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Background: MEDI5395 is a recombinant attenuated Newcastle disease virus engineered to express a human granulocyte-macrophage colony-stimulating factor transgene. Preclinically, MEDI5395 demonstrated broad oncolytic activity, augmented by concomitant programmed cell death-1/programmed cell death ligand-1 (PD-L1) axis blockade. Durvalumab is an anti-PD-L1 immune checkpoint inhibitor approved for the treatment of various solid tumors. We describe the results of the first-in-human study combining intravenous MEDI5395 with durvalumab in patients with advanced solid tumors., Methods: This phase I, open-label, multicenter, dose-escalation, dose-expansion study recruited adult patients with advanced solid tumors, who had relapsed or were refractory or intolerant to ≥1 prior line of standard treatment. MEDI5395 was administered intravenously as six doses over 15-18 days. The dose-escalation phase assessed four-dose levels (10 8 , 10 9 , 10 10 , 10 11 focus forming units (FFU)) of MEDI5395, with sequential or delayed durvalumab. Durvalumab 1500 mg was administered intravenously every 4 weeks up to 2 years. The dose-expansion phase was not initiated. The primary objectives were to evaluate safety and tolerability, dose-limiting toxicities (DLTs) and the dose and schedule of MEDI5395 plus durvalumab administration. Secondary objectives included the assessment of the efficacy, pharmacokinetics, pharmacodynamics, and immunogenicity of MEDI5395., Results: 39 patients were treated with MEDI5395; 36 patients also received durvalumab. All 39 patients experienced ≥1 treatment-emergent adverse event (TEAE), most commonly fatigue (61.5%), nausea (53.8%) and chills (51.3%). Grade 3-4 TEAEs occurred in 27 (69.2%) patients; these were deemed MEDI5395-related in 12 (30.8%) patients. Two patients experienced a DLT, and the maximum tolerated dose of MEDI5395 with sequential and delayed durvalumab at study termination was 10 11 and 10 10 FFU, respectively. Four patients (10.3%) achieved a partial response (PR). Patients with PR or stable disease tended to have higher baseline PD-L1 and CD8+ levels in their tumor tissue. A tendency to dose-dependent pharmacokinetics of the viral genome was observed in whole blood and a tendency to dose-dependent viral shedding was observed in saliva and urine. Neutralizing antibodies were observed in all patients but did not appear to impact efficacy negatively., Conclusion: This study demonstrates the feasibility, safety and preliminary efficacy of MEDI5395 with durvalumab in patients with advanced solid tumors., Trial Registration Number: NCT03889275., Competing Interests: Competing interests: DD reports Grants/Research Support (institutional) from Arcus, CellSight Technologies, Immunocore, Merck, Regeneron Pharmaceuticals, Tesaro/GSK; Consultant fees from ACM Bio, Ascendis Pharma, Clinical Care Options (CCO), Gerson Lehrman Group (GLG), Merck, Medical Learning Group (MLG), Xilio Therapeutics. CE Speakers’ Bureau from Castle Biosciences; reports Intellectual Property for US Patent 63/124,231, “Compositions and Methods for Treating Cancer”, December 11, 2020; US Patent 63/208,719, “Compositions and Methods For Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer”, June 9, 2021. BAC reports Institutional Research Support from AstraZeneca, AbbVie, Actuate Therapeutics, Astellas, Agenus, Bayer, Dragonfly Therapeutics, Mink Therapeutics, Pfizer, Pyxis Oncology, Repare Therapeutics, Regeneron; Advisory Boards for Seattle Genetics. GKD reports Consulting Fee from Amgen, AstraZeneca, Bayer, Eli Lilly, Janssen, Meru, Mirati, Novartis, Regeneron. SS reports Employment from University of North Carolina; Honoraria from Naveris, Exelixis, Eisai, Medscape, Association of Community Cancer Centers; Consulting fees from Exelixis. Grants or Funds from Merck, AstraZeneca, Exelixis, Regeneron, Inovio, ASCO. MJB reports Grants, Funds, Trial support to institution (Mayo Clinic). KJH reports Scientific Advisory Board Membership for Oncolys, PsiVac, Replimune (paid to institution); Consulting Fees for PsiVac, Replimune, VacV (paid to institution); Grants or Funds from Replimune (paid to institution). SPP reports Consulting Fees from Amgen, AstraZeneca, BeiGene, Bristol-Myers Squibb, Certis, Eli Lilly, Jazz, Genentech, Illumina, Merck, Pfizer, Signatera, Tempus; Grants or Funds (research funding to university) from Amgen, AstraZeneca, A2bio, Bristol-Myers Squibb, Eli Lilly, Fate Therapeutics, Gilead, Iovance, Merck, Pfizer, Roche/Genentech. EG reports Consulting Fee from Kiyatec (personal compensation), Karyopharm Therapeutics (Data Safety Monitoring Board, compensation to employer), Boston Scientific (Data Monitoring Committee, compensation to employer), Servier Pharmaceuticals (Advisory Board, compensation to employer), Boehringer Ingelheim (Advisory Board, compensation to employer); Grants or Funds from Servier Pharmaceuticals (formerly Agios Pharmaceuticals), Denovo Biopharma, Celgene, MedImmune. AS reports Consulting fees from Roche and Chugai; Academic grants from Replimune, Histosonics, Oncolytics Biotech, Transgene (all paid to institution). SA: AstraZeneca employee and stock holder. ZC reports no potential conflict of interest. CF: AstraZeneca employee and stock holder. MG: AstraZeneca employee and stock holder. JBu: AstraZeneca employee and stock holder. ET: AstraZeneca employee and stock holder. ND: AstraZeneca employee and stock holder. KL: AstraZeneca employee and stock holder. FA: AstraZeneca employee and stock holder. DZ reports Institutional Grants from Merck, Genentech, AstraZeneca, Plexxikon, and Synthekine; Personal Fees; AstraZeneca, Xencor, Memgen, Takeda, Synthekine, Immunos, Tessa Therapeutics, Miltenyi, and Calidi Biotherapeutics. DZ is a holder of a patent on use of oncolytic NDV for cancer therapy (unrelated to the agent under study)., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Engagement of programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) can interfere with the CD28 signaling requisite for T-cell activation. While immune checkpoint inhibitors (ICIs) can relieve this suppression, they are unable to drive CD28 costimulation that may mechanistically contribute to ICI resistance. Thus, CD28 costimulation in the context of checkpoint inhibition may activate immunosuppressed T-cells in the tumor microenvironment. Davoceticept (ALPN-202) is an Fc fusion of a CD80 variant immunoglobulin domain (vIgD) designed to mediate PD-L1-dependent CD28 costimulation while inhibiting the PD-L1 and CTLA-4 checkpoints. PD-L1-restriction of davoceticept's CD28 