that expresses human GM-CSF and the fusogenic protein GALV-GP R-. IGNYTE is a multicohort phase 1/2 study that evaluates the safety and efficacy of RP1 in combination with nivo (NCT03767348) in a range of tumor types. Preliminary data demonstrated a durable anti-tumor activity and tolerability for RP1+nivo. Here, we present updated results from the initial and melanoma (mel) and anti-PD1 naïve non-melanoma skin cancer (NMSC) cohorts with RP1+nivo. Methods: RP1 is administered via intratumoral injection Q2W, up to 10 mL/visit, first alone at a dose of 106 PFU/mL and then starting with the 2nd dose at 107 PFU/mL in combination with nivo (240 mg IV Q2W for 4 months (mos) then 480 mg IV Q4W up to 2 yrs) for up to 8 doses, with the option to re-initiate RP-1. Eligible patients (pts) must have at least one measurable & injectable tumor of ≥ 1 cm, ECOG 0-1, and no prior oncolytic therapy. For mel, both anti-PD1 naïve and failed pts were eligible, for NMSC pts who were anti-PD1 naïve. Results: As of data extraction on January 31, 2022, 13/36 pts with mel (36.1%) and 19/31 pts with NMSC (61.3%) had a best response of PR or CR. For mel this was 5/8 (62.5%), 6/16 (37.5%), 0/6 and 2/6 (33.3%) for pts with anti-PD1 naïve cutaneous, anti-PD1/anti-PD1+anti-CTLA-4 failed cutaneous, uveal and mucosal mel respectively. For the anti-PD1 naïve NMSC this included 11/17 (64.7%), 1/4 (25%), 3/4 (75%) and 4/6 (66.6%) patients with CSCC, BCC, MCC and angiosarcoma respectively, including 8/17 (47.1%) being CR for CSCC. Current immature median DOR was 13.27 mos (current range 3.67-16.93 mos) for mel, and 7.32 mos (current range 1.88-23.11mos) for anti-PD1 naïve NMSC. Any grade TEAE ( > 25%) in all cohorts combined were fatigue, nausea, pyrexia, chills, diarrhoea, pruritus, and influenza-like illness. TEAE ≥grade 3 ( > 5%) were disease progression and fatigue. No deaths related to RP1 was observed, with one death related to nivo (myocarditis). Biomarker data from paired biopsies indicated robust T cell infiltration and an increase in tumor inflammation gene signature post-treatment. Clinical responses observed were independent of baseline tumor PD-L1 expression status. Conclusions: RP1 in combination with nivo provides a durable anti-tumor activity in pts with skin cancers, including anti-PD1 failed and anti-PD1/anti-CTLA-4 failed mel. The combination continued to be generally well tolerated with no new safety signals identified. Based on this data, enrollment into both a registration-directed cohort of pts who have anti-PD1 failed cutaneous mel (n = 125) and a cohort of pts with anti-PD1 failed NMSC (n = 30) is ongoing. Up-to-date data from this ongoing trial will be reported at the conference. A randomized Ph2 trial of RP1+cemiplimab vs. cemiplimab alone in anti-PD1 naïve NMSC is also underway (NCT04050436). Clinical trial information: NCT03767348.

9584 Background: All immune therapies that rapidly activate T cells, including T cell engagers, can induce cytokine release syndrome (CRS). Tebentafusp (tebe), a T cell receptor bispecific (gp100 x CD3) can also induce skin adverse events (AEs), due to gp100+ cutaneous melanocytes. CRS and skin AEs may require management with short term corticosteroids, which may also be used as premedication for subsequent tebe doses. Here we report the first analysis of systemic corticosteroid use and correlation with efficacy from a Phase (Ph) 3 trial for any T cell engager. Methods: Post hoc analyses were performed on the tebe arm of the Ph3 [NCT03070392] study in previously untreated HLA-A*02:01+ metastatic uveal melanoma (mUM) (N = 245). Due to the low rate of severe AEs in Ph1 trials, prophylactic corticosteroids were not mandated. The association between overall survival (OS) and corticosteroid use (new start within 30 days of first tebe dose) was investigated using landmark analyses in the safety population. Multivariate analyses were adjusted for key patient characteristics and AEs of special interest: CRS, rash, and liver function test (LFT) elevation. Steroid type (hydrocortisone vs. others) and treatment duration (1 vs. > 1 day) were also investigated. Results: In the Ph3 trial, 64/245 (26%) patients received new systemic corticosteroid within 30 days after the first dose of tebe, mostly for treatment of AEs (56/64, 88%) or pre-medication due to previous AE (14/64, 22%). 25 of the 64 patients received corticosteroids only for a single day. The most frequent AEs (≥15%) were rash (18/64, 28%), CRS (15/64, 23%), and hypotension (12/64, 19%). In a logistic regression model, elevated baseline LDH, the dominant prognostic marker, was most strongly associated with use of corticosteroids (p = 0.01). In the multivariate analysis, corticosteroids were not associated with any significant OS difference (HR 1.41, 95% CI 0.83-2.4, p = 0.2) and this effect did not differ in patients with or without CRS, rash or LFT elevation (all interaction tests p > 0.2). There was no difference in OS according to corticosteroid type or whether administered for 1 vs > 1 day. Conclusions: This is the first analysis from a phase 3 trial of the impact of systemic corticosteroids on survival for a T cell engaging cancer therapy. The vast majority of tebe-treated patients (84%) either did not require corticosteroids (74%) or only received them on a single day (10%). The most frequent reason for corticosteroid use was an emergent AE, including CRS and rash. Corticosteroid use following the pre-specified AE guidelines was not associated with any significant impact on OS. Clinical trial information: NCT03070392.

