Immunotherapy Bridge 2019 and Melanoma Bridge 2019: meeting abstracts

Immunotherapy Bridge 2019 and Melanoma Bridge 2019: meeting abstracts Naples, Italy. December 4 December 7, 2019 Published: 11 February 2020 © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Immunotherapy Bridge 2019 SITC session—mechanisms of success and failure in immunotherapy

well characterized, however, the contribution of NK cells is not well understood. This is partly due to the paucity of NK cell-specific markers that can identify NK cells in the tumor microenvironment (TME).
We developed an NK cell-specific transcriptional signature to estimate the NK cell abundance in the TME. This signature, together with NKchemokines shown to modulate the priming of adaptive immunity 1 were investigated in patients with advanced non-small cell lung cancer (NSCLC) treated with a PD-L1 inhibitor, durvalumab. Methods: Peripheral blood mononuclear cells (PBMCs) and Fluorescence-Activated Cell Sorted (FACS) NK/CD8 populations from three heathy donors were subjected to single cell RNA sequencing (scRNAseq, 10X Genomics) and transcriptome analysis (Affymetrix), respectively. Fresh frozen tumor biopsies from 97 NSCLC were profiled with RNA sequencing prior to durvalumab treatment; 29 of these had paired tumors procured 29 days following treatment with durvalumab. Kaplan-Meier (KM) analyses were performed to identify predictive effects of the NK cell-specific signature. Clinical trial: 1108/ NCT01693562 Results: Transcripts over-expressed in sorted NK relative to CD8 cells were first identified (p < 0.01; fold > 3) and intersected with 28 mRNAs up-regulated in the NK cell cluster determined by scRNAseq, providing an 8 gene NK cell-specific transcriptional signature defined as MEDI-NK. MEDI-NK correlated with NK signatures recently described 2 , and included chemokines shown to induce an effective NK-response 1 .
When evaluated in TCGA, higher expression of MEDI-NK was associated with good prognosis (Overall Survival, OS) of patients with melanoma and breast cancer (p value = 0.03 and = 0.001, respectively). At baseline, MEDI-NK was highly correlated with the previously identified IFNγ signature 3 and was associated with Progression Free Survival (PFS p value < 0.02) of NSCLC patients treated with durvalumab. Following treatment with durvalumab, the increased expression of MEDI-NK and of additional genes leading to NK-priming of adaptive immunity 1 was observed to be associated with patients' overall survival (OS p value < 0.01). Similar findings were not observed prior to durvalumab treatment.
Conclusions: Using single cell analysis, an NK cell-specific signature was developed to better define the role of NK cells in anti-PDL1 therapy. The increased expressions of the NK cell-specific gene signature and of genes leading to NK-cell priming of adaptive immune response were associated with clinical benefit to durvalumab.
Background: Ipilimumab (Ipi), an anti-cytotoxic T-lymphocyte-associated antigen4 (CTLA-4) monoclonal antibody, has been shown to improve survival in patients (pts) with advanced melanoma [1][2][3]. Several retrospective studies have shown how the combination of radiotherapy (RT) and Ipi in the treatment of melanoma brain metastases (MBMs) pts improves the outcomes, without however clarifying the exact timing of the two modalities [3][4][5][6][7][8][9][10].  Table 1. We divided the pts into 3 different groups based on therapies timing: 18 in RT PRE-IPI, 20 in RT CONCOMITANT (CONC) IPI, 15 in RT POST-IPI group. Ipi was administered intravenously at a dose of 3 mg/kg over 90 min every 3 weeks for 4 doses. A total of 127 lesions, were treated with SRS/SRT performed by CK. We evaluated the local response according to RECIST criteria. We assessed LC as the sum of complete response, partial response and stable disease, IC and median OS from the date of the SRS/SRT procedure. Results: The median follow-up was 10 [1]. One factor that limits the recognition of these neoantigens by T cells is the level of expression of the mutated gene product in cancer cells. In the BALB/c-derived 4T1 mouse model of ICB-refractory metastatic breast cancer, we have previously shown that tumor-targeted radiation therapy (RT) combined with CTLA4 blockade induces CD8 + T cell-mediated regression of irradiated tumors and inhibits lung metastases [2]. Analysis of the T-cell receptor (TCR) repertoire indicated that unique clonotypes expand in treated tumors, suggesting that tumor rejection involves T cells reactive to a set of tumor antigens that are made available to the immune system by RT [3]. Therefore, we hypothesize that RT increases the expression of genes containing immunogenic mutations and hence promotes priming of neoantigen-specific T cells.