costimulatory activity may minimize systemic T-cell activation and avoid untoward systemic toxicities. At the same time, preclinical studies have suggested that treatment with davoceticept during PD-1 inhibition may enhance antitumor activity by upregulating PD-L1, potentially synergizing with davoceticept's PD-L1-dependent costimulatory mechanism. This report details two cases of fatal cardiac events following treatment with davoceticept in combination with pembrolizumab (anti-PD-1) in the phase 1 study, NEON-2. Both events occurred in females in their 60s; one with choroidal melanoma and prior immunotherapy, the other with ICI-naïve microsatellite stable colorectal cancer. The clinical courses were fulminant with symptom onset at 2 weeks, followed by rapid decline. Cardiac autopsy from one patient confirmed immune-related myocarditis, and immunosequencing revealed expansion of a single T-cell clone that was not present in the pretreatment tumor. These cases highlight the importance of understanding risk factors that may contribute to immune-related myocarditis and other severe immune-related adverse events when CD28 agonism is targeted in the context of checkpoint inhibition.NEON-2 (NCT04920383)., Competing Interests: Competing interests: LC: Consultant: Janssen, AstraZeneca, Pliant Therapeutics, CDR-Life, Actuate TherapeuticsSRC: Research Support (institutional): Alpine Immune SciencesKL: Nothing to disclose. FS: Grants or Contracts: NIH P01 (Deciphering LKB1-associated immunotherapy resistance in lung adenocarcinoma). NL: Support for the present manuscript (institutional): Alpine Immune Sciences; Consulting Fees: SK Life Sciences; Research Funding (institutional): Alexo Therapeutics, Ascentage Pharma, BeiGene, Constellation Pharmaceuticals, Forty Seven, Loxo, Macrogenics, Merck, Pfizer, Regeneron, Apexian Pharmaceuticals, Coordination Therapeutics, Symphogen, CytomX Therapeutics, InhibRx, Incyte, Jounce Therapeutics, Livzon, Northern Biologics, Tesaro, Innovent Biologics, LAM Therapeutics, Ikena, Celgene, Shattuck Labs, Alpine Immune Sciences, Genmab, Odonate, Mersana, Seagen, Alpine Biosciences, Astellas Pharma, Celgene, Helsinn, Therapeutics, Ikena Oncology, Lilly, Sapience Therapeutics, Epizyme, Gilead, Glaxo Smith Kline, Tizona, Servier, Alkermes, KSQ, Repare Therapeutics, Biosplice/Samumed, Sapience Therapeutics, SK Life Sciences, Janssen, Arcus, Artios, BioNTech SE, Alkermes/Mural OncologyAE: Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune Sciences; Support for attending meetings: Alpine Immune Sciences; Patents planned, issued or pending: Tempest Therapeutics, University of CaliforniaHL: Former employee of Alpine Immune Sciences; Holds stock or stock options in Alpine Immune SciencesJS: Employee of Adaptive Biotechnologies; Holds stock in Adaptive Biotechnologies. HT: Support for present manuscript: Biostatistician supporting work on the manuscript; Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune SciencesMJC: Employee of Merck Sharp & Dohme, a subsidiary of Merck & Co., Rahway, New Jersey, USA; Owns stock in Merck & Co. SLP: Employee of Alpine Immune Sciences; Holds stock or stock options in Alpine Immune Sciences. AN: Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune SciencesDD: Support for present manuscript: Alpine Immune Sciences; Grants/Research Support (institutional): Arcus, CellSight Technologies, Immunocore, Merck, Regeneron Pharmaceuticals, Tesaro/GSK. Consulting Fees: ACM Bio, Ascendis Pharma; Clinical Care Options (CCO), Gerson Lehrman Group (GLG), Merck, Medical Learning Group (MLG), Xilio Therapeutics. Payment or honoraria for lectures, presentations, speaker’s bureaus, manuscript writing or educational events: Castle Biosciences. Patents planned, issued or pending: US Patent 63/124,231, “Compositions and Methods for Treating Cancer”, December 11, 2020 US Patent 63/208,719, “Compositions and Methods For Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer”, June 9, 2021., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Davoceticept (ALPN-202) is an Fc fusion of a CD80 variant immunoglobulin domain designed to mediate programmed death-ligand 1 (PD-L1)-dependent CD28 co-stimulation while inhibiting the PD-L1 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) checkpoints. The safety and efficacy of davoceticept monotherapy and davoceticept and pembrolizumab combination therapy in adult patients with advanced solid tumors were explored in NEON-1 and NEON-2, respectively., Methods: In NEON-1 (n=58), davoceticept 0.001-10 mg/kg was administered intravenous either once weekly (Q1W) or once every 3 weeks (Q3W). In NEON-2 (n=29), davoceticept was administered intravenously at 2 dose levels (0.1 or 0.3 mg/kg) Q1W or Q3W with pembrolizumab (400 mg once every 6 weeks). In both studies, primary endpoints included incidence of dose-limiting toxicities (DLT); type, incidence, and severity of adverse events (AEs) and laboratory abnormalities; and seriousness of AEs. Secondary endpoints included antitumor efficacy assessed using RECIST v1.1, pharmacokinetics, anti-drug antibodies, and pharmacodynamic biomarkers., Results: The incidence of treatment-related AEs (TRAEs) and immune-related adverse events (irAEs) was 67% (39/58) and 36% (21/58) with davoceticept monotherapy, and 62% (18/29) and 31% (9/29) with davoceticept and pembrolizumab combination, respectively. The incidence of ≥grade (Gr)3 TRAEs and ≥Gr3 irAEs was 12% (7/58) and 5% (3/58) with davoceticept monotherapy, and 24% (7/29) and 10% (3/29) with davoceticept and pembrolizumab combination, respectively. One DLT of Gr3 immune-related gastritis occurred during davoceticept monotherapy 3 mg/kg Q3W. During davoceticept combination with pembrolizumab, two Gr5 cardiac DLTs occurred; one instance each of cardiogenic shock (0.3 mg/kg Q3W, choroidal melanoma metastatic to the liver) and immune-mediated myocarditis (0.1 mg/kg Q3W, microsatellite stable metastatic colorectal adenocarcinoma), prompting early termination of both studies. Across both studies, five patients with renal cell carcinoma (RCC) exhibited evidence of clinical benefit (two partial response, three stable disease)., Conclusions: Davoceticept was generally well tolerated as monotherapy at intravenous doses up to 10 mg/kg. Evidence of clinical activity was observed with davoceticept monotherapy and davoceticept in combination with pembrolizumab, notably in RCC. However, two fatal cardiac events occurred with the combination of low-dose davoceticept and pembrolizumab. Future clinical investigation with davoceticept should not consider combination with programmed