Introduction Axatilimab (AXA), a high-affinity monoclonal antibody targeting colony-stimulating factor 1 receptor (CSF-1R), depletes CSF-1R–dependent monocytes and macrophages that potentiate inflammation and fibrosis in chronic graft-versus-host disease (cGVHD). Following the pivotal AGAVE-201 study (NCT04710576), AXA 0.3 mg/kg every 2 weeks (Q2W) was approved in the US for cGVHD after failure of ≥2 prior lines of therapy. Objectives To assess the pharmacodynamic (PD) changes in circulating monocytes and serum proteome of patients treated with AXA in AGAVE-201. Methods AGAVE-201 enrolled 241 patients (0.3 mg/kg Q2W, n=80; 1 mg/kg Q2W, n=81; 3 mg/kg every 4 weeks, n=80). Whole blood and serum were collected pre- and post-infusion. Monocytes were analyzed by flow cytometry and defined as CD45+ → CD14+ HLA-DR+, with subtypes defined as classical (CD14high/CD16low), nonclassical (NC; CD14low/CD16high), and intermediate (CD14high/CD16high). CSF-1R ligands CSF-1 and IL-34 were quantitated by enzyme-linked immunosorbent assays. Proteomic analysis was performed using OLINK Explore HT (>5000 proteins). Differentially expressed proteins (DEPs) in the 1 mg/kg and 3 mg/kg cohorts were identified at Cycle 1 Day 8 (C1D8) and Cycle 2 Day 1 (C2D1) and compared with baseline based on a fold change ≥1.5 and P≤0.05. Results AXA induced time- and dose-dependent increases in CSF-1 and IL-34 serum levels and decreases in NC monocytes. Interpretation of PD effects at the 0.3 mg/kg dose was limited due to infrequent sampling; however, consistent with prior studies, transient reductions in NC monocytes were observed at Day 3–4 after dose, returning to baseline by Day 8 in a healthy volunteer study conducted in Japan. Changes across the dose range were consistent with the extent of AXA exposure and clearance. DEPs were identified at 1 mg/kg (C1D8, 193 DEPs; C2D1, 47 DEPs) and 3 mg/kg (C1D8, 504 DEPs; C2D1, 304 DEPs) relative to baseline. Proteins impacted by AXA included soluble cellular markers expressed by CSF-1R+cells (eg, monocytes, macrophages, dendritic cells); CLEC10A, CD163, CD300E, EB13, SIGLEC-10, and ECM1 were significantly reduced by AXA. Notably, skin matrix proteins (keratin 5 and collagen types V and IX) were upregulated and inversely correlated with skin tightening clinical scores, suggesting a potential mechanistic link to improvements in skin fibrosis. Conclusion Proteomic analysis of AGAVE-201 revealed changes in soluble markers associated with CSF-1R–expressing cells and reductions in the number and cellular function of CSF-1R–regulated monocyte lineage, contributing to disease resolution. Biomarkers that correlate with skin tightening improvement may indicate potential involvement in cGVHD skin disease and/or resolution post-AXA. These findings support the mechanism of action of AXA and provide hemodynamic evidence of target engagement and tissue-specific effects.

