Comprehensive Secretome Profiling Elucidates Novel Disease Biology and Identifies Pre-Infusion Candidate Biomarkers to Predict the Development of Severe Cytokine Release Syndrome in Pediatric Patients Receiving CART19

Introduction: The most common severe toxicity associated with chimeric antigen receptor T-cells targeting CD19 (CART19) is cytokine release syndrome (CRS; PMID: 29972754). Our group and others have published seminal observations on the biology of CRS through cytokine profiling, measuring a small number of analytes (PMID: 27076371, 33434058). Multiple biomarkers including interferon gamma (IFNG), IL-6, and IL-10 have been associated with the development of severe CRS in previous studies (PMID: 33434058). To date, the only biomarker predictive of the development of CRS prior to infusion has been disease burden. To obtain a more robust understanding of CRS biology, we performed comprehensive secretome profiling to measure more than 1400 serum analytes on serial serum samples collected from patients treated with the 41BB-containing CTL019 on two clinical trials. Methods: Serum from patients enrolled on two clinical trials of the CART19 product CTL019 (NCT01626495 & NCT02906371) were obtained serially from pre-infusion to one month post infusion. Patients were categorised as having "minimal" (no CRS, Grade 1, or Grade 2) or "severe" (Grade 3 or 4) CRS. The serum secretome was profiled using the Olink Explore 1536 Analysis platform (Olink, Upsala, Sweden). 1484 proteins were measured from serum via proximity extension assay (PEA) high-multiplex immunoassay. Differential expression analysis, correlation analyses and receiver operating characteristic (ROC) calculations were performed using R (version 4.0.4) in RStudio. Significance was based on a fold change of greater than 2 or less than -2 and a false discovery rate of less than 0.05 calculated using a Benjamini-Hochberg correction. Results: 26 patients (10 NCT01626495 & 16 NCT02906371) were included comprising 128 unique datapoints from baseline to 35 days post-infusion. Thirteen patients had minimal and 13 had severe CRS. Differentially expressed proteins between minimal and severe CRS at the peak timepoint are shown in (A; green represents IFNG responsive proteins). Not surprisingly, proteins involved in IL-6 and IFNG signalling were increased, including biomarkers of hemophagocytic lymphohistiocytosis (HLH) such as VSIG4, CXCL9, CXCL10, CD163. The IL-18 signalling axis was dysregulated at peak CRS in severe patients with markedly elevated IL18 and IL18BP, despite prior reports suggesting IL-18 up-regulation is unique to the late CRS seen with CART22 (PMID: 32925169). Soluble markers of checkpoint inhibition, including soluble PDL1 (CD274) and LAG3 were also highly elevated. Finally, biomarkers of endothelial damage, such as PLAT, TMSB10 and CALCA were significantly elevated in patients with severe CRS. Pathway analysis revealed significant dysregulation in targetable cytokine, chemokine, and signalling pathways (B). A volcano plot of differentially expressed proteins at pre-infusion (C) identified a single protein, MILR1, as a candidate biomarker that was highly differentially expressed in patients who would subsequently develop severe CRS. MILR1 expression decreased over time (D). An ROC of MILR1 as a predictor for development of severe CRS (E) demonstrated pre-infusion elevated MILR1 could accurately predict development of severe CRS (sensitivity 88%, specificity 97%, AUC=0.977). We identified correlates of MILR1 at pre-infusion and found that MILR1 correlated most highly with soluble FLT3 (R=0.86, p<0.01). Elevated levels of serum FLT3 at pre-infusion also predict severe CRS (F) with similar ROC as MILR1 (sensitivity 79%, specificity 93%, AUC=0.897). Interestingly, FLT3 decreased over time (F) and in an inverse pattern to FLT3 ligand (FLT3LG). Conclusions: With comprehensive secretome profiling we made multiple novel insights into the biology of CRS after CART19 and identified several potentially targetable proteins and pathways that could mitigate severe CRS. Similar secretome profiling in patients who developed neurotoxicity will also be shown. We identified two novel pre-infusion biomarkers that demonstrate significant capacity to predict the development of severe CRS following CART19 infusion. The inverse relationship apparent between FLT3 and FLT3LG that persists over time is an important finding that implies a potential biological role for FLT3/FLT3 ligand in the development of severe CRS. Mechanistic studies exploring the role of MILR1 and FLT3 in the initiation of CRS are ongoing. Figure 1 Figure 1. Disclosures Lambert: Novartis, shionogi, argenx, Rigel, octapharma: Consultancy; Rigel, Novartis, Sysmex, octapharma: Research Funding. Bassiri: Kriya Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company; Guidepoint Global: Consultancy. Levine: Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Other: Co-Founder and equity holder; Ori Biotech: Membership on an entity's Board of Directors or advisory committees; Immusoft: Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Avectas: Membership on an entity's Board of Directors or advisory committees; Akron: Membership on an entity's Board of Directors or advisory committees; In8bio: Membership on an entity's Board of Directors or advisory committees. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Barrett: Tmunity Therapeutics: Current Employment. Grupp: Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Teachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