death-1-inhibitor anticancer mechanisms, until its safety profile is more fully elucidated., Trial Registration Number: NEON-1 (NCT04186637) and NEON-2 (NCT04920383)., Competing Interests: Competing interests: DD: Support for the present manuscript: Alpine; Grants/Research Support (institutional): Arcus, CellSight Technologies, Immunocore, Merck, Regeneron Pharmaceuticals, Tesaro/GSK. Consultant: ACM Bio, Clinical Care Options (CCO), Gerson Lehrman Group (GLG), Merck, Medical Learning Group (MLG), Xilio Therapeutics. CE Speakers’ Bureau: Castle Biosciences. Stockholder: None. Patents planned, issued or pending: US Patent 63/124,231, “Compositions and Methods for Treating Cancer”, December 11, 2020; US Patent 63/208,719, “Compositions and Methods for Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer”, June 9, 2021. LC: Honoraria/Consultant: Pliant Therapeutics, Janssen, CDR-Life, Actuate Therapeutics. NL: Support for the present manuscript (institutional): Alpine Immune Sciences; Consulting Fees: SK Life Sciences; Research Funding (institutional): Alexo Therapeutics, Ascentage Pharma, BeiGene, Constellation Pharmaceuticals, Forty Seven, Loxo, Macrogenics, Merck, Pfizer, Regeneron, Apexian Pharmaceuticals, Coordination Therapeutics, Symphogen, CytomX Therapeutics, InhibRx, Incyte, Jounce Therapeutics, Livzon, Northern Biologics, Tesaro, Innovent Biologics, LAM Therapeutics, Ikena, Celgene, Shattuck Labs, Alpine Immune Sciences, Genmab, Odonate, Mersana, Seagen, Alpine Biosciences, Astellas Pharma, Celgene, Helsinn, Therapeutics, Ikena Oncology, Lilly, Sapience Therapeutics, Epizyme, Gilead, Glaxo Smith Kline, Tizona, Servier, Alkermes, KSQ, Repare Therapeutics, Biosplice/Samumed, Sapience Therapeutics, SK Life Sciences, Janssen, Arcus, Artios, BioNTech SE, Alkermes/Mural Oncology. JM: Support for the present manuscript (institutional): Alpine Immune Sciences; Grants or contracts (institutional): NovoCure, Genentech, Alpine Immune Sciences, Amgen, Trishula Therapeutics, BioEclipse Therapeutics, FujiFilm, ImmuneSensor, Simcha, Repertoire Immune Sciences, Nektar Therapeutics, Synthorx, Istari Oncology, Ideaya Biosciences, Rubius, University of Arizona, Senwha, Storm Therapeutics, Werewolf Therapeutics, Fate Therapeutics, Y-Mab, Agenus, T-Scan, Lovance; Consulting Fees: BMS, Amunix, Thirona Bio, Adagene, Imaging Endpoints, Boxer Capitol, Oberland Capital, IQVIA, Genome Insight, Incyte, Novotech, Red Arrow Therapeutics; Payment or honoraria for lectures, presentations, speaker’s bureaus, manuscript writing or educational events: Caris Life Sciences, Daiichi-Sankyo, TGen; Participation on a data safety monitoring board or advisory board: Topotecan Episcleral Plaque for Treatment of Retinoblastoma; Other: Board member: Caris Molecular Tumor Board, Caris Consultant; Speakers Bureau: Caris Life Sciences, Immunocore, Castle Biosciences. MMi: Support for the present manuscript (institutional): Alpine Immune Sciences; Consulting Fees (Advisory Board): The Limbic, Bristol Myers Squibb Pty Ltd, Guardant Health, Beigene Australia Pty Ltd, Merck Pte Ltd, AstraZeneca Pty Ltd, Pfizer Australia Pty Ltd; Honoraria (meeting chair): Roche Products Pty Ltd, The Limbic; Participation on a Data Safety Monitoring Board: Novartis Pharma AG (Europe); Leadership or fiduciary role on a Scientific Advisory Board: Thoracic Oncology Group Australia, Melanoma and Skin Cancer Trials Australia. MMc: Support for the present manuscript (institutional): Alpine Immune Sciences; Grants or contracts (institutional): Aadi Biosciences, Alpine Immune Sciences, Arcus Biosciences, Arvinas, Ascentage Pharma Group, ASCO, Astellas, Aulos Bioscience, Bayer, Bicycle Therapeutics, BioMed Valley Discoveries, BioNTech, Bristol-Myers Squibb, C4 Therapeutics, Dragonfly Therapeutics, EMD Serono, Epizyme, Erasca, Exelixis, Foghorn Therapeutics, GI Therapeutics, Genentech/Roche, Gilead Sciences, GlaxoSmithKline, IDEAYA Biosciences, Ikena Oncology, ImmVira Pharma, Infinity Pharmaceuticals, Jacobio Pharmaceuticals, Kechow Pharma, Kezar Life Sciences, Kinnate BioPharma, MedImmune, Mereo BioPharma, Metabomed, Moderna, NBE Therrapeutics, Nektar, Novartis, NucMito Pharmaceuticals, OncoC4, Oncorus, OnKure, PACT Pharma, Pfizer, Plexxikon, Poseida, Prelude Therapeutics, Pyramid Biosciences, Regeneron, Sapience Therapeutics, Scholar Rock, Seattle Genetics, Synthrox, Teneobio, Tempest Therapeutics, Tizona Therapeutics, TMUNITY Therapeutics, TopAlliance Biosciences, Xilio; Consulting Fees (institutional): Castle Biosciences, IQVIA, Merck, Moderna, Pfizer. MV: Support for the present manuscript: Alpine Immune Sciences (institutional); Honoraria: MSD; Consulting or Advisory Role: AstraZeneca, MSD; Grants or Contracts (Research Funding, institutional): AstraZeneca/MedImmune, AstraZeneca, MSD, Alpine Immune Sciences, Virocure, Hinova Pharmaceutics, Atridia, Antengene, BeiGene, Hengrui Pharmaceutical. RES: Grants and Contracts: AstraZeneca, Merck; Consulting (steering committee): GlaxoSmithKline, Janssen Oncology, Daiichi, BeiGene; Consulting (advisory board): AstraZeneca, Macrogenics, Sanofi, Gilead, Regeneron, Targeted Oncology, GI Therapeutics, GE Health Care, Lily Oncology; Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events (educational presentations: Illumina, GameOn!, OncLive, Binay Foundation, APP Oncology, Masters in Thoracic Oncology Summit; consulting for manuscript: EMD Serono. JSG: Grants or contracts: Novartis; Consulting: Amgen, Arcus Biosciences, AI Proteins, AstraZeneca, Beigene, Blueprint Medicines, Bristol Myers Squibb, Genentech/Roche, EMD Serono, InterVenn Biosciences, Gilead Sciences, iTeos Therapeutics, Jounce Therapeutics, Karyopharm Therapeutics, Lilly, Loxo, Merus, Mirati Therapeutics, Pfizer, Sanofi, Silverback Therapeutics, Merck, Moderna Therapeutics, Mariana Oncology, Takeda; Payment or Honoraria: Merck, Pfizer, Novartis, Pfizer, Takeda; Leadership or fiduciary role: SAB Happy Lungs; Stock or Stock Options: Immediate family member is an employee with equity in Ironwood Pharmaceuticals. AN: Payment for honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Dava Oncology. AP: Honoraria: Texas Society of Clinical Oncology (TxSCO); Consulting or Advisory Role: Bayer, Novartis, Merck, Seattle Genetics, Silverback Therapeutics, Shenzhen IONOVA Life Sciences, Gilead, Daiichi Sankyo, HalioDx, Janssen; Consulting or Advisory Role (immediate family member): Genentech/Roche, Merck, Bristol-Myers Squibb; Research Funding (institutional): Merck, Pfizer, Lilly, Plexxikon, Corvus Pharmaceuticals, Tesaro, AbbVie, Forty Seven, Five Prime Therapeutics, Infinity Pharmaceuticals, Pieris