Materials and methods:
We performed whole-exome sequencing and RNA sequencing of untreated and irradiated (8GyX3) 4T1 cells in vitro to identify tumor-specific neoantigens and determine which ones are upregulated by RT. These mutations were also documented in vivo, in 4T1 tumors harvested before and after treatment (8GyX3 + anti-CTLA4). Dedicated algorithms were used to predict MHC-I and MHC-II-binding epitopes from these mutated genes. Peptides with a predicted affinity < 500 nM were synthesized and tested in vitro for binding in a MHC stabilization assay. The best candidates were used to vaccinate BALB/c mice, followed by challenge with 4T1 cells to test for the induction of protective anti-tumor immunity. Results: Out of 309 total mutations initially identified in 4T1 cancer cells, two MHC-I and one MHC-II neoepitopes were immunogenic in vaccination experiments as assessed by IFNγ/TNFα response after T cell re-stimulation. These neoepitopes were encoded by genes upregulated by RT. Vaccination with these three neoantigens induced a significant tumor growth delay in mice only when vaccination was combined with tumor-targeted RT. We observed significant changes in the intratumoral TCR repertoire in vaccinated mice. In addition, in vivo killing experiments demonstrated a potent cytolytic activity of T cells from vaccinated mice towards one of these neoepitopes. These results were confirmed in vitro after MHC-I blockade of the peptide-loaded target cells. Mass-spectrometry analyses of MHC-I-bound peptides are currently ongoing to assess the differences in presented antigens between untreated and irradiated cancer cells.

Conclusions:
Overall, our data demonstrate the potential of RT to modulate the expression of antigenic mutations in tumors which could enhance responses to immunotherapy.  The successful deployment of immune checkpoint inhibitors (ICI) in cancer immunotherapy relies on the responsiveness of an individual's immune system for relief of that particular blockade in the cancer immunity cycle [1][2][3]. As most patients fail to respond to ICI, there is a need for biomarkers that can predict patient's clinical benefit thereby identifying the patient population most likely to respond [4,5]. The goal of this study was to augment the prediction accuracy by identifying and testing novel candidate biomarkers that could envisage response to ICI in patients with metastatic melanoma. The analysis had two specific features: validation against previously published predicting biomarkers and characterization of patients' transcriptomes at individual gene and pathway levels, where network enrichment analysis (NEA) integrated disparate genes into pathway scores [6]. Materials and methods: Gene expression profiles were obtained using NanoString ® panels (IO 360 ™ beta or UIO) on formalin fixed paraffin embedded biopsies (FFPE) obtained from 30 stage IV metastatic melanoma patients treated with ipilimumab (anti-CTLA4) and 50 patients treated with Nivolumab (anti-PD1) of which 22 were first-line and 28 pretreated with ipilimumab. The samples originated from the pathological anatomy department of Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli, Italy. All patients have appropriately signed informed consent. Statistical associations between treatment response and either gene or pathway score variables were estimated in linear models, which included covariates of known importance to ICI, such as mRNA expression of the checkpoint proteins and their ligands.

