Faculty Bibliography

Background mRNA-2752 is a first-in-class mRNA-based therapeutic agent encoding T cell co-stimulator OX40L, and proinflammatory cytokines IL-23 and IL-36g. Preclinical data demonstrated activity in a range of tumor microenvironments (TMEs), including immune checkpoint inhibitor (CPI)- refractory cancer models, and synergy when combined with alpha-PDL1 antibodies. Data from the dose escalation phase of the study was previously reported and the recommended mRNA- 2752 dose for expansion (RDE) was up to 8mg ITu. Methods We evaluated the safety and efficacy of ITu mRNA- 2752 administered as monotherapy (Arm A; previously reported ASCO 2020) and in combination with the PD-L1 inhibitor durvalumab (Arm B) in patients (pts) with tumors that were palpable or accessible with image guidance. Here we report preliminary data for the expansion cohorts in TNBC, CPI-refractory melanoma, and HNSCC. Biomarker analyses included IHC/F-IHC of immune status markers and whole transcriptome assessments of paired tumor biopsies. Protein quantification of IL-23, IL-36g and other pro-inflammatory cytokines were performed in plasma and tumors. Results As of 01JUL 2022, 88 pts were treated, 69 in Arm B with mRNA-2752 dosed by tumor size ranging from 0.25-8 mg. The most common treatment related adverse events occurring in >= 10% of pts in Arm B included grade 1/2 injection site erythema/pain/swelling, fever, chills, fatigue, nausea, and flushing. Grade 3 events included injection site pain/erythema, and fever. There were no grade 4/5 related events. Objective responses were observed in immune refractory tumors by RECIST and iRECIST, including a confirmed PR in a PD-L1 low TNBC and confirmed iCR in an immune checkpoint- refractory melanoma pt, respectively. Biomarker analyses of plasma and tumor show mRNA-2752 treatment was associated with elevated pro-inflammatory cytokines, including IL- 23, IL-36g, IFNgamma, and TNFalpha. F-IHC of paired tumor biopsies showed increase in proliferating CD8+ T cells. Transcriptional profiling of the TME demonstrates a pronounced immune response including dendritic cell recruitment and T cell activation, which remained elevated in longitudinal samples. The greatest increases in immune response and markers of cytolytic activity were observed in pts deriving clinical benefit. Conclusions ITu mRNA-2752 + IV durvalumab is safe, tolerable, and shows preliminary efficacy in immune refractory tumors, including TNBC and melanoma. Biomarker analyses indicate mRNA-2752 drives cytokine responses. Consistent with the expected mechanism of action, a productive and sustained inflammatory response is observed in the TME in response to treatment, including signatures of increased innate and adaptive immune cell abundance and effector response

Background Though FDA-approved for use after surgery for stage III/IV melanoma in the adjuvant setting, ipilimumab (anti-CTLA-4) and interferon-a are not typically used because of the improved toxicity profile and relapse-free survival (RFS) benefits seen with anti-PD-1-based regimens in this patient population. However, it is important to study samples derived from anti-CTLA-4 treated patients from the mechanistic perspective to understand its contribution to ipilimumab/ nivolumab therapy and to study single agent CTLA4 therapy in the adjuvant cases of BRAF wild-type patients who were re-resected and previously received anti-PD-1 therapy. Recently published data from our group indicated that nitric oxide (NO) increased in immune effector cells among patients treated with longer RFS, whereas NO also increased in immune suppressor cells among patients with shorter RFS. Given this unexpected dichotomy, we measured the posttranslational modification in the immune cells that is a direct result of increased NO levels on the interferon response protein STAT1 important for response to anti-CTLA-4 therapy/interferon responsiveness. Nitration is a stable post-translational modification of NO metabolism and STAT1 is nitrated at the same position (Y701) as it is phosphorylated. Methods Peripheral blood mononuclear cells (PBMCs) collected from 35 patients at day 0 and day 155 of ipilimumab therapy were analyzed for phosphorylated STAT1 (pSTAT1; using flow cytometry) and nitrated STAT1 at position 701 (nSTAT1; using liquid chromatography with tandem mass spectrometry). Patients were divided into groups based on RFS post ipilimumab therapy. Results Pre- and post-therapy PBMCs were available from 35 patients with stage IIIC/IV melanoma. The median patient age was 58 years (range, 21-78 years), and 63% of patients were male. pSTAT1 levels had the least variability in the tercile of patients with the longest RFS (P=0.002). Higher pSTAT1 levels in PBMCs were associated with worse RFS before (P = 0.007) and after therapy (P = 0.036) with ipilimumab. Expectedly, pSTAT1 levels in PBMCs increased following ipilimumab therapy. Patients whose STAT1 remained nitrated had decreased RFS, but intriguingly, increases in nitration of STAT1 following ipilimumab therapy was associated with favorable outcomes (P=0.01). Conclusions These data suggest that interferon responsiveness is regulated by changes in nitration of STAT1. Nitration of Y701 on STAT1 may regulate excessive interferon responses by limiting pSTAT1 phosphorylation for successful ipilimumabbased therapy in melanoma and deserves further study in the adjuvant and metastatic settings.