Pharmaceuticals, Surface Oncology, Livzon, Vigeo Therapeutics, Astellas Pharma, Klus Pharma, Symphogen, Syndax, Arcus, Fochon, Upsher-Smith, Exelixis, Seattle Genetics, Bolt, Ionova, Daiichi Sankyo, Sanofi, Gilead Sciences, Seagen, Shenzhen Ionova Life Science, Pionyr Immunotherapeutics, Loxo Oncology, Inc. On behalf of Eli Lilly and company, Nektar Therapeutics, Alpine Immune Sciences, Amgen, Institut de Recherches Internationales Servier (I.R.I.S.), 1200 Pharma, Arcus Biosciences, Genentech, Aadi Bioscience, Prelude, KSQ Therapeutics, Carrick Therapeutics. JFG: Payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events: Bristal Myers Squibb, Curio Sciences. MS: Consulting fees: Adagene, Adaptimmune, Alkermes, Alligator, Anaptys, Asher, Biond, Biontech, Boston Pharmaceuticals, Bristol-Myers, Dragonfly, Evaxion, Evolveimmune, Gilead, Glaxo Smith Kline, Ichnos, Immunocore, Incyte, Innate pharma, Iovance, iTEOS, Jazz Pharmaceuticals, Kanaph, Merck, Molecular Partners, Nextcure, Nimbus, Normunity, Numab, Ocellaris-Lilly, Oncohost, Ontario Institute for Cancer Research, Partner Therapeutics, Pfizer, Pierre-Fabre, PIO Therapeutics, Pliant, Regeneron, Rootpath, Sapience, Simcha, Sumitomo, Targovax, Teva, Turnstone, Verastem, Xilio; Leadership or fiduciary role in other board, society, committee or advocacy group: Society for Immunotherapy of Cancer, past President (unpaid); Stock options: Actym, Adaptive Biotechnologies, Amphivena, Asher, Evolveimmune, Intensity, Nextcure, Normunity, Oncohost, Thetis; Stock: Johnson and Johnson, Glaxo-Smith Kline. AE: Employee of Alpine Immune Sciences; Support for attending meetings and/or travel: Alpine Immune Sciences; Patents planned, issued, or pending: Tempest Therapeutics, University of California; Holds stock or stock options in Alpine Immune Sciences. LB: Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune Sciences. HL: All support for the present manuscript: Former employee of Alpine Immune Sciences; Holds stock or stock options in Alpine Immune Sciences. HT: All support for the present manuscript: Alpine Immune Sciences biostatistician supporting work on the manuscript; Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune Sciences. MJC: Merck Sharp & Dohme, a subsidiary of Merck & Co., Rahway, New Jersey, USA; Owns stock in Merck & Co, Rahway, New Jersey, USA. SLP: Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune Sciences. AN: Employee of Alpine Immune Sciences; Holds stock options in Alpine Immune Sciences., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Toll-like receptor 9 (TLR9) agonists induce inflammatory responses that promote the killing of infectious micro-organisms, cancer cells and develop adaptive immune responses. Their ability as immunomodulators to enhance the activity of checkpoint inhibitors (CPI) in treating liver tumors is limited in part by the distinctive biology of intrahepatic myeloid-derived suppressor cells (MDSC) and challenges with tumor-specific therapeutic delivery. We have shown that the regional delivery of type C TLR9 agonist via pressure-enabled drug delivery (PEDD) system improves delivery to the tumor, enhances depletion of MDSCs and overall, stimulates the immune system in combination with or without CPI. Currently, CPIs are delivered intravenously, although there is a growing interest in its subcutaneous (SQ) administration. We compared nelitolimod formerly known as SD-101 administered using PEDD in combination with systemic (Sys) or SQ CPI in murine liver metastases (LM)., Methods: The LM model was developed by injecting MC38-Luc cells via the spleen of 8-12 week old male C57/BL6 mice followed by splenectomy. After a week, fluorescently labeled nelitolimod (10 µg/mouse) was delivered via PEDD and co-administered anti-programmed cell death-1 (α-PD-1) either via Sys or SQ. Tumor burden was monitored by in vivo imaging system. Serum cytokine levels were analyzed by Luminex. Tissues were harvested on Day 3 (D3) or Day 10 (D10) post-PEDD to enrich CD45 + cells and were analyzed via NanoString targeted transcriptomics (D3) or flow cytometry (FC, D10) to interrogate immune cell populations (D10). For NanoString analysis, the innate immune panels were selected, and for FC, MDSCs (CD11b + Gr1 + ), B cells (B220 + ), dendritic cells (DC, CD11c + ), T (CD3 + ) cells, and M1-like macrophages (F4/80 + CD38 + Egr2 - ) were quantified., Results: Nelitolimod delivered via PEDD resulted in changes in innate and adaptive immune cells within LM, including depletion of liver MDSC and increased M1-like macrophages in the liver, which are supportive of antitumor immunity. While CPI monotherapy failed to control tumor progression, nelitolimod and CPI combination improved LM control, survival and antitumor immunity beyond the nelitolimod monotherapy effect, irrespective of CPI delivery route., Conclusion: The SQ route of CPI delivery was equivalent to Sys in combination with nelitolimod, suggesting SQ-CPI may be a rational choice in combination with PEDD of nelitolimod for liver tumor treatment., Competing Interests: Competing interests: CCG, YL, AB, JL, MM, KF, BFC, PG, SCK are employee of Trisalus Life Sciences. LC, IBL, SP, EY, DOT, TM have no conflict of interest. Conflict of interests of DD are: Grants/Research Support (institutional): Arcus, CellSight Technologies, Immunocore, Merck, Regeneron Pharmaceuticals, Tesaro/GSK. Consultant: ACM Bio, Ascendis Pharma, Clinical Care Options (CCO), Gerson Lehrman Group (GLG), Merck, Medical Learning Group (MLG), Xilio Therapeutics. CE Speakers’ Bureau: Castle Biosciences. Intellectual Property: US Patent 63/124,231, “Compositions and Methods for Treating Cancer”, December 11, 2020 US Patent 63/208,719, “Compositions and Methods For Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer”, June 9, 2021., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Intratumorally delivered immunotherapies have the potential to favorably alter the local tumor microenvironment and may stimulate systemic host immunity, offering an alternative or adjunct to other local and systemic treatments. Despite their potential, these therapies have had limited success in late-phase trials for advanced cancer resulting in few formal approvals. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to determine how to design clinical trials with the greatest chance of demonstrating the benefits of intratumoral immunotherapy for patients with cancers across all stages of pathogenesis., Methods: An Intratumoral Immunotherapy Clinical Trials Expert Panel composed of international key stakeholders from academia and industry was assembled. A multiple choice/free response survey was distributed to the panel, and the results of this survey were discussed during a half-day consensus meeting. Key discussion points are summarized in the following manuscript., Results: The panel determined unique clinical trial designs tailored to different stages of cancer development-from premalignant to unresectable/metastatic-that can maximize the chance of capturing the effect of intratumoral immunotherapies. Design elements discussed included study type, patient stratification and exclusion criteria, indications of randomization, study arm determination, endpoints, biological sample collection, and response assessment with biomarkers and imaging. Populations to prioritize for the study of intratumoral immunotherapy, including stage, type of cancer and line of treatment, were also discussed along with common barriers to the development of these local treatments., Conclusions: The SITC Intratumoral Immunotherapy Clinical Trials Expert Panel has identified key considerations for the design and implementation of studies that have the greatest potential to capture the effect of intratumorally delivered immunotherapies. With more effective and standardized trial designs, the potential of intratumoral immunotherapy can be realized and lead to regulatory approvals that will extend the benefit of these local treatments to the patients who need them the most., Competing Interests: Competing interests: JJL: Researcher: AbbVie, Astellas, Astrazeneca, Bristol-Myers Squibb, Corvus, Day 1, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Merck, Moderna, Nektar, Next Cure, Numab, Palleon, Pfizer, Replimmune, Rubius, Servier, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, XencorConsultant/Advisor/Speaker: Abbvie, Agenus, Alnylam, Atomwise, Bayer, Bristol-Myers Squibb, Castle, Checkmate, Codiak, Crown, Cugene, Curadev, Day One, Eisai, EMD Serono, Endeavor, Flame, G1 Therapeutics, Genentech, Gilead, Glenmark, HotSpot, Kadmon, KSQ, Janssen, Ikena, Inzen, Immatics, Immunocore, Incyte, Instil, IO Biotech, Macrogenics, Merck, Mersana, Nektar, Novartis, Partner, Pfizer, Pioneering Medicines, PsiOxus, Regeneron, Replimmune, Ribon, Roivant, Servier, STINGthera, Synlogic, Synthekine, 7 Hills, Affivant, Bright Peak, Exo, Fstar, Inzen, RefleXion, Xilio (stock) Actym, Alphamab Oncology, Arch Oncology, Duke Street Bio, Kanaph, Mavu, NeoTx, Onc.AI, OncoNano, physIQ, Pyxis, Saros, STipe, Tempest, Abbvie, Agenus, Amgen, Immutep, Evaxion. DD: Grants/Research Support (institutional): Arcus, Immunocore, Merck, Regeneron Pharmaceuticals, Tesaro/GSK. Consultant: ACM Bio, Ascendis, Castle, Clinical Care Options (CCO), Gerson Lehrman Group (GLG), Immunitas, Medical Learning Group (MLG), Replimmune, Trisalus, Xilio Therapeutics. CE Speakers’ Bureau: Castle Biosciences. Stockholder: None. Intellectual Property: US Patent 63/124,231, "Compositions and Methods for Treating Cancer", December 11, 2020 US Patent 63/208,719, "Compositions and Methods For Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer", June 9, 2021. AM: Owner: HiFiBio, Deka Bio, Hotspot Therapeutics, Shattuck Labs, Researcher: Astra Zeneca, BMS, Sanofi, Consultant/Advisor/Speaker: Gritstone, Innate Pharma, Neogene, Deka Bio, Hotspot therapeutics, J&J, Medicxi, Depth Charge, BiolineRx, Clover Pharma, Grey Wolf, Lytix, RedX, HiFiBio, ImCheck, Applied Materials, Takeda, Shattuck Labs, Marengo therapeutics, Pierre Fabre, Third Rock Ventures, SotioPublicly Traded Stocks: Centessa. RHA–Employee: Seven & Eight Biopharmaceuticals. Ownership Interest Less Than 5 Percent: Seven & Eight Biopharmaceuticals. NB: Consulting Fees: Novartis, Apricity, Rome Therapeutics, BreakBio, Carisma Therapeutics, CureVac, BioNTech, Gilead, Tempest Therapeutics, Boehringer Ingelheim, Contracted Research: Regeneron Pharmaceutics, Dragonfly Therapeutics, Harbour Biomed Sciences, Ownership Interest Less Than 5 Percent: BreakBio, Apricity. JDB: Researcher: Merck, Genentech, Astrazeneca, Kite/Gilead, BMS, Celldex, Oncovir, Seattle Genetics, ADC Therapeutics, Epizyme, Consultant/Advisor/Speaker: Merck, Genentech, Astrazeneca, Kite/Gilead, BMS, Seattle Genetics, ADC Therapeutics, Epizyme, Asgaard Therapeutics, Global BioAccess, SIRPant Immunotherapeutics. JC: Executive Role: UofL Health, Researcher: Amgen, Iovance, Fate, Replimune, Consultant/Advisor/Speaker: Replimune advisory board 2020-21, Royalties or Patent Beneficiary: US Patents: University of Louisville. RC: Employee: Replimune. TdB: Consultant/Advisor/Speaker: Terumo, Boston Scientific, Astra Zeneca, Nanobiotix, Quantum Surgical, Guerbet, Cook medical, Johnson & Johnson. TDdG: Patents: (1) The use of cytostatics for the accelerated differentiation of DC; WO2009019320-A2; WO2009019320-A3; AU2008285598-A1; EP2281030-A2; CA2724018-A1; US2011117051-A1. US8,470,789B2; DCprime BV. (2) Immunoglobulins binding human Vγ9VÎ’2 T cell receptors; P31885NL00. Consulting Fees: Mendus (formerly Immunicum, formerly DCPrime BV), Partner Therapeutics, GE Health, LAVA Therapeutics, Contracted Research: Idera Pharmaceuticals, Macrophage Parma, Ownership Interest Less Than 5 Percent: LAVA Therapeutics. MGF: Employee: Regeneron. GVG: Employee: Merck. Publicly Traded Stocks: Immunogen, Aveo, Beta Bionics. KJH: Researcher: AstraZeneca, Boehringer-Ingelheim, Replimune, Consultant/Advisor/Speaker: Arch Oncology, AstraZeneca, BMS, Boehringer-Ingelheim, Codiak, Eisai, Inzen, Merck-Serono, MSD, Oncolys, Pfizer, Replimune. HK: Employee: Ankyra Therapeutics. Consultant/Advisor/Speaker: Castle Biosciences, Marengo Therapeutics. CMK: Researcher: Amgen, Merck & Co., Kartos Pharmaceuticals, EMD Serono, Deciphera Pharmaceuticals, Eily lilly, Blueprint Medicines, Incyte Corporation, Clovis Oncology, BMS, Aadi Bioscience, Pfizer, Iterion Therapeutics, Springworks Therapeutics, Nektar Therapeutics, GSK, Adaptimmune, Medimmune, Bioatla, Oncternal Therapeutics, Daiichi- Sankyo Co. Traycon Pharm, Eisai. Ascentage Pharma, Hutchison Medipharma, Salarius Pharmaceuticals, Loxo Oncology, -BTG Specialty Pharmaceuticals, AStex Pharmaceuticals, Xencor, bayer healthcare, Athenex pharmaceuticals, servier, K- group Beta, Trilliium Pharmaceuticals, Ayala Pharmaceuticals, Ningbo Newbay Technology Development, Rain Therapeutics, Inhibrx, C4 Therapeutics, Foghorn Therapeutics, Theseus Pharmaceuticals, Cogent Therapeutics, Curadev PharmaRegeneron, Consultant/Advisor/Speaker: Chemocentryx, Kartos, Servier, Immunicom, Other: Spouse employed by Daichii Sankyo with stock options. ADK: Employee: Merck. KL: Employee: Marengo Therapeutics. SL: Executive Role: Big