TPS2704 Background: RP2 is a selective replication-competent herpes simplex virus 1 (HSV-1) that contains a codon-optimized sequence for human granulocyte-macrophage colony-stimulating factor (GM-CSF), the gibbon ape leukemia virus surface glycoprotein (GALV-GP) with the R- sequence deleted (R-), GM-CSF, and an anti–cytotoxic T lymphocyte antigen 4 (CTLA-4) antibody-like molecule. These modifications are intended to increase oncolytic potency, cell-to-cell spread, tumor antigen release, and systemic anti-tumor immune activation. RP2 has demonstrated antitumor activity in preclinical models using viruses similar to RP2 expressing murine versions of anti-CTLA-4 and GM-CSF, both alone and in combination with PD1-1 blockade. Methods: This is a Phase 1 multicenter, open-label, study evaluating RP2 ± nivolumab (NCT04336241). Part 1 evaluated escalating doses of monotherapy RP2 with a modified 3 + 3 design. Patients (pts) received intratumoral (IT) injections of up to 10 mL of RP2 into superficial or visceral tumors Q2W x5 across two dose levels (DL1: 105 plaque-forming units (PFU)/mL once, then followed by 106 PFU/mL x4; DL 2: 106 PFU/mL once, then followed by 107 PFU/mL x4). The recommended Phase 2 dose of RP2 was identified as 106 PFU/mL once, followed by subsequent doses of 1 × 107 PFU/mL. In Part 2, up to 60 evaluable pts will be enrolled, including 30 pts in Cohort 2a and 30 in Cohort 2b. Cohort 2a was designed to evaluate the safety and antitumor activity of the combination in pts with solid tumors, and is fully enrolled. Cohort 2b is open for enrollment of pts with advanced or metastatic uveal melanoma, NSCLC, breast cancer, head and neck cancer or GI cancers (NSCLC, breast or GI cancers must have at least one liver tumor suitable for injection). Pts in Part 2 receive intra-tumoral RP2 at the RP2D Q2W for up to 8 doses in combination with 240 mg nivolumab IV Q2W or 480 mg nivolumab IV Q4W for up to two years from the first RP2 dose. In Part 3 (not yet open for enrollment), up to 15 evaluable pts are able to be enrolled to evaluate RP2 monotherapy including at least 10 pts with NSCLC, breast or GI cancers with at least one liver tumor suitable for injection. Reinitiation of up to 8 additional RP2 doses is permitted if prespecified criteria are met. Pts are dosed with RP2 by direct visualization or ultrasound-guided injection into superficial, subcutaneous, or nodal tumors and by image-guided injection into deeper lesions, including visceral lesions. Key inclusion criteria include histologically confirmed advanced or metastatic non-neurological solid tumors, at least one measurable and injectable tumor of diameter ≥1 cm, ECOG ≤1, and adequate hematologic, hepatic, and renal function. Exclusion criteria include prior treatment with oncolytic viruses, acute or chronic hepatitis B or C infection, and HIV infections. Clinical trial information: NCT04336241.