Skin is the most frequently involved organ in chronic graft-versus-host disease (cGVHD) and presents as superficial lichenoid lesions and deep, fibrotic sclerosis that may arise sequentially. Preclinical models have demonstrated cGVHD involves colony stimulating factor-1 receptor (CSF-1R)-dependent tissue macrophage infiltration. However, spatial and temporal analysis of immune responses at high resolution has not been possible in human tissue to date, a major limitation to our understanding of disease pathophysiology and responses to therapy. We undertook sub-cellular spatial transcriptomics with the 10x Xenium platform to dissect tissue immune networks involved in human patient skin, first from lichenoid cGVHD biopsies relatively early after HCT (n=8), then before and after treatment with the CSF-1R-blocking antibody axatilimab in later sclerotic phases in patients enrolled in the AGAVE-201 trial that led to FDA approval (Wolff D et al, NEJM 2024) (n=5 responders + 5 non-responders). Skin with confirmed lichenoid cGVHD histopathology segregated into high (HI) and low (LI) immune cell-infiltrated tissue that reflected macrophage and T cell abundance. Non-cGVHD biopsies collected before or after HCT served as controls. While CSF1Rhi macrophages were equally abundant irrespective of cGVHD, an alternatively activated CSF1RintLYZhi population was the predominant subset differentially infiltrating HI skin (p=0.0011) and resided closest to basal keratinocytes (BKs) by proximity analysis. CD8 T cells were the dominant infiltrating lymphocytes distinctive to HI skin (p=0.0016) and in close bidirectional proximity to CSF1RintLYZhi macrophages (p<0.0001). These immune cells were, in turn, located adjacently to BKs, forming a putative pathogenic triad. Highly aberrant spinous and basal keratinocyte (BK) differentiation was seen deep within cGVHD epidermis. Closer BK proximity of CSF1RintLYZhi macrophages and type-1 and type-17 T cells (expressing IFNG, TBX21 and IL17A, RORC respectively) was associated with increased BK transcripts associated with immune cell recruitment (GJB2) and antigen presentation (CD74), suggesting immunomodulatory functions of keratinocytes in cGVHD. These HI keratinocytes bore high TGFB1 and ZEB2 and low CDH1 expression, consistent with epithelial-mesenchymal transition. Within the dermis, major fibroblast subsets were ablated during cGVHD, including the papillary fraction abutting the epidermal-dermal border and the dominant reticular LGR5+-equivalent subset (p=0.0376), pivotal to tissue organization and homeostasis. A proportion (5-10%) of residual skin fibroblasts were donor origin and expressed features of macrophage-mesenchymal transition. We next analyzed pre- and post-treatment skin biopsies from patients with steroid-refractory sclerosis enrolled on the AGAVE-201 study. As opposed to lichenoid cGVHD, cellular infiltration, including CSF-1R+ macrophages, was relatively sparse in this late phase of disease. Nevertheless, overall response to axatilimab was associated with a significant depletion of macrophages, including CSF-1Rhi and CSF1RintLYZhisubsets and concurrent increases in FoxP3+ regulatory T cells in the skin (p<0.05), suggesting that a systemic regulatory response may mediate clinical response. These immune subsets were unchanged in non-responders. LGR5+ fibroblasts, the main reticular population, demonstrated increased baseline collagen, M2 and myofibroblast-polarizing and collagen-amplifying gene transcripts in axatilimab non-responders. Similarly, papillary fibroblasts from non-responders expressed high baseline levels of profibrogenic TGF-β target genes. In contrast, papillary fibroblasts were characterized by lower activation states in responders. These data highlight a specific, aberrant CSF1R+ tissue macrophage population in lichenoid skin cGVHD and its colocalization in a putative pathogenic triad with CD8 T cells at the BK interface culminating in profound, previously unrecognized abnormalities in keratinocyte and fibroblast differentiation and mesenchymal transition—potential harbingers of fibrosis. The deletion of CSF-1R+ macrophages with axatilimab in more sparsely infiltrated sclerotic phases of cGVHD was associated with Treg expansion and clinical response. These data confirm the central role of macrophages in human cGVHD, provide a putative mechanism of response to CSF-1R inhibition, and suggestion treatment at the lichenoid stage may represent an optimal therapeutic window.