Against Cancer (Belgium), Breast Cancer Trials (Australia), International Breast Cancer Study Group (Switzerland) Researcher: Research funding to institution from Novartis, Bristol Meyers Squibb, Merck, Puma Biotechnology, Eli Lilly, Nektar Therapeutics, Astra Zeneca, Roche-Genentech and Seattle Genetics, Consultant/Advisor/Speaker: Consultant (not compensated) to Seattle Genetics, Novartis, Bristol Meyers Squibb, Merck, AstraZeneca, Eli Lilly, Pfizer, Gilead Therapeutics and Roche-Genentech. Consultant (paid to institution) to Aduro Biotech, Novartis, GlaxoSmithKline, Roche-Genentech, Astra Zeneca, Silverback Therapeutics, G1 Therapeutics, PUMA Biotechnologies, Pfizer, Gilead Therapeutics, Seattle Genetics, Daiichi Sankyo, Merck, Amunix, Tallac Therapeutics, Eli Lilly and Bristol Meyers Squibb, Role: Presenter (Speaker). GVL: Consultant/Advisor/Speaker: Consultant Advisor for Agenus, Amgen, Array Biopharma, AstraZeneca UK, Boehringer Ingelheim International, Bristol Myers Squibb, Evaxion Biotech A/S, Hexal AG (Sandoz Company), Highlight Therapeutics S.L., Innovent Biologics USA, Merck Sharpe & Dohme (Australia), Merck Sharpe & Dohme, Novartis Pharma AG, PHMR, Pierre Fabre, Provectus Australia, Qbiotics Group Limited, Regeneron Pharmaceuticals. IM: Consulting Fees: Bristol-Myers Squibb, F-STAR, Alligator, Pharma Mar, AstraZeneca, Numab Therapeutics, Roche, Amunix, Gossamer, Molecular Partners, Merck-Serono, Genmab, PharmaMar, Contracted Research: Roche, Bristol-Myers Squibb, Highlight Therapeutics, Alligator, Genmab, Astrazeneca. MRM: Researcher : Roche, Astrazeneca, Novartis, Immunocore, BMS, Pfizer, Merck/MSD, Regeneron, BiolineRx, Replimune, GRAIL, Alkermes, iOx, Vaccitech, Consultant/Advisor/Speaker: Infinitopes. BN: Researcher: As a principal investigator for clinical trials sponsored by UZ Brussel, my institution received support from Novartis, Pfizer, Bayer, BMS, MSD, Pierre-Fabre, Amgen, Consultant/Advisor/Speaker: Novartis, BMS, MSD, Pfizer, Pierre-Fabre, Amgen. DJP: Consulting Fees: ViiV Healthcare, Bayer, Hoffman La Roche, EISAI, H3B, MiNa Alpha Therapeutics, DaVolterra, Fees for Non CE Services: Hoffmann La Roche, EISAI, Contracted Research: Merck Sharpe and Dohme, Bristol Myers Squibb (to institution). RAS: Researcher: Boston ScientificConsultant/Advisor/Speaker: Cook Medical, TriSalus, Replimune, Medtronic. SBS: Owner: Aperture Medical Technology, Executive Role: Aperture Medical Technology, Researcher: GE Healthcare, Johnson & Johnson, Elesta, Consultant/Advisor/Speaker: GE Healthcare, XACT Robotics, Microbot, Candel Therapeutics, Varian, Merck & Co., Royalty and Patent Beneficiary: Aperture Medical Technology, Publicly Traded Stocks: Johnson & Johnson, Poseidx Therapeutics, Motus GI, Sientra, Avadel, Lantheus. PS: Employee: Johnson & Johnson, Publicly Traded Stocks: Johnson & Johnson. SITC staff members SMW, CG, AK, NL, EG and KJ have no disclosures., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Since the first approval for immune checkpoint inhibitors (ICIs) for the treatment of cutaneous melanoma more than a decade ago, immunotherapy has completely transformed the treatment landscape of this chemotherapy-resistant disease. Combination regimens including ICIs directed against programmed cell death protein 1 (PD-1) with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) agents or, more recently, anti-lymphocyte-activation gene 3 (LAG-3) agents, have gained regulatory approvals for the treatment of metastatic cutaneous melanoma, with long-term follow-up data suggesting the possibility of cure for some patients with advanced disease. In the resectable setting, adjuvant ICIs prolong recurrence-free survival, and neoadjuvant strategies are an active area of investigation. Other immunotherapy strategies, such as oncolytic virotherapy for injectable cutaneous melanoma and bispecific T-cell engager therapy for HLA-A*02:01 genotype-positive uveal melanoma, are also available to patients. Despite the remarkable efficacy of these regimens for many patients with cutaneous melanoma, traditional immunotherapy biomarkers (ie, programmed death-ligand 1 expression, tumor mutational burden, T-cell infiltrate and/or microsatellite stability) have failed to reliably predict response. Furthermore, ICIs are associated with unique toxicity profiles, particularly for the highly active combination of anti-PD-1 plus anti-CTLA-4 agents. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to develop this clinical practice guideline on immunotherapy for the treatment of melanoma, including rare subtypes of the disease (eg, uveal, mucosal), with the goal of improving patient care by providing guidance to the oncology community. Drawing from published data and clinical experience, the Expert Panel developed evidence- and consensus-based recommendations for healthcare professionals using immunotherapy to treat melanoma, with topics including therapy selection in the advanced and perioperative settings, intratumoral immunotherapy, when to use immunotherapy for patients with BRAF V600-mutated disease, management of patients with brain metastases, evaluation of treatment response, special patient populations, patient education, quality of life, and survivorship, among others., Competing Interests: Competing interests: CA—Other: Stock Pfizer, Advisory Board: Iovance and Merck, Memorial Sloan Kettering Cancer Center. EIB—Consulting Fees: Nektar, Instilbio, Novartis, Xilio, Sanofi, Merck, and Iovance. DD—IP Rights: US Patent 63/124,231, Compositions and Methods for Treating Cancer, December 11, 2020, US Patent 63/208,719, Compositions and Methods For Determining Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer, June 9, 2021; Consulting Fees: Checkmate Pharmaceuticals, Finch, Shionogi, Vedanta Biosciences; Fees for Non CE Services: Medical Learning Group (MLG), Clinical Care Options (CCO); Contracted Research: Arcus, Checkmate Pharmaceuticals, CellSight Technologies, Immunocore, Merck, Tesaro/GSK. GTG—Consulting Fees: Bristol Myers Squibb, Regeneron, Genentech, Novartis, Merck, Sapience Therapeutics, Exicure, Eisai, Iovance Biotherapeutics, Lyell Immunopharma, and HUYABIO International; Contracted Research: Exelixis (institutional support), Lucerno Dynamics. OH—Researcher: Arcus, Aduro, Akeso, Amgen, Bioatla, BMS, Cytomx, Exelixis, Roche Genentech, GSK, Immunocore, Idera, Incyte, Iovance, Merck, Moderna, Merck Serono, Nextcure, Novartis, Pfizer, Regeneron, Seattle Gen, Torque, Zelluna; Consultant Advisor Speaker: Alkermes, Amgen, Bactonix, Beigene, Bioatla, BMS, Esai, Roche Genentech, Georiamune, GigaGen, Grit