BackgroundTebentafusp, a bispecific fusion protein consisting of an affinity-enhanced T cell receptor fused to an anti-CD3 effector that can redirect T cells to target gp100+ cells, has shown an overall survival benefit for patients with untreated metastatic uveal melanoma (mUM) in a Ph3 trial (NCT03070392). Metastatic uveal melanoma (mUM) is a historically treatment-refractory tumor with 1-year (yr), 2-yr and 3-yr OS rates of 37%, 15% and 9%, respectively, and median OS of 7.8 months in 2L+ patients.1 In the primary analysis of the phase 2 IMCgp100–102 study (NCT02570308) enrolling patients with previously treated mUM, the 1-year overall survival (OS) rate was 62% with median OS of 16.8 months.2 We present updated OS and safety after 2-year follow-up.Methods127 HLA-A*02:01+ 2L+ mUM patients were dosed weekly with tebentafusp following intra-patient dose escalation: 20mcg dose 1, 30mcg dose 2 and 68mcg dose 3+. Primary objective was ORR and secondary objectives included safety, OS and PFS. Here we present the updated OS and safety (data cut-off 31 Mar 2021).ResultsMedian follow-up was 29.9 mos (range 1.8 – 59.9 mos). With extended follow-up, the 1-yr, 2-yr and 3-yr OS rates were 61%, 37% and 24%, respectively (figure 1). Median OS remained unchanged at 16.8 mos (95% CI, 12.8 – 22.5 mos).Mean and median duration of treatment were 9.5 mos and 5.6 mos (0 – 47.4 mos), respectively. As previously reported, most treatment-related AEs (TRAEs) occurred early on treatment. Beyond 6 months, no TRAE led to treatment discontinuation. No new safety signals, changes in the type or treatment-related deaths were reported. Beyond 12 months, there were a total of 7 Grade (G) 3 or 4 events in 3 (7%) patients, all were temporally related to tumor progression and majority included lab abnormalities. Episodes of rash, a common tebentafusp-related AE early on-treatment, were infrequent after 6 months, with no Grade 3 or 4 events.Abstract 538 Figure 1Kaplan-Meier estimate of overall survival at 2-yr follow-up of IMCgp100-102ConclusionsThis study provides the longest follow-up of OS and safety of a soluble TCR therapeutic to date. Tebentafusp continued to show promising survival for 2L+ mUM patients with estimated 2-yr OS rate of 37%. Tebentafusp’s safety profile was as expected and consistent with primary analysis showing that most adverse events occur early on treatment with incidence and severity decreasing with prolonged exposure.Trial RegistrationNCT02570308ReferencesRantala ES, Hernberg M, Kivela TT. Overall survival after treatment for metastatic uveal melanoma: a systematic review and meta-analysis. Melanoma Res 2019;29:561–568.Sacco JJ, Carvajal R, Butler MO, et al. A phase (ph) II, multi-center study of the safety and efficacy of tebentafusp (tebe) (IMCgp100) in patients (pts) with metastatic uveal melanoma (mUM). Ann Oncol 2020;31: S1442–S1143.Ethics ApprovalThe institutional review board or independent ethics committee at each center approved the trial. The trial was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines.

BackgroundRP2 is a first-in-class, enhanced potency oncolytic herpes simplex virus (HSV) -1 expressing GM-CSF, a fusogenic protein (GALV-GP R-), and an anti-CTLA-4 antibody-like molecule that is being tested in an open-label, multicenter, phase 1 study alone and combined with nivolumab (nivo). Preliminary data with RP2 as monotherapy has been presented previously [1–2]. We present updated safety, tolerability, and clinical activity data of RP2 alone and initial data in combination with nivolumab.MethodsUsing a 3+3 dose escalation, patients (pts) received intratumoral injections of up to 10 mL RP2 to superficial and/or visceral tumors Q2W up to 5 times at two dose levels (Dose level 1: 10^5 PFU/mL then 4 doses of 10^6 PFU/mL; dose level 2: 10^6 PFU/mL then 4 doses of 10^7 PFU/mL). Following determination of the RP2D (10^6 PFU/mL, followed by subsequent doses of 10^7 PFU/mL, Q2W X 7), a combination cohort of 30 pts were dosed with RP2 up to 8 times combined with nivo (240 mg Q2W for 4 mos from the second RP2 dose, then 480 mg Q4W for 20 mos). Re-initiation of up to 8 additional RP2 doses is permitted in prespecified circumstances.ResultsNine pts were enrolled into the RP2 monotherapy phase (6 seropositive and 3 seronegative for HSV). Objective responses were observed in 3 pts, 1 ongoing CR for ≥15 months in mucoepidermoid carcinoma, 1 ongoing PR for ≥18 months in esophageal cancer with liver metastases, 1 PR in uveal melanoma with liver metastases that progressed at 15 months. As of June 3rd 2021, 27 patients had been enrolled and ongoing partial responses had been observed in 4/9 anti-PD-1 failed cutaneous melanoma, 1/3 uveal melanoma and 1/3 SCCHN pts. A further 8 patients remained on study with the opportunity for response. Biomarker analyses indicate T cell infiltration, increase in tumor inflammation signature, expansion of existing T cell clones and emergence of new T cell clones, together indicative of local and systemic anti-tumor activity. The combination was well tolerated and no new safety signals were identified.ConclusionsRP2 ± nivo demonstrated good tolerability and durable systemic responses in pts with difficult-to-treat, heavily pretreated and anti-PD-1 failed advanced cancers. These data continue to support the hypothesis that oncolytic delivery of anti-CTLA-4 into tumors, with accompanying antigen release, presentation and immune activation, can provide potent systemic anti-tumor effects. Updated data from the full 30 patient cohort will be presented.Trial RegistrationNCT04336241ReferencesAroldi F, Sacco J, Harrington K, Olsson-Brown A, Nanclares P, Menezes L, Bommareddy P, Thomas S, Kaufman H, Samakoglu S, Coffin R and Middleton M. 421, Initial results of a phase 1 trial of RP2, a first in class, enhanced potency, anti-CTLA-4 antibody expressing, oncolytic HSV as single agent and combined with nivolumab in patients with solid tumors Journal for Immuno Therapy of Cancer 2020;8.Harrington KJ, Aroldi F, Sacco JJ, Milhem MM, Curti B, Vanderwalde A, et al. LB180-Clinical biomarker studies with two fusion-enhanced versions of oncolytic HSV (RP1 and RP2) alone and in combination with nivolumab in cancer patients indicate potent immune activation. Cancer Res 2021. LB-180Ethics ApprovalThe study was approved by institutional review board or the local ethics committee at each site. Informed consent was obtained from patients prior to enrollment.