ABSTRACT:Cytokine release syndrome (CRS) and immune effector cell (IEC)-associated neurotoxicity syndrome (ICANS) are common complications after IEC therapy for hematologic malignancies. This 2-part phase 2 study (INCB 39110-211) investigated the safety and efficacy of itacitinib, a potent, highly selective Janus kinase 1 inhibitor with broad anti-inflammatory activity, for the prevention of CRS and ICANS in patients who received commercial CD19-directed IEC therapy. Patients in part 1 received 200 mg itacitinib once daily 3 days before IEC therapy (axicabtagene ciloleucel [axi-cel], brexucabtagene autoleucel, or tisagenlecleucel) through day 26 with guidelines for use of other CRS/ICANS interventions. In part 2 (double-blind), patients were randomized to receive 200 mg itacitinib twice daily or placebo 3 days before IEC therapy with axi-cel. The primary end point was the proportion of patients with CRS grade ≥2 by day 14 using the American Society for Transplantation and Cellular Therapy consensus grading system. Overall, 111 patients were enrolled (63 in part 1; 48 in part 2); 109 patients were analyzed for efficacy and 110 for safety. By day 14, grade ≥2 CRS occurred in fewer patients on 200 mg twice daily itacitinib (17.4%) than on placebo (56.5%; P = .003). The proportion of patients with grade ≥2 ICANS by day 28 was lower than with placebo (8.7% vs 21.7%). Itacitinib was well tolerated, with pyrexia being the most common treatment-emergent adverse event (200 mg itacitinib twice daily, 43.5%; placebo, 50.0%), and itacitinib-related cytopenias were manageable. Itacitinib did not affect IEC therapy efficacy (objective response rate at 6 months, 39.1% [200 mg itacitinib twice daily] vs 26.1% [placebo]). This study was registered at www.clinicaltrials.gov as #NCT04071366.

BackgroundProphylactic treatment with itacitinib (ITA), a potent, selective oral Janus kinase (JAK)1 inhibitor, to manage onset and severity of cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in response to CAR-T infusion was evaluated in the phase 2 study INCB 39110-211 (NCT04071366). The study had 2 parts: part 1 included patients treated with ITA 200 mg once daily (QD); part 2 was randomized and double-blind and evaluated ITA 200 mg twice daily (BID) vs placebo. Clinical responses relative to placebo in part 2 indicated that ITA reduced onset and severity of CRS and ICANS (Frigault M, et al, abstract accepted at American Society of Hematology [ASH] 2023 Annual Meeting). Based on data from part 1, cytokines were elevated in response to CAR-T treatment as expected and correlated with CRS and ICANS grades. Importantly, CAR-T expansion was not impacted at the ITA QD dose compared with historical values (Pratta M, et al, ASH 2022 Annual Meeting, Poster #4649).ObjectivesTo assess the impact of ITA on CAR-T expansion and on selected circulating cytokines compared with placebo control in study INCB 39110-211.MethodsWhole-blood peripheral mononuclear cells and serum were collected before and during ITA treatment and from the peri–CAR-T (axicabtagene ciloleucel) infusion period from patients with diffuse large B-cell lymphoma. Cytokine analysis was performed using a fully automated microfluidic immunoassay system (Protein Simple Ella, San Jose, CA). CAR-T levels were quantitated using a droplet digital polymerase chain reaction (PCR) assay (Bio Rad, Hercules, CA). Statistical analysis of longitudinal data was performed using the limma package in R. Comparison of individual time points and correlation to CRS and ICANS grade was performed by t-test.ResultsSystemic inflammatory cytokine concentrations, including tumor necrosis factor alpha (TNF-a) and interleukin (IL)-6, were increased 1–5 days after CAR-T infusion and were both significantly reduced by ITA (Figure 1A). ITA caused a dose-dependent reduction in TNF-a concentrations (Figure 1B), and peak TNF-a concentrations appeared to correlate with CRS and ICANS grades based on limited patient numbers (Figure 1C). IL-2RA concentrations were significantly reduced by ITA and were associated with a corresponding significant increase in systemic IL-2 concentrations. CAR-T expansion data will be available upon presentation.ConclusionsJAK1 inhibition with ITA reduced systemic cytokines associated with CAR-T treatment, including TNF-a, IL-6, and IL-2RA, and increased IL-2 concentrations compared with placebo-treated patients receiving CAR-T. A dose-dependent reduction of TNF-a by ITA correlated with CRS and ICANS grade, suggesting TNF-a may represent a suitable biomarker for CAR-T toxicities managed by ITA.