Background Despite significant improvements in treatment outcomes with ICIs, deeper understanding is needed to improve outcomes for patients with melanoma who experience no clinical benefit from ICI therapy and exhibit early disease progression (primary resistance).1 We analyzed clinical and translational factors using data from 9 BMS-sponsored clinical trials of nivolumab (NIVO), ipilimumab (IPI) and their combination in patients with advanced cutaneous melanoma (Check- Mate 003, 037, 038, 064, 066, 067, 069, 511, 742) to predict resistance to ICIs and explore underlying mechanisms. Methods Analyses of pre-treatment clinical covariates and biomarkers (determined by tumor immunohistochemistry, whole exome DNA sequencing, RNA sequencing, and serum cytokine analysis) were performed in patients with melanoma who received NIVO, IPI or NIVO+IPI and were evaluable for resistance. Several methods were used to develop and test models predicting ICI primary resistance, defined as death due to disease or best overall response of progressive disease (excluding pseudoprogression) before the first radiographic scan (<=13 weeks of treatment). Model performance was assessed using cross-validated area under the receiver operating characteristic curve (AUROC) and bootstrap bias-adjusted calibration metrics. Additionally, we evaluated individual biomarker associations with primary resistance. Results Across 1803 patients from 9 clinical trials, 1638 were evaluable for cutaneous melanoma primary resistance and 585 (36%) met the criteria. A multivariable logistic regression model for predicting primary resistance yielded an AUROC of 0.78 (R2=0.29) and included 17 commonly available clinical factors and PD-L1 (table 1). External validation using data from an independent study yielded an AUROC of 0.70 (R2=0.17). Higher levels of acute phase cytokines and expression of genes associated with suppressive tumor microenvironment or stromal factors, including epithelial-mesenchymal transition, cancer-associated fibroblasts, transforming growth factor beta, and angiogenesis, were associated with increased risk of primary resistance. In contrast, higher levels of tumor mutational burden, PD-L1, CD8, mutations in MAPK-pathway genes, and immune gene signature scores were associated with decreased risk (figure 1, table 2). Conclusions This exploratory analysis of clinical and translational factors in patients with advanced cutaneous melanoma identified associations with primary resistance to NIVO, IPI, or NIVO+IPI, with the best model yielding an AUROC of 0.78. Factors associated with primary resistance included higher levels of prognostic cytokines, gene expression indicating an immune suppressive tumor microenvironment, lower levels of anti-tumor immunity, and lack of MAPK-pathway mutations. These findings are supportive of future clinical trial stratification of patients and mechanistic studies targeting the biology of primary resistance to ICI therapy