Uveal melanoma (UM) is a rare aggressive intraocular tumour with a propensity for liver metastases, occurring in ∼50% of patients. The tumour suppressor BAP1 is considered to be key in UM progression. Herein, we present the largest study to date investigating cellular expression patterns of BAP1 protein in 165 UMs, correlating these patterns to prognosis. Full clinical, histological, genetic, and follow-up data were available for all patients. BAP1 gene sequencing was performed on a subset of 26 cases. An independent cohort of 14 UMs was examined for comparison. Loss of nuclear BAP1 (nBAP1) protein expression was observed in 54% (88/165) UMs. nBAP1 expression proved to be a significant independent prognostic parameter: it identified two subgroups within monosomy 3 (M3) UM, which are known to have a high risk of metastasis. Strikingly, nBAP1-positiveM3 UMs were associated with prolonged survival compared to nBAP1-negative M3 UMs (Log rank, p = 0.014). nBAP1 protein loss did not correlate with a BAP1 mutation in 23% (6/26) of the UMs analysed. Cytoplasmic BAP1 protein (cBAP1) expression was also observed in UM: although appearing 'predominantly diffuse' in most nBAP1-negative UM, a distinct 'focal perinuclear' expression pattern - localized immediately adjacent to the cis Golgi - was seen in 31% (18/59). These tumours tended to carry loss-of-function BAP1 mutations. Our study demonstrates loss of nBAP1 expression to be the strongest prognostic marker in UM, confirming its importance in UM progression. Our data suggest that non-genetic mechanisms account for nBAP1 loss in a small number of UMs. In addition, we describe a subset of nBAP1-negative UM, in which BAP1 is sequestered in perinuclear bodies, most likely within Golgi, warranting further mechanistic investigation.

Cervical cancer is the fourth most common cause of cancer-related death in women worldwide and new therapeutic approaches are needed to improve clinical outcomes for this group of patients. Current treatment protocols for locally advanced and metastatic disease consist of ionising radiation and chemotherapy. Chemoradiation induces cytotoxic levels of DNA double-strand breaks, which activates programmed cell death via the DNA damage response (DDR). Cervical cancers are unique given an almost exclusive association with human papillomavirus (HPV) infection; a potent manipulator of the DDR, with the potential to alter tumour sensitivity to DNA-damaging agents and influence treatment response. This review highlights the wide range of therapeutic strategies in development that have the potential to modulate DDR and sensitise cervical tumours to DNA-damaging agents in the context of HPV oncogenesis.

Background Immune checkpoint inhibitors (ICI) improve survival but cause immune-related adverse events (irAE). We sought to determine if CTCAE classification, IBD biomarkers/endoscopic/histological scores correlate with irAE colitis outcomes. Methods A dual-centre retrospective study was performed on patients receiving ICI for melanoma, NSCLC or urothelial cancer from 2012 to 2018. Demographics, clinical data, endoscopies (reanalysed using Mayo/Ulcerative Colitis Endoscopic Index of Severity (UCEIS) scores), histology (scored with Nancy Index) and treatment outcomes were analysed. Results In all, 1074 patients were analysed. Twelve percent (134) developed irAE colitis. Median patient age was 66, 59% were male. CTCAE diarrhoea grade does not correlate with steroid/ infliximab use. G3/4 colitis patients are more likely to need infliximab (p p = 0.008) and Mayo (p = 0.016) scores correlate with severity/infliximab requirement. Patients with higher Nancy indices (3/4) are more likely to require infliximab (p = 0.03). Conclusions CTCAE assessment does not accurately reflect colitis severity and our data do not support its use in isolation, as this may negatively impact timely management. Our data support utilising endoscopic scoring for patients with >grade 1 CTCAE disease, and demonstrate the potential prognostic utility of objective histologic scoring.

