Metastatic uveal melanoma (mUM) is a rare, aggressive malignancy with limited therapeutic options in the metastatic setting. Tebentafusp (IMCgp100) is an immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC), targeting gp100 in HLA-A*0201+ patients. Tebentafusp has demonstrated an overall survival (OS) benefit in clinical trials, usually without marked radiographic tumor regression. We describe a mUM patient who was treated with tebentafusp for more than two years and achieved durable stable disease on imaging but passed away due to sudden cardiac death. Autopsy examination revealed extensive tumor necrosis and dense immune cell infiltration within metastatic liver lesions with minimally viable tumor, providing direct evidence of robust immune activation in the tumor microenvironment. This case highlights the potential for T cell bispecifics to induce profound anti-tumor effects that are not fully captured by conventional radiographic assessment, underscoring the need for integrated clinical, pathologic, and imaging evaluation in this setting.

BACKGROUND:Cancers that do not respond to immunotherapy typically harbor a non-T cell-inflamed tumor microenvironment (TME), characterized by the absence of type I/II interferon signaling and CD8+ T cell infiltration. We previously reported IDH1 somatic mutations were enriched in this phenotype across histologies. Mutant IDH1 (mIDH1) drives immune exclusion via metabolic reprogramming of the TME, and preclinical models show that IDH inhibition can restore anti-tumor immunity. We conducted a Phase II study assessing the preliminary activity of ivosidenib, an IDH1 inhibitor, plus nivolumab, an anti-PD1 antibody, in patients with mIDH1 advanced solid tumors (NCT04056910). METHODS:Patients with advanced or refractory mIDH1 solid tumors and no prior exposure to IDH1 inhibitor were administered ivosidenib 500 mg by mouth daily with nivolumab 480 mg intravenously every 4 weeks. A composite primary endpoint included either six-month progression-free survival (PFS6) or overall response rate (ORR). Translational analyses incorporated pharmacodynamics, proteomics, and spatial transcriptomics. RESULTS:Fifteen patients were enrolled (median age, 54 years; female, 53.3%; ECOG 1, 60%; glioma, 46.7%; R132H, 40%). Three patients (20%) met the primary endpoint. Median PFS was 1.94 months. The most common adverse events were leukopenia (67%), rash (67%), and diarrhea (33%). Treatment reduced plasma (R)-2HG, with greater reductions observed in patients who experienced clinical benefit. Exploratory serum proteomic and tumor spatial transcriptomic analyses suggested treatment-induced immune-modulatory effects. CONCLUSIONS:Ivosidenib plus nivolumab was safe with limited clinical benefit. Translational investigation highlights potential of IDH1 inhibition to alter tumor-immune interactions and provides hypotheses for future immune-checkpoint combinations.

Simultaneous blockade of lymphocyte activation gene-3 (LAG-3) and programmed-death-1 (PD-1) pathways enhance anti-tumor activity in patients with melanoma. It is not known if this immunotherapy induces unique immune modulation or how this may relate to clinical outcomes, compared to the single modalities. We conducted a randomized three-arm phase 2 trial (NCT03743766) in advanced melanoma comparing lead-in treatment with one cycle of relatlimab- (n=14), nivolumab-(n=15), and nivolumab-relatlimab (n=14) as first-line therapy, followed by combination nivolumab-relatlimab, to assess the impact of the lead-in therapy on immune populations, related to objective response rate, progression-free survival (PFS) and major pathologic response on biopsy (MPRbx). Unexpectedly, diminished efficacy was observed when nivolumab or relatlimab was given as a lead-in monotherapy, despite receipt of the combination subsequent to week 4. MPRbx at week 4 was significantly higher with nivolumab- and combination vs. relatlimab- lead-in, mechanistically correlated with higher IFN-g and TCR signaling in CD8+ T cells, and was associated with superior PFS. Relatlimab lead-in was characterized by clustering of CD8+ T and FOXP3+ T regulatory cells in the tumor microenvironment, enrichment in inhibitory receptor pathways and associated with worse PFS. Combination therapy increased the fraction of CD8+TEM cells in association with response to therapy while patients with progressive disease demonstrated decrease in CD14+CD16-HLA-DRlowCD33dim monocytic populations. The analyses of component agents afforded by this novel lead-in trial has identified differential clinical and immunological modulation of anti-PD1 and anti-LAG3 in comparison to the combination therapy.