Introduction: Chronic graft-versus-host disease (cGVHD) is an immune-mediated serious and life-threatening complication after allogeneic hematopoietic stem cell transplantation, occurring in 30%-70% of patients. The pathophysiology of cGVHD may involve inflammation, cell-mediated immunity, humoral immunity, and fibrosis. Axatilimab is a high-affinity humanized IgG4 (kappa light chain) monoclonal antibody targeting colony-stimulating factor 1 receptor (CSF-1R) that is under investigation for the treatment of cGVHD. CSF-1R is a member of the receptor protein tyrosine kinase family of growth factor receptors and is expressed on monocyte precursors, monocytes, and macrophages. Signaling by colony-stimulating factor 1 (CSF-1) or interleukin-34 (IL-34) through CSF-1R has been shown to be the primary regulatory pathway for determining development and differentiation of the mononuclear phagocytic cell lineage. Recent work has established a role for monocyte-derived macrophages as the pathogenic population of cells that establish the fibrotic niche and drive the fibrotic process. Upon entry of monocytes into tissues, the tissue microenvironment regulates their differentiation, resulting in a heterogeneous pool of cells ranging from pro-inflammatory (often referred to as M1 macrophages) to immune suppressive/wound healing (M2 macrophages). An imbalance in the number and activity of these macrophage populations is believed to result in the excess production of fibrotic growth factors, such as transforming growth factor-β and platelet-derived growth factor, leading to uncontrolled fibroblast activation and subsequent fibrosis. Blocking CSF-1R signaling with axatilimab is expected to reduce the circulating levels of pathogenic monocyte-derived macrophage precursors and inhibit their activation in tissues, thereby providing an opportunity to therapeutically intervene in diseases such as cGVHD. The results described herein support this assertion by demonstrating the direct impact of axatilimab exposure to skewing differentiating monocytes away from a pro-inflammatory and pro-fibrotic phenotype. Methods: Primary human monocytes were differentiated in the presence or absence of ascending doses of axatilimab (0.07-7 μM) into distinct phenotypes: activated (M0), pro-inflammatory (M1-like), and suppressive (M2-like) macrophages. Supernatants and cell pellets were then assessed for secreted protein (Luminex Corporation) and gene expression (NanoString Technologies) profiles, respectively. Data were analyzed in R (version 4.1.1). Results: Gene expression profiles of pro-inflammatory cytokines and chemokines (eg, IL-1β, chemokine CC motif ligand 2 [CCL2], CCL7, CCL24, and CCL22) from primary differentiated macrophage subsets were dramatically reduced in the presence of ascending concentrations of axatilimab. Further, macrophages exposed to axatilimab while differentiated under pro-inflammatory conditions showed increased levels of c-MYC expression compared with control cultures. c-MYC is a transcription factor associated with suppressive M2-like macrophages, which suggests that axatilimab interferes with pro-inflammatory macrophage differentiation. Consistent with this, expression of M2-associated cytokines, including IL-10, IL-5, and IL-4, was enhanced in axatilimab-treated macrophage cultures. Conclusions: Blocking CSF-1R signaling in primary, differentiated macrophages dramatically alters the secretomes and gene expression profiles of these cells. Macrophages are key inducers of fibroblast growth factors, which in turn promote fibrotic disease. These data confirm that production of inflammatory and pro-fibrotic factors by pro-inflammatory macrophages is inhibited in the presence of axatilimab, thereby supporting the notion that axatilimab provides a unique opportunity to therapeutically intervene or prevent fibrosis in cGVHD.