Background: Metastatic uveal melanoma (mUM) has a poor prognosis with a 1-yr OS rate of 52%. No systemic treatment has proven an OS benefit in randomized trials. Tebentafusp (tebe), a bispecific consisting of an affinity-enhanced T cell receptor (TCR) fused to an anti-CD3 effector that can redirect T cells to target gp100+ cells, has shown promising activity in previously treated mUM pts. Here, we report the primary analysis of overall survival (OS) in the intention-to-treat population (ITT) of a Ph3 trial of tebe vs. investigator's choice (IC) as first line (1L) therapy in pts with mUM [NCT03070392]. Materials and Methods: In this randomized, open-label, Ph3 trial, 1L HLA-A*02:01+ pts with mUM were randomized 2:1 to receive tebe or IC of pembrolizumab, ipilimumab or dacarbazine, stratified by LDH. The primary endpoint was OS, defined as the time from randomization to death from any cause. Dual primary objectives were to evaluate 1) OS in the ITT population by comparing all tebe-randomized pts to all IC-randomized pts; and 2) OS in tebentafusp-treated patients with rash during week 1 versus all IC-treated patients. Secondary endpoints included safety and RECIST-defined overall response rate (ORR), progression free survival (PFS) and disease control rate (DCR). Here we present the OS in the ITT population. The study was unblinded by an independent data monitoring committee at the first pre-specified interim analysis. Investigator-reported radiographic-based endpoints were not mature at the first interim analysis. This analysis was conducted on the first interim analysis (data extracted Nov 2020). Results: 378 pts were randomized to tebe (252) or IC, including pembrolizumab (103), ipilimumab (15) or dacarbazine (7). Tebe significantly prolonged OS compared to IC (HR 0.51; 95% CI 0.36-0.71; PULN and versus pembrolizumab IC. Most common TRAEs were skin-related (gp100+ melanocytes) or cytokine-mediated (T cell activation) and included pyrexia, pruritus, and rash. These AEs decreased in frequency and severity after the first 3-4 doses and were generally manageable with standard interventions. In the tebe arm, the rate of treatment discontinuation due to TRAEs was low ( Conclusions: In 1L treatment of mUM pts, tebe monotherapy significantly improved OS compared to IC; the first investigational therapy to improve OS in pts with mUM. Tebe had a predictable and manageable AE profile with a low rate of related discontinuation. Tebe is the first TCR therapeutic to demonstrate an OS benefit. Citation Format: Sophie Piperno-Neumann, Jessica C. Hassel, Piotr Rutkowski, Jean-Francois Baurain, Marcus O. Butler, Max Schlaak, Ryan J. Sullivan, Sebastian Ochsenreither, Reinhard Dummer, John M. Kirkwood, Anthony M. Joshua, Joseph J. Sacco, Alexander N. Shoushtari, Marlana Orloff, Richard D. Carvajal, Omid Hamid, Shaad E. Abdullah, Chris Holland, Howard Goodall, Paul Nathan. Phase 3 randomized trial comparing tebentafusp with investigator's choice in first line metastatic uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT002.

Background: The phase 3 IMCgp100-202 trial (NCT03070392) (Study 202) in untreated mUM demonstrated improved overall survival (OS), HR=0.51, at the first pre-specified interim analysis. This OS benefit and the promising estimated 12-month OS rate of 62% observed in previously treated mUM pts on the phase 2 IMCgp100-102 trial (Study 102; NCT02570308) is not explained by RECIST responses alone. In this post hoc analysis of Study 102, we describe the tumor kinetics of pts with prolonged survival (OS ≥ 12 mo). Methods: 127 pts with 2L+ mUM received single agent tebe during the expansion phase of Study 102, where treatment beyond RECIST progression was permitted. Analyses of baseline (BL) and on-treatment tumor measurements, best response and time to progression used in this analysis were adjudicated by an independent radiologic committee. Results: With 19.5 mo median follow-up, 59% (75/127) of pts had OS ≥ 12 mo. Of these 75, a minority had RECIST partial response (PR: 8%; 6/75) and the rest had a best response of stable disease (SD: 57%; 43/75), progressive disease (PD: 33%; 25/75), or not evaluable (NE: 1%; 1/75). In contrast, among 41% (52/127) of pts with OS < 12 mo, 27% (14/52) had SD, 67% (35/52) had PD, and 6% were NE (3/52) as best response. Pts with OS ≥ 12 mo were more likely to have a largest liver metastasis of < 3 cm (43% vs 23%). There were no major differences in the use of prior immunotherapy between the two OS groups. Most pts with OS ≥ 12 mo (59%; 44/75) had at least some reduction in the sum of target lesions, regardless of RECIST response. The majority of these 44 pts (64%; 28/44) had durable tumor reduction defined by PFS > 6 mo. In contrast, among OS < 12 mo pts, reduction in the sum of target lesions was infrequent (13%; 7/52), and none were durable. Most pts with tumor growth as best change in target lesions had OS Interestingly, 42% (25/60) of pts with best response of PD had OS ≥ 12 mo including 4 pts with evidence of tumor shrinkage at 6 months or later. The reason for PD (new lesions or growth in index lesions) was comparable between both OS groups. More pts continued treatment beyond progression in the OS ≥ 12 (81%) compared to OS < 12 mo (56%). Conclusion: Analysis of tumor kinetics while on tebe, the first TCR bispecific to report an OS benefit in a solid tumor, suggests most pts with OS ≥ 12 mo are best described by a new type of immune-related response characterized by durable tumor reduction and slowing rate of tumor growth. RECIST responses therefore capture a minority of patients with OS ≥ 12 mo, while just under half of pts with RECIST PD still had promising OS. Citation Format: Marcus O. Butler, Takami Sato, Richard D. Carvajal, Joseph J. Sacco, Shaad E. Abdullah, Chris Holland, Howard Goodall, Alexander N. Shoushtari. Kinetics of radiographic response for tebentafusp (tebe) in previously treated metastatic uveal melanoma (mUM) patients (pts) achieving prolonged survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT038.

