Faculty Bibliography

Tilsotolimod, an oligodeoxynucleotide TLR9 agonist, administered intra-tumorally, has been clinically evaluated. This compound has demonstrated the ability to induce changes within the tumor microenvironment, to convert non-inflamed cold tumors into inflamed hot tumors, with the hope that these tumors will be more responsive to immune checkpoint blockade.

BACKGROUND:Patients with advanced melanoma have limited treatment options after progression on immune checkpoint inhibitors (ICI). Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, demonstrated an investigator-assessed objective response rate (ORR) of 36% in 66 patients who progressed after ICI and targeted therapy. Herein, we report independent review committee (IRC)-assessed outcomes of 153 patients treated with lifileucel in a large multicenter Phase 2 cell therapy trial in melanoma. METHODS:Eligible patients had advanced melanoma that progressed after ICI and targeted therapy, where appropriate. Melanoma lesions were resected (resected tumor diameter ≥1.5 cm) and shipped to a central good manufacturing practice facility for 22-day lifileucel manufacturing. Patients received a non-myeloablative lymphodepletion regimen, a single lifileucel infusion, and up to six doses of high-dose interleukin-2. The primary endpoint was IRC-assessed ORR (Response Evaluation Criteria in Solid Tumors V.1.1). RESULTS:The Full Analysis Set consisted of 153 patients treated with lifileucel, including longer-term follow-up on the 66 patients previously reported. Patients had received a median of 3.0 lines of prior therapy (81.7% received both anti-programmed cell death protein 1 and anti-cytotoxic lymphocyte-associated protein 4) and had high disease burden at baseline (median target lesion sum of diameters (SOD): 97.8 mm; lactate dehydrogenase (LDH) >upper limit of normal: 54.2%). ORR was 31.4% (95% CI: 24.1% to 39.4%), with 8 complete responses and 40 partial responses. Median duration of response was not reached at a median study follow-up of 27.6 months, with 41.7% of the responses maintained for ≥18 months. Median overall survival and progression-free survival were 13.9 and 4.1 months, respectively. Multivariable analyses adjusted for Eastern Cooperative Oncology Group performance status demonstrated that elevated LDH and target lesion SOD >median were independently correlated with ORR (p=0.008); patients with normal LDH and SOD <median had greater likelihood of response than those with either (OR=2.08) or both (OR=4.42) risk factors. The most common grade 3/4 treatment-emergent adverse events (≥30%) were thrombocytopenia (76.9%), anemia (50.0%), and febrile neutropenia (41.7%). CONCLUSIONS:Investigational lifileucel demonstrated clinically meaningful activity in heavily pretreated patients with advanced melanoma and high tumor burden. Durable responses and a favorable safety profile support the potential benefit of one-time lifileucel TIL cell therapy in patients with limited treatment options in ICI-refractory disease.

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 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:Adjuvant therapy for high-risk resected melanoma with programmed cell-death 1 blockade results in a median relapse-free survival (RFS) of 5 years. The addition of low dose ipilimumab (IPI) to a regimen of adjuvant nivolumab (NIVO) in CheckMate-915 did not result in increased RFS. A pilot phase II adjuvant study of either standard dose or low dose IPI with NIVO was conducted at two centers to evaluate RFS with correlative biomarker studies. METHODS:Patients with resected stages IIIB/IIIC/IV melanoma received either IPI 3 mg/kg and NIVO 1 mg/kg (cohort 4) or IPI 1 mg/kg and NIVO 3 mg/kg (cohorts 5 and 6) induction therapy every 3 weeks for 12 weeks, followed by maintenance NIVO. In an amalgamated subset of patients across cohorts, peripheral T cells at baseline and on-treatment were assessed by flow cytometry and RNA sequencing for exploratory biomarkers. RESULTS:High rates of grade 3-4 adverse events precluded completion of induction therapy in 50%, 35% and 7% of the patients in cohorts 4, 5 and 6, respectively. At a median of 63.9 months of follow-up, 16/56 patients (29%) relapsed. For all patients, at 5 years, RFS was 71% (95% CI: 60 to 84), and overall survival was 94% (95% CI: 88 to 100). Expansion of CD3+CD4+CD38+CD127-GARP- T cells, an on-treatment increase in CD39 expression in CD8+ T cells, and T-cell expression of phosphorylated signal-transducer-and-activator-of-transcription (STAT)2 and STAT5 were associated with relapse. CONCLUSIONS:Adjuvant IPI/NIVO at the induction doses used resulted in promising relapse-free and overall survival, although with a high rate of grade 3-4 adverse events. Biomarker analyses highlight an association of ectoenzyme-expressing T cells and STAT signaling pathways with relapse, warranting future validation. TRIAL REGISTRATION NUMBER:NCT01176474 and NCT02970981.

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 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

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 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

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.