TPS9607 Background: The majority of 100,000 annual new U.S. cases of mel consist of localized early-stage disease that undergo wide local excision (WLE) +/- SLN biopsy (SLNB). The tumor draining lymph node is the initial site of immune response including formation of tumor mediated immune suppression and pre-metastatic niches. SLN positivity is a key prognostic factor in early stage mel. Thus, the SLN is a target for local immune intervention to boost the antitumor response. Vusolimogene oderparepvec (RP1) is an intratumorally administered oncolytic immunotherapy with unique potential for neo-adjuvant therapeutic application. RP1 is constructed from a high potency HSV1 strain (RH018A) modified to replicate selectively in tumors (deletion of neurovirulence factors ICP34.5 and ICP47). RP1 encodes GM-CSF and a fusogenic GALV-GP R- protein to maximize oncolytic potency and induce immunogenic cell death. Preclinical and clinical data demonstrate robust antitumor efficacy (including non-injected lesions) of RP1 alone and in combination with checkpoint inhibitors in advanced mel. This trial addresses a crucial gap in understanding the impact of RP1 on SLN dynamics and preventing disease recurrence in high-risk patients (pts). We hypothesize that in pts with high risk, clinically node negative mel (pT3b-T4b), RP1 will reduce rates of SLN positivity as compared to a historic control by favorably reshaping the immune landscape of the primary tumor, SLN, and the peripheral blood. Methods: This is an investigator-initiated, single arm phase 1/2 trial (NCT06216938) designed to assess efficacy and safety of neo-adjuvant RP1 in high-risk, clinically node-negative, non-uveal mel. Eligibility criteria: pT3b, T4a, or T4b non-uveal mel with visible residual tumor or positive biopsy margins, ECOG ≤1, and no prior oncolytic virus therapy. Pts receive 3 doses of neo-adjuvant RP1 (10e6 PFU day 1, 10e7 PFU on days 15 and 21), injected at the primary tumor site followed by standard WLE and SLNB within 35 days of dose 1. Biopsy of residual tumor or archival tumor tissue is obtained pre-RP1 and archival tissue from WLE and SLNB is obtained post-RP1. Blood samples are obtained with each RP1 dose and 3 months post-therapy. Pts are followed for 3 years. Primary endpoint: rate of SLN positivity in the overall cohort. Secondary endpoints: treatment related adverse events (per CTCAE), recurrence free survival, and overall survival. Exploratory endpoints: immunophenotype and microenvironment of the primary tumor, SLN and peripheral blood pre- and post- RP1 (via IHC, IF, and flow cytometry). The observed rate of SLN positivity will be compared to the predicted rate (Melanoma Institute of Australia Prediction Tool for SLN metastatic risk) with a one-sided, one-sample proportion test. Kaplan-Meier estimates will be provided for survival endpoints. The trial is active with 13 of 25 pts enrolled in January 2025. Clinical trial information: NCT06216938 .

Intratumoral TLR9 agonists and anti-PD-1 produce clinical responses and broad immune activation. We conducted a single-arm study of neoadjuvant TLR9 agonist vidutolimod combined with anti-PD-1 nivolumab in high-risk resectable melanoma. In 31 evaluable patients, 55% major pathologic response (MPR) was observed, meeting primary endpoint. MPR was associated with necrosis, and melanophagocytosis with increased CD8+ tumor-infiltrating lymphocytes and plasmacytoid dendritic cells (pDCs) in the tumor microenvironment, and increased frequencies of Ki67+CD8+ T cells peripherally. MPRs had an enriched pre-treatment gene signature of myeloid cells, and response to therapy was associated with gene signatures of immune cells, pDCs, phagocytosis, and macrophage activation. MPRs gut microbiota were enriched for Gram-negative bacteria belonging to the Bacteroidaceae and Enterobacteriaceae families and the small subgroup of Gram-negative Firmicutes. Our findings support that combined vidutolimod and nivolumab stimulates a broad anti-tumor immune response and is associated with distinct baseline myeloid gene signature and gut microbiota. ClinicalTrials.gov identifier: NCT03618641.