Introduction:RP1 and RP2 are novel, enhanced potency oncolytic versions of HSV1 engineered to express human GM-CSF and the gibbon ape leukemia virus membrane R- glycoprotein (GALV-GP R-), providing constitutive fusion activity and increased immunogenic cell death. RP2 further expresses an anti-CTLA-4 antibody-like molecule. Murine versions of RP1 and RP2 exhibited synergy in combination with anti-mouse-PD-1 leading to enhanced regression of both injected and un-injected tumors in mice (Thomas et al JITC 2019). RP1 and RP2 are currently being evaluated in clinical trials in a range of solid tumors alone and combined with anti-PD1 therapy, where deep and durable responses have been demonstrated (SITC 2020). Here we present biomarker data from the Phase 1/2 clinical trial of RP1 alone and combined with nivolumab (NCT03767348) and from the Phase 1 portion of the clinical trial with RP2 alone (NCT04336241). Methods: In the Phase 1/2 studies tumor biopsies and peripheral blood mononuclear samples (PBMCs) were collected at screening and at D43 for biomarker analysis, after combination therapy with nivolumab for RP1 and following single agent treatment for RP2. Immunohistochemistry (IHC) was performed for CD8 (SP57 clone, Ventana) and for PD-L1 (28-8 clone, pharmDx assay). Gene expression was analysed using NanoString to assess effects on a range of genes. The tumor inflammation signature score (TIS) was also calculated. Results:Preliminary Phase 1/2 biomarker data from paired tumor biopsies include the following: Immunohistochemistry for CD8 and PD-L1 (n=30) indicated robust and increased infiltration of CD8+ T cells and PD-L1 expression, both after combined treatment with RP1 and nivolumab and after single agent RP2 across different tumor types, and including reversal of T cell exclusion following prior combined treatment with ipilimumab and nivolumab in melanoma. Gene expression analysis (n=15) demonstrated a significant increase in the expression levels of genes associated with innate and adaptive immune activation and genes previously reported to be associated with responsiveness to anti-PD1 therapy, particularly CD8, CXCL9, CD27 and TIGIT, as well as consistently increased TIS. Conclusion:Consistent with the pre-clinical data, preliminary clinical biomarker data indicate substantial increase in CD8 T cell infiltration and PD-L1 expression, as well as increased TIS score in the majority of patients treated with RP2 alone or RP1 and nivolumab combination. Particularly marked effects were seen in some patients with clinical responses which occurred independent of both baseline PD-L1 and prior anti-PD1 therapy status, which suggests potential broad utility of the RP1/2 treatment approach in igniting an anti-tumor immune response. Tumor mutation burden analysis and T cell receptor sequencing are currently underway and further updates of the dataset will be presented. Citation Format: Kevin J. Harrington, Francesca Aroldi, Joseph J. Sacco, Mohammed M. Milhem, Brendan D. Curti, Ari M. Vanderwalde, Scott Baum, Adel Samson, Anna C. Pavlick, Jason A. Chesney, Jiaxin Niu, Terence D. Rhodes, Tawnya L. Bowles, Robert Conry, Anna Olsson-Brown, Douglas E. Laux, Pablo Nenclares, Lavita Menezes, Alex Deterding, Victoria Roulstone, Joan Kyula, Suzanne Thomas, Praveen K. Bommareddy, Selda Samakoglu, Andrea Pirzkall, Robert S. Coffin, Mark R. Middleton. Clinical biomarker studies with two fusion-enhanced versions of oncolytic HSV (RP1 and RP2) alone and in combination with nivolumab in cancer patients indicate potent immune activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB180.