Results:
First, candidate transcription-based biomarkers were discovered in our cohorts via correlation to clinical benefit and then analyzed for significance by covariate adjustment. Secondly, the candidates performance was validated using a similar previously published NanoString-based gene dataset [7]. In the ICI-naïve anti-PD1 cohort, we identified different genes which were informative on the clinical benefit regardless of the known determinants: F2RL1, ARG1 and ICAM5. In the anti-CTLA4 cohort, the individual gene analysis did not yield any significant and validated associations. However instead, we revealed a number of NEA-based correlates between "progression within 1 year" and pathways e.g. "Cell adhesion molecules", "PECAM1 interactions", as well as a number of immune-related differentially expressed gene lists. Conclusions: NanoString-based transcriptomics and the cohort designs provided high-quality data for discovery of robust biomarkers of ICI response, holding promise for development of clinically useful diagnostic panels in malignant melanoma. Ethics approval: The study was approved by the internal ethics board of the Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli Italy, approval number of registry 17/17 OSS.

Acknowledgements:
The study was supported by the Institutional Project "Ricerca Corrente" of Istituto Nazionale Tumori IRCCS Fondazione "G. Pascale" of Napoli, Italy. Background: SNS-301 is a first-in-class therapeutic cancer vaccine candidate targeting human aspartyl (asparaginyl) β-hydroxylase (ASPH). ASPH is a highly tumor specific antigen that is differentially overexpressed in multiple human cancers but not in healthy adult tissue and is associated with tumor cell growth, motility and invasiveness. SNS-301 is engineered to express an ASPH fusion product within an inactivated λ-bacteriophage viral vector (phage display) to activate J Transl Med 2020, 18(Suppl 1):50 both innate and adaptive arms of the immune system. Extensive preclinical data demonstrated the immunotherapy's ability to overcome tumor self-tolerance and provide anti-tumor immunostimulatory effect including strong activated, functional intra-tumoral CD8+ T cell infiltration. Materials and methods: SNS-301 was tested in a phase I clinical trial via intradermal administration using a 3 M micro-needle injection system in ASPH overexpressing biochemically recurrent prostate cancer patients (pts). Twelve pts with detectable levels of ASPH received 3-23 doses of SNS-301.

Results:
The immunotherapy was well tolerated with only 3 pts. experiencing an adverse event (AE) considered at least possibly related to study drug. All AEs were ≤ grade 3 and no dose-limiting toxicity was observed. All pts. experienced NK cell activation as well as dosedependent ASPH-specific immune responses including CD4+ and CD8+ T-cell and B cell dependent immune responses. Anti-tumor activity and disease stabilization was observed in 8/12 pts. (67%) with declines noted in both overall PSA level and increases in PSA doubling rate.
Conclusions: SNS-301 is a novel immunotherapy that may overcome prior challenges of cancer vaccines and cell therapies. Based on the pre-clinical and phase I results, multiple phase II programs were initiated in ASPH positive patients across many tumor types to evaluate SNS-301 as an active product in the cancer-immunity cycle both as monotherapy and combination therapy with checkpoint inhibitors. A combination phase II study of SNS-301 with pembrolizumab in ASPH positive checkpoint resistant head and neck cancer patients is currently enrolling (NCT04034225). Additionally, ASPH is also in preclinical development as a cell therapy target in both heme and solid malignancies. Background: Tumor mutational burden (TMB) has been shown to be predictive of a good response to immunotherapy in stage IV melanoma and also other tumors, and is starting to be used as an inclusion criterion in ongoing clinical trials. However, the prognostic value of this marker is jet to be validated, also in earlier stages. We analyzed data from primary melanoma of stage II patients from the TCGA database and found that TMB could be prognostic in this collective. In this study, we intended to validate the prognostic value of TMB in a stage II cohort of melanoma patients from our department.