9525 Background: A phase II study of nivo and rela was designed to evaluate the separate antitumor activity of nivo and rela vs. the combination for first-line treatment of pts with advanced mel. We report objective response rate (ORR) at week (wk) 4 and 16, progression-free survival (PFS) for pts receiving lead-in with nivo or rela vs. combination, and correlations with immune-related pathological response (irPR) at wk 4 tumor biopsy. Methods: Pts were randomized (1:1:1) to lead-in treatment with 1 cycle of nivo (480mg IV q4wk), rela (160mg IV q4wk), or nivo-rela followed by combination therapy in all pts. The primary endpoint was ORR to nivo-rela by RECISTv.1.1 at wk 16. Secondary endpoints included progression-free survival (PFS), ORR according to lead-in therapy at wk4, safety, and major pathological response on biopsy at 4wk (MPRbx) per Stein et al, Ann Oncol (2019). Results: The trial enrolled 43 advanced mel pts, median age=67 years, female=15 (35%), ECOG PS 0=34 (79%), BRAFmutant=18 (42%), Stage IV=35 (81%), and LDH >ULN=37 (86%). Pts were randomized to nivo=15, rela=14, and nivo-rela=14 lead-in arms. ORR at wk 4 were 3 (20%), 1 (7.1%), and 0 (0%) in nivo, rela, and nivo-rela lead-in arms, respectively. Among 41 evaluable pts who received at least one cycle of nivo-rela radiological assessment at wk16 was partial response (PR)=14 (34.2%), stable disease (SD)=15 (36.6%), and progressive disease (PD)=12 (29.3%). ORR at wk 16 were 6 (42.9%), 2 (15.4%), and 6 (42.9%) for nivo, rela, and nivo-rela lead-in arms, respectively. Median PFS for the whole cohort was 6.5 mos (95%CI 2.2-not reached [NR]), 4.7 mos, 1.8 mos, and NR in nivo, rela, and nivo-rela lead-in arms, respectively. After adjusting for BRAF status, rela lead-in was significantly associated with worse PFS (HR=3.41, 95% CI 1.11-10.4, p=0.03). Grade ≥3 treatment-related adverse events (TRAEs) were observed in 14 (32.6%) pts. & TRAEs (any grade) leading to treatment discontinuation were observed in 9 (20.9%). After 1 cycle of nivo one pt developed myocarditis leading to death without proceeding to nivo-rela. MPRbx at wk 4 was observed in 11 (31.4%) of 35 evaluable pts and were 50%, 0%, and 43% in nivo, rela, and nivo-rela lead-in arms, respectively. MPRbx was associated with wk16 radiological response (p=0.04) and improved PFS (HR=0.29, 95% CI 0.10-0.87, p=0.03). Nivo-rela resulted in increased CD8 and CD4+FOXP3- cell densities at wk4 (p<0.01 and p=0.03, respectively) and CD8 density at wk 4 was associated with improved PFS (p=0.02). Conclusions: Nivo-rela results in 34.2% ORR and median PFS of 6.5m in pts with advanced mel after lead-in nivo, rela or combination therapy. This first single agent lead-in rela evaluation demonstrated wk 4 ORR of 7%, wk 16 ORR of 15.4% and median PFS 1.8 mos. MPRBx and CD8 density at wk4 are associated with improved PFS. Clinical trial information: NCT03743766 .