Background: Tebentafusp, an investigational TCR–anti-CD3 bispecific fusion protein (ImmTAC) that targets gp100 and activates T cells, has shown monotherapy activity in metastatic melanoma [1-3]. In a phase I/II trial enrolling metastatic uveal melanoma patients we explored biomarkers of clinical outcomes. Methods: The effect of CD163+ macrophages on ImmTAC-mediated T cell proliferation and melanoma cell killing was assessed in vitro. Advanced uveal melanoma HLA-A*02:01+ patients (N = 127) were treated with weekly 68mcg tebentafusp after intra-patient dose escalation (NCT02570308). Serum samples (up to N=123) were profiled for 11 cytokines and chemokines. Digital image analysis of IHC enumerated immune cells per mm2 in tumor biopsies (up to N=112). Median cutoff was used for biomarker correlations with outcomes. Results: In vitro, increasing numbers of CD163+ macrophages resulted in greater inhibition of ImmTAC-mediated T cell proliferation and killing of MEL624 melanoma cells. The numbers of tumor infiltrating T cells (CD3, CD4, CD8) and CD163+ macrophages at baseline were not associated with TS. However, below median CD163:CD3 ratio was associated with greater TS (odds ratio OR=2.9, p=0.014) and OS (hazard ratio HR=0.4, p=0.001), attributable to CD163:CD8 (OR=3.6, p=0.004) but not CD163:CD4 (OR=1.9, p=0.2). Low serum IL-6 levels at baseline trended with TS (OR=2.1, p=0.09) and OS (HR=0.3, p Conclusions: Uveal melanoma patients with low levels of pre-treatment CD163:CD3 in tumor biopsy and serum IL-6 may derive the most benefit from tebentafusp. This hypothesis will be tested in samples from a randomized trial of tebentafusp (NCT03070392). [1] NCT01211262; ASCO 2019 [2] Clinical Cancer Research; 2020[3] #195; ESMO IO; 2020 Subset% with tumor shrinkageMean change in tumor SLD ± S.E.1-year OS %All biomarker evaluable43 (N=95)5.4 ± 3 (N=95)58 (N=105)Low IL-6/Low CD163:CD370 (N=27)-9.4 ± 6.1 (N=27)93 (N=30)Low IL-6/High CD163:CD330 (N=20)10.2 ± 5.0 (N=20)64 (N=22)High IL-6/Low CD163:CD340 (N=20)14.2 ± 7.7 (N=20)41 (N=22)High IL-6/High CD163:CD329 (N=28)9.9 ± 4.0 (N=28)32 (N=31) Citation Format: Jessica C. Hassel, Adel Benlahrech, Sarah Stanhope, Revashnee Naidoo, Emma Leach, Laura Collins, Shaad Abdullah, Koustubh Ranade, Joseph Sacco. Uveal melanoma study patients with low CD163:CD3 ratio in tumor biopsy and low serum IL-6 showed enhanced tumor shrinkage (TS) and overall survival (OS) on tebentafusp [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1673.

9509 Background: Tebentafusp (tebe) is the first T cell receptor (TCR) therapeutic to demonstrate an overall survival (OS) benefit in a randomized Phase 3 (Ph3) study [ NCT03070392 ]. In Ph2, 42% of pts with best overall response (BOR) of progressive disease (PD) survived > 1 year (yr), suggesting RECIST-based radiographic assessments underestimate OS benefit of tebe. Here we analyzed OS in the Ph3 study in a cohort of pts with BOR of PD by comparing tebe to the control arm of investigator’s choice (IC). Methods: 378 pts were randomized in a 2:1 ratio to tebe vs. IC. BOR was assessed by investigators using RECIST v1.1. Treatment beyond first disease progression (TBP) was permitted for both arms. On the IC arm, only patients receiving pembrolizumab (pembro) continued with TBP and were included in the TBP-related analyses. No crossover to tebe was permitted; investigators were free to choose subsequent therapy. This analysis was conducted on the first interim analysis (data extracted Nov-2020). Kaplan-Meier estimates of OS were based on Day 100 landmark to eliminate immortal time bias and to capture majority of the PDs. Results: By Day 100, PD as BOR occurred in 52% (130/252) of tebe pts (PD-tebe) vs. 60% (76/126) of IC pts (PD-IC). Key baseline characteristics including lactate dehydrogenase, alkaline phosphatase, ECOG performance, age, and sex were similar between PD-tebe vs PD-IC. The proportion of pts with PD due to progression of target lesions (TL), non-TL, or new lesions were also similar between the two groups. More pts received TBP among PD-tebe 53% (69/130) vs PD-pembro 16% (10/61). Median duration of TBP was longer for PD-tebe (7 weeks) vs PD-Pembro (3 weeks). The safety profile of PD-tebe pts during TBP was similar to all tebe-treated pts. OS was superior for PD-tebe vs PD-IC, HR = 0.41 (95%CI 0.25-0.66), even when considering key baseline covariates. While some pts had regression of TL despite diagnosis of PD ( < 10% of pts), the OS benefit remained even when limited to pts with best change of tumor growth of TL, HR 0.46 (0.29, 0.73). 58% (75/130) PD-tebe and 52% (40/76) PD-IC pts received subsequent therapies. In a landmark OS analysis of these pts beginning on 1st day of subsequent therapy, prior tebe was associated with better OS vs. prior IC, HR 0.59 (95%CI 0.36-0.96). Conclusions: Tebe is the first TCR therapeutic to demonstrate an OS benefit in a solid tumor. Surprisingly, a strong OS benefit from tebe is observed even in pts with BOR of PD, suggesting that RECIST-based radiographic assessments do not capture the complete benefit from tebe. The safety profile of tebe during TBP was consistent with that for long-term tebe treatment. Clinical trial information: NCT03070392.