Bio, GSK, Idera, Immunocore, Incyte, Instilbio, IO Biotech, Iovance, Janssen, KSQ, Merck, Moderna, Novartis, Obsidian, Pfizer, Regeneron, Sanofi, Seattle Gen, Tempus, Vial Health Tech, Zelluna; Independent Contractor: Alkermes, Amgen, Bactonix, Beigene, Bioatla, BMS, Esai, Roche Genentech, Georiamune, GigaGen, Grit Bio, GSK, Idera, Immunocore, Incyte, Instilbio, IO Biotech, Iovance, Janssen, KSQ, Merck, Moderna, Novartis, Obsidian, Pfizer, Regeneron, Sanofi, Seattle Gen, Tempus, Vial Health Tech, Zelluna; Publically Traded Stocks: Bactonix. TJH—Contracted Research: Genentech, SkylineDX. BI—Consulting Fees: Johnson & Johnson, Volastra Therapeutics, Merck, AstraZeneca, Eisai and Janssen Pharmaceuticals; research funding to Columbia University from Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, and Synthekine. DBJ—IP Rights: MHC-II for use as immunotherapy biomarker, Abatacept as treatment for irAEs; Consulting Fees: BMS, Catalyst, Iovance, Jansen, Mallinckrodt, Merck, Mosaic ImmunoIngineering, Novartis, Oncosec, Pfizer, Targovax; Contracted Research: BMS, Incyte. RPK—Consulting Fees: Regeneron. JJL—Researcher: AbbVie, Astellas, AstraZeneca, Bristol Myers Squibb, Corvus, Day One, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Merck, Moderna, Nektar, Next Cure, Numab, Palleon, Pfizer, Replimmune, Rubius, Servier, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, Xencor; Consultant Advisor Speaker: AbbVie, Agenus, Alnylam, Atomwise, Bayer, Bristol Myers Squibb, Castle, Checkmate, Codiak, Crown, Cugene, Curadev, Day One, Eisai, EMD Serono, Endeavor, Flame, G1 Therapeutics, Genentech, Gilead, Glenmark, HotSpot, Kadmon, KSQ, Janssen, Ikena, Inzen, Immatics, Immunocore, Incyte, Instil, IO Biotech, Macrogenics, Merck, Mersana, Nektar, Novartis, Partner, Pfizer, Pioneering Medicines, PsiOxus, Regeneron, Replimmune, Ribon, Roivant, Servier, STINGthera, Synlogic, Synthekine 7 Hills, Affivant, Bright Peak, Exo, Fstar, Inzen, RefleXion, Xilio (stock) Actym, Alphamab Oncology, Arch Oncology, Duke Street Bio, Kanaph, Mavu, NeoTx, Onc.AI, OncoNano, physIQ, Pyxis, Saros, STipe, Tempest, AbbVie, Agenus, Amgen, Immutep, Evaxion. TCM—Consulting Fees: Merck, BMS, GigaGen, OncoSec; Pfizer – Scientific Advisory Board. MJM—Consulting Fees: AstraZeneca, Nektar Therapeutics, Istari Oncology, Immunai, Xilio Therapeutics; Fees for Non CE Services: Bristol Myers Squibb (served as a speaker). ACP—Consulting Fees: Regeneron, BMS, Merck; Fees for Non CE Services: BMS; Contracted Research: Merck, BMS, Iovance, Regeneron, Takeda, Ideaya, Replimune: All payments to institution. KMR—Consulting Fees: BMS, Merck, Eisai, Immunocore. AKSS—Consulting Fees: Novartis, Pfizer, Regeneron, Iovance. Contracted Research: Ascentage, Ideaya, Regeneron, Bristol Myers Squibb, Immunocore, Merck, Nektar, Olatec, Replimune, Seagen. KS—Contracted Research: Trials at Norris Cotton Cancer Center, Syndax, Provectus, OmniSeq, BrightPath Biotherapeutics, Ludwig Institute of Cancer Research, Hlsinn, AbbVie, BMS, OncoSec, Ultimovacs, Altellas, AstraZeneca, Xencor, Exicure, Numab, Checkmate Pharmaceuticals, Natera. RJS—Reseracher: Merck (research funding); Consultant Advisor Speaker: BMS, Merck, Novartis, Pfizer; Other: Faculty for SCION Workshop. JT—Consulting Fees: GSK, Genentech, BMS, Akoya Biosciences, Merck, AstraZeneca, Compugen, Lunaphore; Contracted Research: BMS, Akoya Biosciences; Other: Akoya Biosciences; Other Details: Patent pending for multiplexing imaging strategy; Ownership Interest Less Than 5 Per Cent: Akoya Biosciences. HAT—Consulting Fees: Genentech, BMS, Novartis, Merck, Eisai, Karyopharm, Boxer Capital, Iovance, Jazz Pharmaceuticals, Medicenna; Contracted Research: Genentech, BMS, Novartis, Merck, GSK, EMD Serono, Eisai, Dragonfly Therapeutics, RAPT Therapeutics. JKT—Nothing to Disclose. CV—Nothing to Disclose. SAW—Consulting Fees: Lyell; Contracted Research: research funds to institution from BMS and Apexigen. MKW—Consulting Fees: Merck, Pfizer, Bristol Myers Squibb, Regeneron, EMD-Serono, ExiCure, Castle Biosciences, Adagene. SITC Staff—CG, AK, NL, SM-W—Nothing to Disclose., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Acral melanoma (AM) has distinct characteristics as compared with cutaneous melanoma and exhibits poor response to immune checkpoint inhibitors (ICIs). Tumor-intrinsic mechanisms of immune exclusion have been identified in many cancers but less studied in AM. We characterized clinically annotated tumors from patients diagnosed with AM at our institution in correlation with ICI response using whole transcriptome RNAseq, whole exome sequencing, CD8 immunohistochemistry, and multispectral immunofluorescence imaging. A defined interferon-γ-associated T cell-inflamed gene signature was used to categorize tumors into non-T cell-inflamed and T cell-inflamed phenotypes. In combination with AM tumors from two published studies, we systematically assessed the immune landscape of AM and detected differential gene expression and pathway activation in a non-T cell-inflamed tumor microenvironment (TME). Two single-cell(sc) RNAseq AM cohorts and 11 bulk RNAseq cohorts of various tumor types were used for independent validation on pathways associated with lack of ICI response. In total, 892 specimens were included in this study. 72.5% of AM tumors showed low expression of the T cell-inflamed gene signature, with 23.9% of total tumors categorized as the non-T cell-inflamed phenotype. Patients of low CD3 + CD8 + PD1 + intratumoral T cell density showed poor prognosis. We identified 11 oncogenic pathways significantly upregulated in non-T cell-inflamed relative to T cell-inflamed TME shared across all three acral cohorts (MYC, HGF, MITF, VEGF, EGFR, SP1, ERBB2, TFEB, SREBF1, SOX2, and CCND1). scRNAseq analysis revealed that tumor cell-expressing pathway scores were significantly higher in low versus high T cell-infiltrated AM tumors. We further demonstrated that the 11 pathways were enriched in ICI non-responders compared with responders across cancers, including AM, cutaneous melanoma, triple-negative breast cancer, and non-small cell lung cancer. Pathway activation was associated with low expression of interferon stimulated genes, suggesting suppression of antigen presentation. Across the 11 pathways, fatty acid synthase and CXCL8 were unifying downstream target molecules suggesting potential nodes for therapeutic intervention. A unique set of pathways is associated with immune exclusion and ICI resistance in AM. These data may inform immunotherapy combinations for immediate clinical translation., Competing Interests: Competing interests: RB declares