292 Ex vivo profiling of PD-1 blockade using an organotypic tissue slice model in solid tumors

BackgroundTumor explant models provide a powerful ex vivo tool to evaluate complex biological mechanisms in a controlled environment. Ex vivo models retain much of the original tumor biology, heterogeneity, and tumor microenvironment, and therefore provide a useful preclinical platform and functional approach to assess drug responses rapidly and directly.MethodsTo explore mechanisms of resistance to cancer immunotherapy, we established an organotypic tissue slice Air-Liquid Interface (ALI) ex vivo system utilizing surgical tumor specimens from patients to assess the impact of the clinically utilized anti-PD-1 antibody nivolumab (OPDIVO). In the present study, we built a real-world patient cohort comprised of six tumor types: non-small cell lung cancer, melanoma, pancreatic ductal adenocarcinoma, breast cancer, prostate cancer, and colorectal cancer. We assessed tissue morphology, histology, PD-L1 IHC (CPS and TPS), CD8 T cell topology, proliferation in the tumor and stromal compartments, and secretome profiling.ResultsOur tumor slice model highly recapitulated features of the original tumor, including tumor architecture, immune phenotypes, and the prognostic markers. To identify responses to aPD-1 treatment, we compared baseline values for the cultured tumor slices with values at different timepoints post treatment. Secretome profiling of tissue explant supernatants using a panel of 94 analytes, revealed alterations to cytokines produced in the tumor microenvironment in response to aPD-1 treatment. We found that soluble expression patterns were associated with T-cell patterns (inflamed, excluded and desert) and PD-L1 score (CPS and TPS) in tumor tissues. These cytokines mediate critical functions across the immune cell cycle. Ongoing efforts to characterize T cell activation, exhaustion, tumor intrinsic responses and microenvironment composition using Imaging Mass Cytometry will be presented.ConclusionsIn this study, we demonstrated the feasibility of using fresh, surgically resected human tumors to test aPD-1 responses in an ex vivo system. Further, this model system has the potential to drive discovery and translational efforts by evaluating mechanisms of resistance to cancer immunotherapy and evaluate new single agent or combination therapies in the ex vivo setting.