Background: Chronic GVHD (cGVHD) occurs in 30-70% of allogeneic hematopoietic cell transplantation patients, and the rate has increased over the past 2 decades. Based on the degree of organ involvement, cGVHD is classified as mild, moderate, or severe. Standard treatment includes systemic corticosteroids (CS), together with immunosuppressive agents. GRAVITAS-309 (NCT03584516) was designed as a 2-part, multicenter, randomized trial to assess efficacy and safety of itacitinib, an orally bioavailable, selective JAK1 inhibitor, in combination with CS as first-line treatment for moderate to severe cGVHD. The dose-finding, open-label, randomized portion of the study initially investigated itacitinib at 200 mg once daily (qd) and 300 mg qd in combination with CS (methylprednisolone or prednisone); both doses were well tolerated. The trial was expanded to test itacitinib 400 mg qd and 300 mg twice daily (bid); the initial 300 mg qd cohort was expanded and a CS monotherapy cohort was added. Results from the expanded portion of the study (n=139 patients) showed improved overall response rate (ORR) at 6 months in patients treated with itacitinib + CS vs CS alone (Im, et al. Blood 2022;140[Suppl 1]:1870). Here, we analyzed samples collected from patients in 4 cohorts (cohort A [300 mg qd], cohort B [400 mg qd], cohort C [300 mg bid], and cohort D [CS alone]) for systemic proteins and immune cell populations, to support investigation into the itacitinib mechanism of action and to correlate with clinical outcomes. Methods: Peripheral blood mononuclear cells (PBMCs) and serum were collected before treatment (baseline, day 1), and day 7, day 14 (PBMCs only), and day 28 following treatment initiation. Serum proteins were analyzed using the OLINK Target 96 platform, and circulating T-cell subsets were measured using flow cytometry of PBMCs and presented as a percentage of parent population. Samples were assessed irrespective of patients' response. Most of the patients (70-100% per cohort) with samples available for analysis were responders (achieved complete or partial responses). Results: Serum levels of proteins, including several proposed as potential prognostic cGVHD biomarkers, changed significantly at day 28 relative to baseline following treatment with itacitinib (Table 1). Levels of elafin, a marker of acute GVHD of the skin, were significantly reduced and levels of dickkopf-3 (DKK3), a marker of sclerotic and nonsclerotic cGVHD, were significantly increased in itacitinib cohorts only. Based on a repeated measures correlation analysis, both markers significantly correlated with overall clinical skin scores (elafin: r=+0.43; DKK3: r=-0.53; both P<0.001). Serum levels of other reported markers of cGVHD, including osteopontin, C-X-C motif chemokine ligand (CXCL) 9 and CXCL10, were significantly decreased exclusively in itacitinib-treated cohorts, and not in the CS only cohort. Some proteins, including matrix metalloproteinase (MMP) 3, increased in all cohorts, suggesting that changes in these proteins cannot be attributed to itacitinib alone, but may reflect response to CS; other proteins, such as suppression of tumorigenicity (ST2), remained unchanged in response to treatment in all cohorts. CS treatment alone resulted in an increase in naive CD4 T cells (CD4 +CD8 ->CD45RA +CCR7 -) and a corresponding reduction of CD4 effector memory T cells (CD4 +CD8 ->CD45RA -CCR7 +) within 28 days (Figure 1, cohort D). Changes in naive CD4 T cells following CS monotherapy were significantly attenuated by itacitinib ( P<0.05 vs CS at day 28), particularly at the 400 mg qd dose (Figure 1, cohort B), which demonstrated the best ORR at 6 months (53% with 400 mg qd vs 36% with CS). Conclusions: Analysis of patient samples from GRAVITAS-309 revealed that serum elafin and DKK3 levels significantly correlated with skin GVHD scores, possibly reflecting the systemic origin of observed skin manifestations. Serum levels of osteopontin, CXCL9, and CXCL10 were specifically reduced by itacitinib in patients with cGVHD and may represent potential pharmacodynamic markers. Observed changes in different peripheral T-cell populations in patients' samples following itacitinib treatment were consistent with those reported in murine GVHD models, suggesting a potential role of naive CD4 + T cells in driving this disease. Planned future analyses will include identification of novel biomarkers that correlate with organ involvement.