PCT/US15/612657 (Cancer Immunotherapy), PCT/US18/36052 (Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic, Prognostic and Therapeutic Uses Thereof), PCT/US63/055227 (Methods and Compositions for Treating Autoimmune and Allergic Disorders); JJL declares DSMB: Abbvie, Immutep; Scientific Advisory Board: (no stock) 7 Hills, Fstar, Inzen, RefleXion, Xilio (stock) Actym, Alphamab Oncology, Arch Oncology, Kanaph, Mavu, Onc.AI, Pyxis, Tempest; Consultancy with compensation: Abbvie, Alnylam, Avillion, Bayer, Bristol-Myers Squibb, Checkmate, Codiak, Crown, Day One, Eisai, EMD Serono, Flame, Genentech, Gilead, HotSpot, Kadmon, KSQ, Janssen, Ikena, Immunocore, Incyte, Macrogenics, Merck, Mersana, Nektar, Novartis, Pfizer, Regeneron, Ribon, Rubius, Silicon, Synlogic, Synthekine, TRex, Werewolf, Xencor; Research Support: (all to institution for clinical trials unless noted) AbbVie, Agios (IIT), Astellas, Astrazeneca, Bristol-Myers Squibb (IIT & industry), Corvus, Day One, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Merck, Moderna, Nektar, Next Cure, Numab, Pfizer (IIT & industry) Replimmune, Rubius, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, Xencor; Patents: (both provisional) Serial #15/612,657 (Cancer Immunotherapy), PCT/US18/36052 (Microbiome Biomarkers for Anti-PD-1/PD-L1 Responsiveness: Diagnostic, Prognostic and Therapeutic Uses Thereof). PL declares equity interest in Amgen. DD declares grants/research support (NIH/NCI and Checkmate Pharmaceuticals) and consulting (Checkmate Pharmaceuticals) during the conduct of the study. DD also reports grants/research support (Arcus, CellSight Technologies, Immunocore, Merck Sharp & Dohme, Tesaro/GSK), consulting (Clinical Care Options (CCO), Finch Therapeutics, Gerson Lehrman Group (GLG), Medical Learning Group (MLG), Xilio Therapeutics), speakers' bureau (Castle Biosciences) and pending provisional patents related to gut microbial signatures of response and toxicity to immune checkpoint blockade (US Patent 63/124,231 and US Patent 63/208,719) outside the submitted work. JMK declares grants/research support (Bristol-Myers Squibb, Amgen) and consulting (Bristol-Myers Squibb, Checkmate Pharmaceuticals, Novartis, Amgen, Checkmate, Castle Biosciences, Immunocore, Iovance, Novartis.) outside the submitted work. HMZ declares grants/research support (NIH/NCI and Checkmate Pharmaceuticals) and consulting (Checkmate Pharmaceuticals) during the conduct of the study, grants/research support (NIH/NCI, Bristol-Myers Squibb and GlaxoSmithKline), personal fees (GlaxoSmithKline and Vedanta) and pending provisional patents related to gut microbial signatures of response and toxicity to immune checkpoint blockade (US Patent 63/124,231 and US Patent 63/208,719) outside the submitted work. Correspondence and requests for materials should be addressed to JJL (lukejj@upmc.edu) and RB (baor@upmc.edu). The remaining authors declare no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: PD-1 and CTLA-4 inhibitors are associated with several adverse events including a spectrum of immune-related adverse effects (irAEs). Neurologic irAEs are uncommon occurrences with varied presentations. We describe two separate cases of ipilimumab associated meningoencephalomyelitis and demyelinating polyneuropathy with unusual presentations., Case Presentation: Two melanoma patients were treated with ipilimumab in the adjuvant setting. The first patient developed a meningoencephalitis following 3 doses of ipilimumab. MRI imaging of the brain confirmed leptomeningeal enhancement although cerebrospinal fluid (CSF) analyses were negative for malignant cells consistent with meningoencephalomyelitis. Although she initially improved following treatment with steroids and intravenous immunoglobulin, she subsequently relapsed. She was successfully treated with infliximab and made a complete neurological recovery. A second patient developed progressive lower extremity weakness following two doses of ipilimumab. MRI imaging of the spine confirmed diffuse nerve root enhancement consistent with acute inflammatory demyelinating polyneuropathy (AIDP). He was treated with high dose steroids with resolution of neurological symptoms. Both patients remain disease free., Conclusions: Neurological irAEs are uncommon adverse events in the context of CTLA-4 and/or PD-1 inhibitor therapy. Care must be taken to distinguish these from leptomeningeal disease. Early recognition of neurological irAEs is critical for the initiation of specific anti-inflammatory agents to prevent and potentially reverse neurological sequelae.

Background: Durable remissions are observed in a fraction of metastatic melanoma patients treated with high-dose interleukin-2 (HD IL-2). Early studies reported overall (OR) and complete response (CR) rates of 16% and 8% respectively. Toxicity limited use to specialized centers with standardized protocols. We report on 243 patients treated at the University of Pittsburgh in a non-intensive care unit (ICU) oncology specialty setting., Methods: Clinical and radiological data were collected on 243 patients treated between 1992 and 2015. Each HD IL-2 cycle was given over 5 days, cycles were repeated after 9 days and courses (2 cycles) were repeated every 6-9 weeks in patients with stable or responding disease, for up to 3 courses total. Influence of baseline characteristics on outcomes was assessed using Kaplan-Meier estimates and Cox proportional hazards analysis., Results: Two hundred forty-three patients received 692 cycles (5270 doses) between 1992 and 2015. Two hundred thirty-seven patients were evaluable for response: OR rate 18.1% with CR rate 8.0%. Median overall survival (OS) 9.6 months in the entire cohort but 64.9 months in responders. Median number of cycles delivered was 2,and median number of doses per cycle was 8. Toxicity was consistent with prior reports. HD IL-2 required ICU transfers in 11 patients and 1 death was attributed to HD IL-2. Pre-treatment lactate dehydrogenase (LDH) levels correlated significantly with progression-free survival [1-2× upper limit normal (ULN) HR 1.95; >2× ULN HR 2.32] and overall survival (1-2× ULN HR 1.67; >2× ULN 2.49). Response to HD IL-2 and site of metastatic disease also correlated significantly with progression-free and overall survival., Conclusions: In this large series of patients spanning more than two decades, OR/CR rates with HD IL-2 were 18.1%/8.0% respectively. Toxicity data was consistent with prior reports. Pre-treatment LDH values and site(s) of metastatic disease may be useful markers to select patients at greater likelihood of benefit to HD IL-2 therapy.