AB0030 BETA BOSWELLIC ACID BLOCKS INNATE IMMUNE RESPONSES IN MULTIPLE OA JOINT CELLS

Background:Osteoarthritis (OA) incidence has skyrocketed in the last decade and yet a definitive treatment has still to be found. This worldwide disease is depriving our society from their life quality and has become a grave economic burden. Research on anti-inflammatory tools has been done on traditional Asian medicine. Boswellic acid is a plant-derived molecule from the Boswellia species that has shown to prevent cartilage loss in an OA mouse model[1]. However, the specific mechanism of action is still unclear. The activation of innate immune receptors, such Toll-like receptor 4 (TLR4) has been involved in chondrocyte-mediated inflammatory responses and OA development. Although, boswellic acid has shown an inhibitory effect on TLR4-mediated inflammatory responses little is known about its role on TLR4-mediated chondrocyte inflammatory and catabolic responses.Objectives:Determine the ability of beta boswellic acid (BBA) to block TLR4-mediated innate immune responses in chondrocytes and synoviocytes.Methods:In silicoThe binding affinity of beta boswellic acid (BBA) to TLR4 complex signalling was determined by optimized docking algorithm in the BIO-HPC Research Group facilities.In vitroCellular proteome and secretome profiling (LC-MALDI/TOFF) was used to study inflammatory pathways induced by the agonist of TLR4 (LPS [100ng/ml]) and IL1R (IL1β [0.1ng/ml]). The effect of BBA on TLR4-mediated innate immune responses was determined by RT-PCR, Western Blot and ELISA in primary human OA chondrocytes (hOC), murine ATDC5 chondrocytes, human synoviocytes (SW982) and primary human osteoblasts (hOB). Cell viability was tested using the methyl-thiazolyl-tetrazolium (MTT) reagent. Nitric oxide production in cell culture media was assessed by Griess reaction. Green Malachite Assay was used to semi-quantify the whole phosphoproteome.EthicsThis study was approved by the CEIC (CAEIG 2014/310).Results:Cellular proteome and secretome profiling validated the activation of TLR4 and IL1R signalling by LPS and IL1β ligands and revealed an enrichment in innate immune responses (NF-Kβ, NLRP3, MMPs, Interleukins, etc).Non-toxic doses of BBA [0.5-1000nM] prevented the activation of TLR4 in multiple articular joint cells and inhibited TLR4 & IL1R-dependant innate immune responses at the mRNA and protein level such as inflammatory factors IL6, NOS2, COX2, LCN2, MMP1, -3, -9, -13 and ADAMTS4, among others. Furthermore, NF-Kβ/IKBα and NLRP3/PYCARD/IL1β axis were also severely inhibited after BBA treatment. Moreover, these results were validated by in silico docking analysis that showed BBA interacted with TLR4/NF-Kβ.Conclusion:We prove that BBA inhibit TLR4 & IL1R -dependent innate immune responses in multiple human joint cells (Figure 1). We show that NF-Kβ & NLRP3 signalling, both associated to OA, are blocked (mRNA and protein) after BBA treatment (Figure 1).Our data support previous studies showing the prevention of cartilage loss in an OA animal models by BBA might come from its ability to inhibit TLR4 signalling. In the clinical practice of rheumatologists, Boswellia Serrata could be a useful nutraceutical to manage OA inflammation due to its content in BBA.Figure 1.References:[1]Wang, Q.; Pan, X.; Wong, H.H.; Wagner, C.A.; Lahey, L.J.; Robinson, W.H.; Sokolove, J. Oral and topical boswellic acid attenuates mouse osteoarthritis. Osteoarthr. Cartil.2014, 22, 128–132, doi:10.1016/j.joca.2013.10.012.Acknowledgements:Eloi Franco-Trepat and Ana Lois-Iglesias contributed equally to this work.This research has been funded by the non-profit FER (Fundación Española de Reumatologia /Spanish Foundation of Rheumatology) through the project “Búsqueda de nuevos fármacos bloqueantes de la inflamación asociada a TLR4 en condrocitos humanos artrósicos”.Disclosure of Interests:None declared

Cell type-selective secretome profiling in vivo

ABSTRACTSecreted polypeptides are a fundamental biochemical axis of intercellular and endocrine communication. However, a global understanding of composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection, and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte, and myeloid cell secretomes by direct purification of biotinylated secreted polypeptides from blood. Our secretome atlas validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types, and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by increased unconventional secretion of the cytosolic enzyme BHMT. This secretome profiling strategy enables dynamic and cell-type dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Differential Secretome Profiling of Human Osteoarthritic Synoviocytes Treated with Biotechnological Unsulfated and Marine Sulfated Chondroitins

Symptomatic slow-acting drugs (SYSADOA) are increasingly used as effective therapies for osteoarthritis, representing an attractive alternative to analgesics or non-steroidal anti-inflammatory drugs to relieve disease symptoms. Pharmaceutical preparations of chondroitin sulfate, derived from animal sources, alone or in combination with glucosamine sulfate, are widely recognized for their beneficial effect on osteoarthritis treatment. A growing interest has also been devoted to understanding the molecular mechanisms modulated by SYSADOA using -omic strategies, most of which rely on chondrocytes as a model system. In this work, by using an integrated strategy based on unbiased proteomics and targeted cytokine profiling by a multiplexed protein array, we identified differences in the secretomes of human osteoarthritic synoviocytes in response to biotechnological unsulfated, and marine sulfated chondroitins treatments. The combined strategy allowed the identification of candidate proteins showing both common and distinct regulation responses to the two treatments of chondroitins. These molecules, mainly belonging to ECM proteins, enzymes, enzymatic inhibitors and cytokines, are potentially correlated to treatment outcomes. Overall, the present results provide an integrated overview of protein changes in human osteoarthritic synoviocytes secretome associated to different chondroitin treatments, thus improving current knowledge of the biochemical effects driven by these drugs potentially involved in pathways associated to osteoarthritis pathogenesis.