Supplementary Methods from Integrative Genomics Identifies Molecular Alterations that Challenge the Linear Model of Melanoma Progression

Skin sulforaphane concentration post-BSE-SFN administration by dose group and day

The gut microbiome acts as a tumor-extrinsic regulator of responses to immune-checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4 receptors. Primary resistance to anti-PD-1 ICI can be reversed via responder-derived fecal microbiota transplant (FMT) in patients with refractory melanoma. Efforts to create stool banks for FMT have proved difficult. Therefore, we aimed to establish a novel donor-screening program to generate responder-derived FMT for use in PD-1 refractory melanoma. Candidate PD-1 responder donors and PD-1 refractory recipients were recruited via clinic-based encounters at the University of Pittsburgh Medical Center hospitals. Eligible donors and recipients underwent physician assessment and screening of serum, stool and nasopharynx for transmissible agents, which included SARS-CoV-2 modification. The cost of donor and recipient screening was calculated. Initially, 29 donors were screened with 14 eligible donors identified after exclusion; of the 14 donors, eight were utilized in clinical trials. The overall efficiency of screening was 48%. Seroprevalence rates for cytomegalovirus, Epstein-Barr virus, HSV-2, HHV-6, HTLV-1, HTLV-2, and syphilis were similar to published statistics from healthy blood donors in the USA. Donor stool studies indicated a 3.6% incidence of E. histolytica and norovirus, 3.7% incidence of giardia and 7.1% incidence of C. difficile. A single donor tested positive for SARS-CoV-2 in stool only. The cost for finding a single eligible donor was $2260.24 (pre-COVID) and $2,460.24 (post-COVID). The observed screening efficiency suggests that a well-resourced screening program can generate sufficient responder-derived donor material for clinical trial purposes. Eliminating testing for low-prevalence organisms may improve cost-effectiveness.

Plasma sulforaphane concentrations by day, dose, and pre- or post-BSE-SFN administration

Representative gating strategy illustrating (A) CD4 and CD8 T cell identification (B) expression of ICOS on CD4 positive T cells (C) expression of PD-1 and Ki-67 within PD-1 positive cells and (D) expression of CD38/HLA-DR, PD-1 and KI-67 within CD8+ PD-1+ T cells is shown.

Treatment significantly affects the circulating immune compartment.

9540 Background: The majority of unresectable melanoma patients eventually progress on first-line checkpoint blockade therapy. Duvelisib is a potent phosphoinositide 3-kinase (PI3K) δ and γ isoform inhibitor approved for relapsed/refractory CLL. PI3Kγ inhibition has been shown to restore anti-PD1 activity in preclinical tumor models through conversion of tumor associated macrophages (TAMs) from an immunosuppressive to a pro-inflammatory state. PI3Kδ inactivation has been associated with impaired T regulatory cell (Treg) function and reduced intratumoral Treg numbers. We report study design and results of a phase I study to evaluate the safety and efficacy of duvelisib and nivolumab in anti-PD1 refractory patients. Methods: Utilizing a modified 3+3 design with escalating doses of duvelisib (15 mg daily, 25 mg daily, and 25 mg BID), patients with unresectable melanoma were treated with nivolumab and duvelisib until unacceptable toxicity or disease progression. Primary endpoints for the phase I portion were maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of duvelisib. Secondary endpoints include early and late toxicities and anti-tumor activity based on RECIST v1.1. Exploratory endpoints were to evaluate dose-dependent changes in the immune cell populations of peripheral blood and tumor tissue with duvelisib and nivolumab. Results: Ten patients have been enrolled (7 at dose level I, 3 at dose level II) with mean age 64. Two patients had BRAF V600E mutation and were previously treated with BRAF/MEK inhibitors. No dose limiting toxicities (DLT) were observed. Grade 3 or higher adverse events (AEs) attributable to study treatment occurred in 4 patients (40%), including G3 neutropenia (10%), G3 diarrhea (10%), G3 vomiting (10%) and G4 hepatitis (20%). Both instances of hepatitis resolved rapidly with 1-2mg/kg of prednisone. In one patient, hepatitis and nausea/vomiting led to discontinuation of treatment. Out of 6 patients who had follow-up imaging, 4 exhibited progressive disease, one stable disease, and one had partial response of 11 months duration. Given the published data on the regulation of macrophages and Tregs by PI3Kδγ inhibitors, we are utilizing multispectral imaging to measure the spatial interactions of key immune cells within the tumor microenvironment (T cells, B cells, and macrophages) in all matched pre-/post-treatment paraffin-embedded tissues, for which results will be presented. Conclusions: The combination of duvelisib and nivolumab was well-tolerated in patients with advanced unresectable melanoma refractory to ICI without DLTs to date at 15 and 25mg daily dosages. Significant anti-tumor activity was noted in one patient. Enrollment at dose level II is ongoing. Clinical trial information: NCT04688658 .