Background Despite improved outcomes in advanced (unresectable or metastatic) melanoma, many patients progress after ICI,1-3 and have low response rates to subsequent therapy.4-7 Lifileucel, a one-time autologous TIL cell therapy, demonstrated an investigator-assessed ORR of 36% in Cohort 2 (C2), which enrolled 66 patients who progressed post-ICI and appropriate targeted therapy.8,9 We now report outcomes of 153 patients enrolled across C2 and C4 (NCT02360579), representing the largest phase 2 study of cell therapy in melanoma. Methods Eligibility criteria were identical for C2 and C4. Patients had >=1 lesion(s) resected (~1.5 cm in diameter postresection) and shipped to a central GMP facility for 22-day lifileucel manufacturing. All patients received a nonmyeloablative lymphodepletion (NMA-LD) regimen, a single lifileucel infusion, and up to 6 doses of high-dose IL-2. Primary endpoint was IRC-assessed ORR (RECIST v1.1). Results The full analysis set included 153 patients (C2: n=66; C4: n=87) treated with lifileucel, with a median of 3 prior lines of therapy (range: 1-9) and substantial baseline disease burden (>3 target and non-target lesions: 76%; median target lesion SOD: 97.8 mm; LDH >ULN: 54%). ORR was 31% (95% CI: 24.1%-39.4%) (C2: 35%; C4: 29%), with 8 CRs and 40 PRs (figure 1). At median study follow-up of 27.6 months, median DOR was not reached (NR). Forty-two percent of responses extended >=18 months, and 40% (19/48) of responses were ongoing at time of datacut (figure 2). In multivariable analyses adjusted for ECOG PS, elevated LDH and target lesion SOD >median were independently correlated with ORR (p=0.008); normal LDH and SOD < median were associated with higher odds of response than either (OR=2.08) or both (OR=4.42) risk factors. The median OS was 13.9 months (95% CI: 10.6-17.8. In an analysis of survival by response at first assessment (1.5 months post-lifileucel infusion), median OS in responders was NR (95% CI: 22.5 months-NR). The most common (>=30%) G3/4 TEAEs were thrombocytopenia (77%), anemia (50%), and febrile neutropenia (42%). TEAEs were consistent with known safety profiles of NMA-LD and IL-2, and their incidence decreased within the first 2 weeks post-lifeucel infusion, characteristic of onetime treatment. Conclusions Lifileucel demonstrated clinically meaningful and durable activity (ORR: 31%; mDOR: NR) in heavily pretreated patients with advanced melanoma and high tumor burden after ICI (and targeted therapy, where appropriate). Favorable safety profile and sustained responses support the potential benefit of one-time lifileucel TIL cell therapy as a novel treatment option for patients without approved therapies post-ICI. (Table Presented)

Background Tumor Infiltrating Lymphocyte (TIL) Adoptive Cell Transfer (ACT) is effective in treating malignant melanoma and other solid tumors. The success of TIL ACT relies on the adequate expansion of TIL, with previous studies showing a positive association between number of TIL infused and patient response. To identify characteristics important for TIL expansion, we performed a multiomic analysis of patients' TIL product. Methods Expanded TIL products from metastatic melanoma patients enrolled in clinical trials at Moffitt Cancer Center were collected before infusion. CD4+ and CD8+ were isolated and analyzed by RNA-seq (n=13) and pan-acetyl histone 3 ChIP-seq (n=20). The number of TIL, percentage of CD4/ CD8 infused, progression free survival (PFS), and overall survival (OS) were recorded. To more evenly divide both RNASeq and ChIP-seq samples between groups, patients were categorized into TIL high vs TIL low based on division at the geometric mean of the number of TIL infused (geometric mean= 4.6e10, range= 9.05e+09 - 1.13e+11). Log2 fold changes of +/-0.5 and q-values of <0.1 were considered significant. Results Individuals in the TIL high group had longer (PFS) (median=92 vs 4, p< 0.0001) and OS (OS median=92 vs 10 months , p<0.0001) than those in the TIL low group, and the percentage of CD4+ infused was negatively correlated with the number of TIL infused (R2=-0.72, p=7.7e-05). RNA-seq revealed 30 differentially expressed genes (DEGs) in CD4+ between groups. The upregulated genes in the TIL low group included TNFRSF4, PTGDR2, IL5, and CCL4, which are associated with Th2 and Treg phenotypes. Pathway analysis of the RNA-seq data further suggested increased Th2 and Th17 polarization in the TIL low group. ChIP-seq showed 18 differentially acetylated genes (DAGs) between TIL high and TIL low CD4+, including increased acetylation at known inducers of Tregs CD200R1, IL12RB2. RNA-seq revealed 3 DEGs between CD8+ in the TIL high and TIL low groups. ChIP-seq revealed 18 DAGs between TIL high and TIL low CD8+, with increased acetylation at genes known to be upregulated during CD8+ activation (e.g., SLC4A10, TIGIT and P2RY1). Conclusions Our results and those of other groups have shown that increased number of TIL infused are associated with positive patient outcomes. Our results further indicate that CD4+ Th2, Th17 and Treg phenotypes in the TIL product are associated with decreased numbers of TIL achieved during expansion. Consequently, we hypothesize that approaches to directing T-cell polarization during TIL expansion may increase patient response rates