Materials and methods:
We included patients with stage II melanoma diagnosed between 2000 and 2018 in the University Hospital of Tuebingen and for whom formalin-fixed and paraffine embedded normal and tumor tissue were available. Tumor and normal DNA sequencing was performed using a next generation sequencing (NGS) panel that covers 693 genes, 7 promotor regions and the intronic region of 26 genes with known fusion partners. TMB was expressed in mutations per megabase (mut/Mb) and the median TMB was used as cut-off to define high and low-TMB sub-groups. Descriptive analysis of patients' characteristics and survival analysis were performed. The follow-up time was defined as the time between diagnosis and relapse or death.       Background: Recent introduction of anti-PD-1 (Nivolumab and Pembrolizumab) and anti-PD-L1 (Atezolizumab, Darvalumab) immune checkpoint inhibitors revolutionized oncological guidelines. IrAEs reported in clinical trials account to a maximum of 85%, while grade 3/4 of toxicity were reported in 10% of patients. Quality of AEs reporting in RCTs is satisfactory, but methods for data collection and analysis are unclear. The purpose of the study is to establish a cohort of cancer patients treated with immune checkpoint inhibitors (PD-1/PD-L1 inhibitors) in order to determine incidence and characteristics of irAEs in a real-world setting and improve clinical management.

Materials and methods:
We conducted a prospective cohort study in patients receiving anti-PD-1/PDL1 drugs for treatment of metastatic or locally advanced non-small cell lung cancer, renal cell carcinoma, squamous cell carcinoma of the head and neck, Hodgkin lymphoma starting from Jan 2019. We created a clinical pathway aimed to improve management of patients at risk for IRAEs. In particular, definite recommendations have been implemented for cases fulfilling criteria for suspected irAEs. They concern procedures for evaluation and diagnosis, specific treatments and rules for drug discontinuation. Background: Acute Lymphoblastic Leukemia (ALL) patients is the most common malignancy in children and represents 75-80% of leukemia cases. The most frequent immunophenotype is B-cell precursor ALL (B-ALL) in which, signaling via the B cell receptor (BCR) and its precursor (pre-BCR), play a crucial role in tumor promotion. It has been reported that Leukemias originate from cells with stem characteristics (LSC) well described in Acute Myeloid Leukemia (AML) but controversial in ALL. We propose to identify these cells by their dysregulated signaling pathway, using a combination of phosphoflow (SNCP) and cell surface markers in a high dimension flowcytometric approach in pediatric ALL.

Methods:
We enrolled a cohort of 20 B-ALL pediatric patients and adult healthy donors (HD) for a pilot study in order to set up the Single SCNP method. To evaluate the activation of ERK and STAT signal pathways, in addition to the phosphoprotein activation markers we developed a high-dimensional multicolor panel of 20 extracellular markers and applied it to 5 HD and 1 blood samples at baseline and after stimulation with Phorbol Myristate Acetate (PMA). The Spectraviewer Cytometer Aurora has been used to perform the experiments and the data have been analyzed with Cytobank using visualization tools like SPADE and viSNE algorithms. Results: In this pilot study, we show that it is possible to perform high dimension phenotypic and functional panels using fluorescently labeled antibodies, and that this constitutes a major advantage for the study of pediatric samples where sample-size is limiting. By defining SPADE trees clustered on cell surface markers, we traced multiple phosphorylation events monitored with ERK1,2 (pT202/ pY204), p38MAPK (pT180/pY182), STAT1 (pY701), STAT3 (pY705) and STAT5 (pY694) in HD and B-ALL sample at basal levels and after stimulation.
Conclusions: This study shows that this approach to characterize the activation pathways in different leukemia subpopulations, is feasible and potentially powerful enough to identify LSC. It can also be used as model for cancer patients were the sample size, as like pediatric samples, is very limited.