Supplementary Data from Melanoma MicroRNA Signature Predicts Post-Recurrence Survival

An open reading frame (ORF1) located upstream of the urease structural gene ureA in Rhizobium meliloti strain AK631 was cloned and characterized by DNA sequencing. Comparison of the amino acid sequence revealed partial homology with the urease accessory gene ureD of Klebsiella aerogenes and Proteus mirabilis. Mutational analysis of ORF1 showed that the gene is necessary for urease activity. Its function is still unknown.

Background Neoadjuvant PD-1 blockade produces major pathological responses (MPR) in ~30% of patients (pts) with high-risk resectable MEL with durable relapse-free benefit, and increased circulating activated CD8+ T cells.1,2 Vidutolimod (vidu) comprises a CpG-A oligodeoxynucleotide packaged within a virus-like particle (VLP) and is designed to activate tumor-associated plasmacytoid dendritic cells (pDC) via TLR9, inducing an IFN-rich tumor microenvironment and anti-tumor CD8+ T cell responses. Vidu/anti-PD-1 resulted in durable tumor responses in PD-1 refractory MEL.3 This phase II study evaluated the pathological, clinical and immunological activities of neoadjuvant vidu/nivo in high-risk stage III B/C/D resectable MEL. Methods Vidu/nivo was administered over 7 weeks (vidu 10mg IT Q1W x7, nivo 240mg Q2W x3) pre-surgery. Post-surgery, vidu/nivo (vidu SC 5mg, nivo 480mg Q4W) was continued for 48 weeks. Primary endpoints included MPR rate, and incidence of DLT. Secondary endpoints were radiographic response, relapse-free survival (RFS), distant metastasis-free survival (DMFS) and overall survival (OS). Pathological response assessment was performed to evaluate % residual volume of tumor (RVT) per consensus criteria4-6 by 3 blinded dermatopathologists: 0% (pCR); 0%50% (pNR). Radiographic response assessed using RECIST v1.1. Serial blood, tumor and stool were collected for correlative analyses. Results 31 pts were enrolled, of whom 30 evaluable for per-protocol (PP) analyses as 1 pt progressed pre-surgery. No DLTs were observed. 8 Gr3 TRAE, including hypertension (7/8) and colitis (1/8) were observed; no delays in surgery occurred. ORR by BICR was 45% (all) and 47% (PP). In PP population, MPR was observed in 57% (17/30) including 47% pCR (14/30) and 10% pMR (3/30). With median follow-up of 26.5 months, median RFS was not reached (table 1). Post-treatment, MPR was associated with increased CD8+ tumor infiltrating lymphocytes (p<0.0001), and peripheral immune activation and pDC activation by multiparameter flow cytometry (p < 0.001). Spatial investigation of immune cell infiltrates by mIHC revealed significantly immune cell infiltrates (p<0.05) and higher pDC (p=0.053) within tumor (but not stroma) of MPRs post-treatment (figure 1a,b). Deconvoluted RNAseq confirmed these findings compared to a control cohort of PD-1 treated MEL. Conclusions Neoadjuvant vidu/nivo has minimal tox and demonstrated promising activity with 47% pCR rate and 57% MPR rate. MPR was associated with improved 1-/2- year RFS (94%/88%), and 1-/2- year DMFS (94%/94%) and 2-year OS (100%). MPR is associated with pDC and immune infiltrate (figure 1c). Further evaluation of this combination is ongoing in an ongoing randomized phase II trial (EA6194, NCT04708418). Acknowledgements We thank and Checkmate Pharmaceuticals for funding and vidutolimod. This research was supported in part by the University of Pittsburgh Center for Research Computing through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. This research was supported by the Melanoma Research Foundation Breakthrough Consortium (MRFBC) Award (Davar, Stein); NIH R01 CA257265 (Zarour, Davar); and NIH P50 CA254865 (Zarour). Trial Registration Clinical trial information: NCT03618641. References Amaria RN, Reddy SM, Tawbi HA, et al. Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma. Nat Med. 2018 Nov;24(11):1649–1654. Huang AC, Orlowski RJ, Xu X, et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nat Med. 2019 Mar;25(3):454–461. doi: 10.1038/s41591-019-0357-y. Ribas A, Medina T, Kirkwood JM, et al. Overcoming PD-1 Blockade Resistance with CpG-A Toll-Like Receptor 9 Agonist Vidutolimod in Patients with Metastatic Melanoma. Cancer Discov. 2021 Dec 1;11(12):2998–3007. doi: 10.1158/2159-8290.CD-21-0425. Tetzlaff MT, Messina JL, Stein JE, et al. Pathological assessment of resection specimens after neoadjuvant therapy for metastatic melanoma. Ann Oncol. 2018 Aug 1;29(8):1861–1868. Cottrell TR, Thompson ED, Forde PM, et al. Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC). Ann Oncol. 2018 Aug 1;29(8):1853–1860. doi: 10.1093/annonc/mdy218. Stein JE, Soni A, Danilova L, et al. Major pathologic response on biopsy (MPRbx) in patients with advanced melanoma treated with anti-PD-1: evidence for an early, on-therapy biomarker of response. Ann Oncol. 2019 Apr 1;30(4):589–596. doi: 10.1093/annonc/mdz019. Ethics Approval The study was approved by University of Pittsburgh9s Institutional Review Board, approval number MOD19040237-002. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Background Immune checkpoint inhibitors (ICI) directed against PD-(L)1 are associated with improved response rates in melanoma (MEL), and squamous/non-squamous non-small cell lung cancer (NSCLC).1-4 Conversely, anti-PD(L)1 blockade is minimally efficacious in microsatellite stable (MSS) colorectal carcinoma (CRC). Pixatimod (P) is a heparan sulfate (HS) mimetic that is a cholestanol-sulfotetrasaccharide conjugated small molecule compound with unique NK- and T cell- dependent immunomodulatory properties. P does not possess CpG ODN motifs and hence does not activate TLR9 directly. Rather, P increases CpG ODN accumulation in lysosomal compartment of DCs, leading to enhanced production of IL-12 and NK cell activation.5 Preclinically, in combination with anti-PD-1, P led to increased infiltration of both central and effector memory CD4 and CD8 T cells in a IL-12 and TLR9 dependent fashion in multiple tumor modes.5-9 Low dose Cy has immunostimulatory and antiangiogenic properties and has synergy with CpG,10, 11 and PD-1 ICI.12 In MSS mCRC, P/N demonstrated objective response rate (ORR) 12% in a Phase Ib study.13 We hypothesized that P and nivolumab (N) combination may overcome resistance in PD-1 relapsed/refractory (R/R) tumors; and that the addition of Cy may facilitate P+N activity in MSS CRC. Methods This is a nonrandomized, open-label, multichort, phase IIA study (NCT05061017) evaluating several pixatimod combinations in 3 cohorts (figure 1). The recommended phase 2 dose (RP2D) of P is 25mg IV Q1W and N is dosed at 480mg IV Q4W. Immunomodulatory dose of Cy is 50 mg twice daily, 1-week-on, 1-week-off. P+N+low-dose Cy will be evaluated in PD-1 naïve MSS CRC (cohort 1). P+N will be evaluated in PD-1 R/R melanoma (cohort 2) and NSCLC (cohort 3). In 1st stage of each cohort, 9-13 patients will be enrolled. If ≥1 response(s) are seen, 8-14 additional patients will be enrolled in the 2nd stage. The primary endpoint in this Simon two-stage trial is objective response rate (ORR) using RECIST v1.1. Key secondary endpoints include ORR by iRECIST, median and landmark survival (PFS, OS) and safety. Exploratory endpoints will characterize pharmacokinetics, immune contexture, immunophenotypic analyses and gut microbiome pre- and post- treatment blood, tumor and stool samples. Acknowledgements We would like to thank all the participating patients, and their families. We would like to thank the research staff of the Hillman Cancer Center's (HCC) Immunotherapy and Drug Development Center (IDDC). Trial Registration Clinical trial information: NCT05061017. References Borghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373(17):1627–39. Brahmer J, Reckamp KL, Baas P, Crino L, Eberhardt WE, Poddubskaya E, et al. Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. N Engl J Med. 2015;373(2):123–35. Robert C, Long GV, Brady B, Dutriaux C, Maio M, Mortier L, et al. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med. 2015;372(4):320–30. Robert C, Schachter J, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med. 2015;372(26):2521–32. Brennan TV, Lin L, Brandstadter JD, Rendell VR, Dredge K, Huang X, et al. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation. J Clin Invest. 2016;126(1):207–19. Barash U, Lapidot M, Zohar Y, Loomis C, Moreira A, Feld S, et al. Involvement of Heparanase in the Pathogenesis of Mesothelioma: Basic Aspects and Clinical Applications. J Natl Cancer Inst. 2018;110(10):1102–14. Boyango I, Barash U, Naroditsky I, Li JP, Hammond E, Ilan N, et al. Heparanase cooperates with Ras to drive breast and skin tumorigenesis. Cancer Res. 2014;74(16):4504–14. Katz A, Barash U, Boyango I, Feld S, Zohar Y, Hammond E, et al. Patient derived xenografts (PDX) predict an effective heparanase-based therapy for lung cancer. Oncotarget. 2018;9(27):19294-306. Ostapoff KT, Awasthi N, Cenik BK, Hinz S, Dredge K, Schwarz RE, et al. PG545, an angiogenesis and heparanase inhibitor, reduces primary tumor growth and metastasis in experimental pancreatic cancer. Mol Cancer Ther. 2013;12(7):1190-201. Huang XM, Zhang NR, Lin XT, Zhu CY, Zou YF, Wu XJ, et al. Antitumor immunity of low-dose cyclophosphamide: changes in T cells and cytokines TGF-beta and IL-10 in mice with colon-cancer liver metastasis. Gastroenterol Rep (Oxf). 2020;8(1):56-65. Leong WI, Ames RY, Haverkamp JM, Torres L, Kline J, Bans A, et al. Low-dose metronomic cyclophosphamide complements the actions of an intratumoral C-class CpG TLR9 agonist to potentiate innate immunity and drive potent T cell-mediated anti-tumor responses. Oncotarget. 2019;10(68):7220-37. Italiano A, Bessede A, Pulido M, Bompas E, Piperno-Neumann S, Chevreau C, et al. Pembrolizumab in soft-tissue sarcomas with tertiary lymphoid structures: a phase 2 PEMBROSARC trial cohort. Nat Med. 2022;28(6):1199-206. Kuo JC, Bampton D, Lemech C, Brown M, Stanley A, Chojnowski G, et al. Preliminary results from a phase 1b study of pixatimod (PG545) in combination with nivolumab in patients with advanced solid tumors with an expansion cohort in patients with metastatic pancreatic cancer. Ann Oncol. 2018;2018. Ethics Approval The study was approved by University of Pittsburgh's Institutional Review Board, approval number MOD21060203-002. Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Background The p38 mitogen-activated protein kinase (MAPK) pathway limits dendritic cell priming and we have discovered a novel tumor-intrinsic immune-exclusion role for p38 MAPK across multiple tumor types. ARRY-614 (pexmetinib) is a p38 MAPK inhibitor that enhances immune-checkpoint blockade (ICB) in murine models. Here we report on the safety and anti-tumor activity of ARRY-614 with nivolumab (N), ipilimumab (I), or N+I in human subjects with advanced solid tumors. Methods Subjects received daily, oral ARRY-614 (de-escalated from 800 to 200mg) with N (480 mg), I (3 mg/kg), or N+I (1 mg/kg) on either 28- or 21-day cycles. The primary objec-tive was safety and tolerability of ARRY-614 with N, I, or N +I and selection of the recommended phase II dose (RP2D) using a Bayesian dose-finding strategy. Preliminary anti-tumor activity per RECISTv1.1 or irRECIST were secondary objectives.