Background Therapies targeting T-cell checkpoints have resulted in anti-tumor responses leading to FDA approval of multiple immunotherapies for metastatic melanoma and an expanding list of other malignancies. Despite having unprecedented efficacy, PD1 and CTLA4 antagonist antibodies still fail to benefit many patients. Critically, an understanding of mechanisms of resistance to immunotherapies is lacking. We recently identified and validated a novel population of peripheral blood T-cells predictive of resistance in nivolumab (alphaPD1) treated metastatic melanoma patients. This population is defined by the marker set CD3+CD4+CD127-GARP-CD38 +CD39+. Based on the co-expression of CD38 and CD39, we have termed the population ectoenzyme expressing T-cells (Teee). Methods We utilized high-dimension flow cytometry to assess Teee frequencies in adjuvant immunotherapy treated metastatic melanoma patient peripheral blood samples. To characterize the phenotype of Teee, we utilized flow cytometry and CITESeq on melanoma patient tumor specimens. We also evaluated the presence and phenotype of Teee in several l syngeneic murine tumor models. Results We found that Teee were of low frequency in demographic matched healthy donors and increased in both resected (p=0.018) and active disease metastatic melanoma patients (p=0.003). Circulating Teee frequencies positively correlated with frequencies of Tregs (R²=0.4367, p<0.0001), MDSCs (R²=0.2706, p=0.0004) and M2-like monocytes (R²=0.1514, p=0.0109). Increases in circulating Teee were associated with relapse in resected melanoma patients treated with adjuvant combination ipilimumab (alphaCTLA4) and nivolumab (p=0.0213). Human tumors showed high frequencies of Teee in tumor infiltrate. We also demonstrated the existence of this population in MC38 and YUMM mouse tumor models. Intratumor frequencies of Teee positively correlated with tumor volume (R²=0.96, p=0.0006) and inversely correlated with overall immune infiltrate (R²=0.3198, p=0.0280). Our characterization of this population showed an enhanced adenosine generating phenotype (i.e. CD73^high, CD26^low), a terminal exhaustion phenotype (i.e. TOX^high, TCF1^low), expression of inhibitory receptors (e.g. CTLA4, TIM3) and ligands (e.g. PDL1, B7-H4), and expression of immunosuppressive cytokines (e.g. IL-8, TGFbeta). Supporting a mechanistic relationship to resistance, Teee suppressed autologous T-cell proliferation (p=0.0278) and inflammatory function. Conclusions We have identified a novel population of ectoenzyme expressing T-cells associated with immunotherapy resistance in metastatic melanoma patients. This population of cells had phenotypes associated with immune suppression and suppressed autologous T-cells in vitro. Collectively, our data support evaluation of targeting Teee to augment the efficacy of immunotherapy

Helper (CD4⁺) T cells perform direct therapeutic functions and augment responses of cells such as cytotoxic (CD8⁺) T cells against a wide variety of diseases and pathogens. Nevertheless, inefficient synthetic technologies for expansion of antigen-specific CD4⁺ T cells hinders consistency and scalability of CD4⁺ T cell-based therapies, and complicates mechanistic studies. Here we describe a nanoparticle platform for ex vivo CD4⁺ T cell culture that mimics antigen presenting cells (APC) through display of major histocompatibility class II (MHC II) molecules. When combined with soluble co-stimulation signals, MHC II artificial APCs (aAPCs) expand cognate murine CD4⁺ T cells, including rare endogenous subsets, to induce potent effector functions in vitro and in vivo. Moreover, MHC II aAPCs provide help signals that enhance antitumor function of aAPC-activated CD8⁺ T cells in a mouse tumor model. Lastly, human leukocyte antigen class II-based aAPCs expand rare subsets of functional, antigen-specific human CD4⁺ T cells. Overall, MHC II aAPCs provide a promising approach for harnessing targeted CD4⁺ T cell responses.