Total RNA-transcriptomics for identification of predictors of overall survival in metastatic melanoma patients treated with anti-PD-1
Qingyang Xiao, Javier Oliver, Juan Luis Onieva, Pilar Piñeiro, Alicia Garrido-Aranda, Aurora Laborda-Illanes, Elena Gallego, Cynthia Sample collection (tissue, blood, urine and feces) for diagnosis, biomarker and molecular analysis will be collected at baseline, after each cycle (except tissue) surgery and afterwards in the adjuvant setting. Results: Proteomic analysis of sera of treated patients, with particular emphasis on cytokines and chemokines, are being performed in order to identify possible markers associated with a better clinical outcome. The antitumor immune response in peripheral blood lymphocytes has been monitored, in order to evaluate whether the combination of antiCTLA4 and anti-PD1 is able to increase the number and/or the repertoire of melanoma-specific T-cells after treatments. Gene sequencing analysis and expression profiling of genes involved in immune response by different means will also be evaluated in order to detect possible variations induced by the treatment on a molecular level. Finally, data on the modification induced by the disease and treatment on the microbioma and microbiota at different time points, showed interesting influences in maintaining or creating a beneficial equilibrium. All these preliminary data will be presented and discussed together with efficacy/toxicity, based on percentages of pathological complete responses reached at surgery. Conclusion: Understanding the molecular mechanisms of metastatic spread and exploiting such knowledge in prevention will likely have a profound impact on melanoma prognosis in advanced stages.
In a melanoma patient's population including stage IIIB-C, or IV with potentially resectable disease, neoadjuvant immunotherapy was feasible, while identification of biomarkers of response and prognosis is ongoing in order to allow a better patient's selection.  Background: T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Identification of the specific extracellular contacts between the T cell receptor (TCR) and CD3 subunits upon recognition of peptide-major histocompatibility complexes (pMHC) gives more precise guidance for immunotherapeutic strategies that modulate T-cell immunity by targeting signaling through the TCR-CD3 complex. Previous studies that targeted the antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity.

Materials and methods:
Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCR-CD3 assembly [1]. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function.
Results: Based on our TCR-CD3 structural model, we mutated specific TCR-residues (Fig. 1A) that resulted in decreased TCR-CD3 binding (as evident from CD3γε tetramer binding- Fig. 1B) as well as lower cytokine responses (Fig. 1C). However, one Cβ helix 4-F strand mutant, NP202203AA showed higher T cell response (Fig. 1B). This mutant also showed enhanced TCR-pMHC bond lifetime in BFP assays leading to prolonged T cell signaling. Collectively, this data places us in a unique position to translate our findings towards improved immunotherapy strategies. Conclusion: Our hypothesis is that by modulating TCR-CD3 interactions in specific ways, immune-mediated cytotoxicity can be increased without losing specificity for the cancer antigen. To test our hypothesis, we sought to mutate specific TCR-residues that interact with CD3 to increase the affinity of the TCR-CD3 interaction, resulting in better CD3 tetramer binding as well as higher cytokine responses. Previously, we have used structure-based modeling to redesign the antigen binding region of DMF5 TCR (a TCR specific for Mart-1 melanoma antigen) to increase T cell signaling potency [2]. A TCR library for DMF5 TCR was created using site-specific mutagenesis in the Cβ helix 3 and helix 4-F strand regions of the TCR (Fig. 1A) by in vitro combinatorial retroviral TCR display to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for efficacy and toxicity to develop more effective T cell therapies for human targets.    Background: Continuous combination of MAPKi and anti-PD-(L)1 is currently tested in several trials to improve outcome of BRAFV600 mutated melanoma patients (pts). However, a major obstacle for continuous combination is the high frequency of grade 3/4 treatmentrelated adverse events (TRAE). In a preclinical model we showed that short-time MAPKi induces T cell infiltration and is synergistic with anti-PD-1. In pts we found increased T cell infiltration upon D + T after short-term MAPKi, while this was frequently below baseline levels after > 2 weeks (W) MAPKi. The aim of this phase 2b study was to identify the optimal duration of D + T in combination with PEM. Methods: Treatment-naïve BRAFV600E/K mutant advanced melanoma pts (n = 32) started PEM 200 mg Q3W and were randomized in W6 to continue PEM only (cohort 1), or to receive in addition intermittent D 150 mg BID + T 2 mg QD for 2× 1 W (cohort 2), 2× 2 W (cohort 3), or continuous for 6 W (cohort 4). All cohorts continued PEM for up to 2 years. Primary endpoints were safety and treatment-adherence. Secondary endpoints were objective response rate (ORR, RECIST 1.1) at week 6, 12, 18 compared to baseline and PFS.