e21513 Background: Tebe is a bispecific gp100-targeted T cell receptor fusion protein that can redirect polyclonal T cells to target gp100+ cells leading to T cell activation and release of inflammatory mediators. Hepatocytes do not express gp100 and tebe did not redirect T cells against normal hepatocytes in preclinical in vitro studies. However, since most mUM pts have liver metastases, tebe may result in secondary effects from localized tumor-related inflammation. Here we describe LFT kinetics and outcomes for pts in the IMCgp100-102 study (NCT02570308). Methods: 127 HLA-A*02:01+ pts with 2L+ mUM received tebe, administered weekly at the RP2D following intra-patient dose escalation (C1D1: 20μg; C1D8: 30μg; C1D15+: 68μg). Pts were eligible if ALT/AST ≤ 3 x ULN and bilirubin ≤ 1.5 x ULN. LFTs were measured at baseline (BL) and weekly prior to each dose using local laboratories. AE grading was based on CTCAE v4.03. This analysis was conducted on the primary analysis snapshot dated 04Jun20. Results: At BL,125/127 (98%) pts had ALT/AST ≤ grade(G)1 and 122/127 (96%) had liver metastasis. 68/127 (54%) had an increase in post-BL grade for ALT, AST or both. Of these 48/68 (71%) increased to G1, 9/68 (13%) to G2, 7/68 (10%) to G3, and 4/68 (6%) to G4. 67 of these 68 pts (99%) had liver metastasis and most had largest liver metastasis > 3cm (38 pts > 3 cm, 29 pts < 3cm and 1 pt without). ALT/AST increases occurred early in treatment in 36/68 (53%) including at Dose 1 (12/68; 18%), Dose 2 (10/68; 15%), or Dose 3 (14/68; 21%). In the other 32/68 pts (47%), ALT/AST increases occurred at or after Dose 4 (4-65), and most of these events (21/32; 66%) were associated temporally with increase in size of liver metastases. Among the 11 pts with G3/4 ALT/AST increases post-BL, most pts experienced these events early (Doses 1-3) (8/11; 73%) and in the context of either increase in size of liver metastases / disease progression or biliary obstruction (9/11; 82%). Most pts, 60/68 (88%), continued treatment despite an increase in ALT/AST grade. Among 8 pts who discontinued treatment, 3 were due to disease progression and 3 were due to adverse events. Median time for ALT/AST to return to BL was 9 days and there were no temporal increases in albumin or INR. Conclusions: Approximately 1/2 of tebe treated pts experienced an increase in post-BL CTCAE grade for ALT/AST. Among these pts, most ALT/AST increases were mild. Most of the pts with G3/4 ALT/AST increase (9/11) were explained by increase in size of liver metastases or biliary obstruction. Increase in LFT are not unusual at time of disease progression in mUM given high frequency of liver metastases. In summary, ALT/AST increases occurred early in about half of the pts, were generally self-limiting, permitted treatment to continue, and did not appear to impact liver synthetic function (INR/Albumin). Clinical trial information: NCT02570308.

9527 Background: Tebe is a bispecific consisting of an affinity-enhanced T cell receptor fused to an anti-CD3 effector that can redirect T cells to target gp100+ cells. In this Phase (Ph) 3, randomized trial of first line (1L) metastatic uveal melanoma (mUM) [NCT03070392], tebe significantly improved overall survival (OS) vs. investigator’s choice (IC) in the intention-to-treat population (ITT). In previous trials, tebe-related skin adverse events (AEs), hypothesized to be on-target, off-tumor activity against gp100-expressing melanocytes, were associated with improved OS. This association was tested prospectively as a co-primary endpoint in the Ph3 study. Methods: 378 1L HLA-A*02:01+ mUM pts were randomized 2:1 to tebe (n = 252) or IC (n = 126). Co-primary endpoints were 1) OS in all randomized pts (ITT) and 2) OS in tebe-randomized pts who develop any grade rash in week (wk) 1 vs. all receiving IC. Rash was defined as composite of preferred AE terms. Melanocyte-related AEs (MRAEs) were defined as pigment change AEs in the skin or hair. Overall study-wide alpha was controlled at 0.05, with 90% assigned to ITT and 10% to rash. This analysis was conducted on the first interim analysis (data extracted Nov-2020). Results: In the 245 tebe treated pts, the characteristic skin related AEs included most frequently rash (at any time) in 201 pts (82%), pruritis in 167 pts (68%), MRAEs in 109 pts (45%) and erythema in 69 pts (28%). While rash, erythema and pruritis mostly occurred in the first 4 weeks, MRAEs occurred after a median of 2.7 mo. Rash captures most pts, 201/227 (89%), who have any of these skin related AEs. Rash occurred in 146 pts (60%) by wk 1; 179 pts (73%) by wk 2; and 195 pts (80%) by wk 3. Tebe pts with wk 1 rash had significantly longer OS vs. the IC arm, HR 0.35 (95% CI 0.23, 0.53), p < 0.0001. The estimated 1-yr OS rates were 83% vs 58%, respectively. When expanded to include tebe pts with rash through wk 3, the 1-yr OS rate of 75% was still numerically higher than IC. The 50 (20%) tebe pts who did not experience rash by week 3 had 1-yr OS rate of 55%. Conclusions: In 1L mUM pts, tebe significantly improved OS compared to IC in the ITT analysis. Week 1 rash, presumed due to tebe redirection of T cells to gp100+ skin melanocytes, was associated with a very strong OS benefit. Therefore, rash may be a marker that the immune system can be mobilized by tebe to target gp100+ cells. The vast majority of tebe pts will develop a rash at some point, and tebe pts without rash may still derive benefit. Clinical trial information: NCT03070392.