Checkpoint immunotherapies (CPIs) have improved outcomes for metastatic melanoma patients, with objective response rates to combination ipilimumab and nivolumab of ~58%. Preclinical data suggest that histone deacetylase (HDAC) inhibition enhances antitumor immune activity and may augment CPI. In a phase Ib open-label pilot trial (NCT03565406), patients with therapy-naive metastatic melanoma were treated with the class I/IV HDAC inhibitor mocetinostat orally three times a week in combination with nivolumab and ipilimumab every 3 weeks for 12 weeks followed by 12-week maintenance cycles of nivolumab every 2 weeks and mocetinostat at the same dose and schedule as induction. The endpoints of the trial were safety, definition of a recommended phase 2 dose, preliminary assessment of response, and correlative marker determination. Patient PBMC and serum samples collected at baseline and on-treatment were assessed by flow cytometry and Luminex assays for immune correlates. Ten patients were treated: nine with 70-mg and one with 50-mg mocetinostat. In the 70-mg cohort, eight patients had objective responses. The patient in the 50-mg cohort had an early progression of disease. All patients had grade 2 or higher toxicities, and six had grades 3 and 4 toxicities. Patient PBMC showed significant decreases in myeloid-derived suppressor cells and trends towards reduced anti-inflammatory monocyte phenotypes. Patient serum showed significant upregulation of granzyme A and TNF and trends towards increased granzyme B and IFNγ. Collectively, combining CPI and mocetinostat had favorable response rates but with high levels of toxicity. Assessment of immune correlates supports a shift away from immunosuppressive phenotypes towards enhanced immune responses.

BACKGROUND:Recent data suggest that patients with stage III melanoma are at high risk for developing central nervous system (CNS) metastases. Because a subset of patients with stage II melanoma experiences worse survival outcomes than some patients with stage III disease, the authors investigated the risk of CNS metastasis in stage II melanoma to inform surveillance guidelines for this population. METHODS:test, the cumulative incidence, and Cox multivariable regression analyses were performed to evaluate the association between baseline characteristics and the development of CNS metastases. RESULTS:Patients with stage III melanoma had a higher rate of developing brain metastases than those with stage II melanoma (100 of 468 patients [21.4%] vs. 82 of 586 patients [14.0%], respectively; p = .002). However, patients who had stage IIC melanoma had a significantly higher rate of isolated first recurrences in the CNS compared with those who had stage III disease (12.1% vs. 3.6%; p = .002). The risk of ever developing brain metastases was similarly elevated for patients who had stage IIC disease (hazard ratio [HR], 3.16; 95% CI, 1.77-5.66), stage IIIB disease (HR, 2.83; 95% CI, 1.63-4.91), and stage IIIC disease (HR, 2.93; 95% CI, 1.81-4.74), and the risk was highest in patients who had stage IIID disease (HR, 8.59; 95% CI: 4.11-17.97). CONCLUSIONS:Patients with stage IIC melanoma are at elevated risk for first recurrence in the CNS. Surveillance strategies that incorporate serial neuroimaging should be considered for these individuals until more accurate predictive markers can be identified.

PURPOSE/OBJECTIVE:Ipilimumab and nivolumab have each shown treatment benefit for high-risk resected melanoma. The phase III CheckMate 915 trial evaluated adjuvant nivolumab plus ipilimumab versus nivolumab alone in patients with resected stage IIIB-D or IV melanoma. PATIENTS AND METHODS/METHODS:In this randomized, double-blind, phase III trial, 1,833 patients received nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks (916 patients) or nivolumab 480 mg once every 4 weeks (917 patients) for ≤ 1 year. After random assignment, patients were stratified by tumor programmed death ligand 1 (PD-L1) expression and stage. Dual primary end points were recurrence-free survival (RFS) in randomly assigned patients and in the tumor PD-L1 expression-level < 1% subgroup. RESULTS:= .269) or in patients with PD-L1 expression < 1% (hazard ratio, 0.91; 95% CI, 0.73 to 1.14). In all patients, 24-month RFS rates were 64.6% (combination) and 63.2% (nivolumab). Treatment-related grade 3 or 4 adverse events were reported in 32.6% of patients in the combination group and 12.8% in the nivolumab group. Treatment-related deaths were reported in 0.4% of patients in the combination group and in no nivolumab-treated patients. CONCLUSION/CONCLUSIONS:Nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks did not improve RFS versus nivolumab 480 mg once every 4 weeks in patients with stage IIIB-D or stage IV melanoma. Nivolumab showed efficacy consistent with previous adjuvant studies in a population resembling current practice using American Joint Committee on Cancer eighth edition, reaffirming nivolumab as a standard of care for melanoma adjuvant treatment.