Conclusion:
The ESMO 2018 IMPemBra data indicated that PEM + intermittent D + T for 2× 1 W or 2× 2 W are promising combinations in terms of safety and feasibility, warranted to be tested in subsequent trials.   Background: Although targeted therapies (TT) and immunotherapies (IMT) have improved survival for pts with BRAF V600 mutated stage IV MM, many pts progress and will ultimately die from this disease. Preclinical data has shown that BRAF inhibition (BRAFi) in BRAF-mutated tumors is associated with increased T cell infiltration, supporting the rationale for a clinical combinatorial approach with IMT. Although there are multicentered trials ongoing evaluating this combinatorial approach for pts with untreated MM, there are no approved therapies for pts after TT and IMT failure. Notably, pts with untreated brain metastases (BM) are often excluded from such trials. We hypothesized that N in combination with DT is safe and will demonstrate clinical activity in BRAF-mutated pts refractory to PD1 therapy and in pts with BM.

Methods:
We report a single arm phase II study (NCT02910700) of NDT in pts with BRAF-mutated, unresectable stage III or stage IV MM. Prior IMT is allowed, but pts who have received BRAF/MEKi are ineligible. Pts with untreated BM and asymptomatic or mildly symptomatic/requiring stable or decreasing steroids (up to PO dexamethasone of 8 mg or equivalent) are also allowed. Pts received 3 mg/kg Q2wks of N (later amended to 480 mg q4wks), 150 mg BID of D and 2 mg QD of T, all starting on Day 1. The primary objective of this study is to determine safety and efficacy (ORR by RECIST 1.1) of the NDT combination. This study was continuously monitored for safety and futility. Tissue and blood-based samples to assess for correlative studies are also collected. Results: Following a 6 pts safety run-in with no observed DLTs, 26 pts received NDT-16 pts were PD1 refractory, 10 were PD-1 naive. 9 of these 26 pts had BM. Of the 22 pts evaluable for response, 17 achieved PR and 3 CR (ORR 91%). 12 PD1 refractory were evaluable for response; 2 achieved CR and 9 PR (ORR 83%). 67% of the evaluable pts with BM achieved an intracranial response, including 2 CRs. Although the median PFS for all pts was ~ 8 months, the median OS was not reached. 65% of pts experienced treatment related grade 3/4 AEs, but only 3 pts discontinued due to toxicities. Conclusions: NDT is well-tolerated and shows promising clinical activity in pts with IMT refractory disease and with BM. There were no significant differences in outcomes between pts with and without BM. Further translational investigation to better delineate mechanisms of response are ongoing.

Melanoma Bridge 2019
Poster Background: Bispecific antibodies have shown activity in hematologic (heme) but not solid tumors. ImmTAC molecules are unique TCR-anti-CD3 bispecifics that redirect T cells against intracellular antigens. Tebentafusp (IMCgp100), an ImmTAC targeted against melanocyte-associated lineage antigen gp100, has shown monotherapy responses in advanced melanoma with associated immune changes. Tebentafusp causes rash and cytokine-mediated AEs, hypothesized to be on-target (gp100) or effector (CD3) mediated. We explored clinical and biological characteristics of pts associated with treatment benefit. Materials and methods: 84 HLA-A2 positive advanced melanoma pts received tebentafusp on study IMCgp100-01 in 13 dose escalation cohorts. Efficacy was assessed by Kaplan-Meier survival and treatment related AEs (TRAE) reported by CTCAE v4.0. Serum samples evaluated changes in cytokines. A multivariate analysis investigated the relationship between efficacy and safety variables.