BACKGROUND:A first-in-human, randomized pilot phase II clinical trial combining vaccines targeting overexpressed, non-mutated tumor blood vessel antigens (TBVA) and tyrosine kinase inhibitor dasatinib was conducted in human leukocyte antigen (HLA)-A2+ patients with advanced melanoma.METHODS:Patient monocyte-derived type-1-polarized dendritic cells were loaded with HLA-A2-presented peptides derived from TBVA (DLK1, EphA2, HBB, NRP1, RGS5, TEM1) and injected intradermally as a vaccine into the upper extremities every other week. Patients were randomized into one of two treatment arms receiving oral dasatinib (70 mg two times per day) beginning in week 5 (Arm A) or in week 1 (Arm B). Trial endpoints included T cell response to vaccine peptides (interferon-γ enzyme-linked immunosorbent spot), objective clinical response (Response Evaluation Criteria in Solid Tumors V.1.1) and exploratory tumor, blood and serum profiling of immune-associated genes/proteins.RESULTS:Sixteen patients with advanced-stage cutaneous (n=10), mucosal (n=1) or uveal (n=5) melanoma were accrued, 15 of whom had previously progressed on programmed cell death protein 1 (PD-1) blockade. Of 13 evaluable patients, 6 patients developed specific peripheral blood T cell responses against ≥3 vaccine-associated peptides, with further evidence of epitope spreading. All six patients with specific CD8+ T cell response to vaccine-targeted antigens exhibited evidence of T cell receptor (TCR) convergence in association with preferred clinical outcomes (four partial response and two stabilization of disease (SD)). Seven patients failed to respond to vaccination (one SD and six progressive disease). Patients in Arm B (immediate dasatinib) outperformed those in Arm A (delayed dasatinib) for immune response rate (IRR; 66.7% vs 28.6%), objective response rate (ORR) (66.7% vs 0%), overall survival (median 15.45 vs 3.47 months; p=0.0086) and progression-free survival (median 7.87 vs 1.97 months; p=0.063). IRR (80% vs 25%) and ORR (60% vs 12.5%) was greater for females versus male patients. Tumors in patients exhibiting response to treatment displayed (1) evidence of innate and adaptive immune-mediated inflammation and TCR convergence at baseline, (2) on-treatment transcriptional changes associated with reduced hypoxia/acidosis/glycolysis, and (3) increased inflammatory immune cell infiltration and tertiary lymphoid structure neogenesis.CONCLUSIONS:Combined vaccination against TBVA plus dasatinib was safe and resulted in coordinating immunologic and/or objective clinical responses in 6/13 (46%) evaluable patients with melanoma, particularly those initiating treatment with both agents.TRIAL REGISTRATION NUMBER:NCT01876212.