Background: Treatment with chimeric antigen receptor T-cell (CAR-T) therapies has been associated with durable clinical responses in patients with relapsed/refractory B-cell malignancies. However, these treatments can be complicated by cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Janus kinase (JAK) pathways are important for the cytokine signaling involved in CRS and ICANS pathogenesis. Itacitinib, a potent, selective, oral JAK1 inhibitor, was evaluated to manage onset and severity of CRS and ICANS in response to CAR-T infusion in a phase 2 clinical trial (NCT04071366). Preliminary results demonstrated that prophylactic itacitinib treatment in patients receiving CAR-T therapy resulted in reduced onset and severity of CRS and ICANS (Frigault M et al, abstr #OS15-07, European Society for Blood and Marrow Transplantation Annual Meeting [EBMT] 2022). To assess whether JAK inhibition affects CAR-T expansion and function, circulating CAR-Ts were analyzed from patient samples from the phase 2 trial. Methods: Whole-blood peripheral blood mononuclear cells were collected before and during itacitinib treatment (200 mg once daily) and from the peri-CAR-T infusion period. CAR-T levels were quantitated using a droplet digital polymerase chain reaction (PCR) assay (Bio Rad, Hercules, CA). CAR-Ts were visualized using RNAScope technology (Advanced Cell Diagnostics [ACD], Newark, CA). Functional readouts were based on interferon (IFN)-g and granzyme B expression and were quantitated by HALO® image analysis platform (ACD). Serum cytokine analysis was performed using a fully automated microfluidic immunoassay system (Protein Simple Ella, San Jose, CA). Results: Thirty-one patients received itacitinib before infusion with axicabtagene ciloleucel (axi-cel); of these, 28 were diagnosed with diffuse large B-cell lymphoma (DLBCL). Expansion of CAR-Ts was quantitated and normalized to total white blood cell (WBC) counts and plotted over time (Figure 1A). Peak levels of CAR-Ts were observed between 7 and 14 days postinfusion in the presence of a JAK inhibitor, consistent with published CAR-T expansion data. Kinetic analysis of patients with DLBCL (Figure 1B) resulted in area under the curve and maximum observed concentration levels comparable to historical values. RNAScope technology allowed visualization of CAR-Ts in patient samples at Day 7 or 14 postinfusion, and the cells expressed functional markers IFN-g and granzyme B. Systemic inflammatory cytokine levels, including interleukin (IL)6, IFN-g, IL2, and granulocyte-macrophage colony-stimulating factor, were increased as early as 1 to 3 days after CAR-T infusion and correlated with CRS and ICANS grades. Conclusion: Patients receiving commercial CAR-T products co-treated with itacitinib demonstrated kinetic parameters of CAR-T expansion similar to historical values. Functional marker expression and cytokine production suggest that JAK inhibition at the current study dose has no deleterious effect on CAR-T function. The current study will expand with a randomized, double-blind, placebo-controlled portion that will enroll patients with DLBCL receiving axi-cel with itacitinib (200 mg twice daily) vs placebo. The assays described will be used to further investigate CAR-T proliferation, persistence, and function in the presence of either itacitinib or placebo. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract In the tumor microenvironment, dying cells release ATP which gets converted to AMP by CD39. AMP is subsequently converted to immunosuppressive adenosine by the extracellular 5'-nucleotidase CD73. CD73 is linked to the cell surface by a GPI-anchor and can be released into the plasma as soluble CD73. In order to lower extracellular levels and reverse the immunosuppressive activity of adenosine in the tumor microenvironment, an antibody was researched and discovered that antagonizes CD73 function. INCA00186 is a humanized monoclonal antibody that binds and inhibits CD73 function. INCA00186 binds human and cynomolgus CD73 with sub-nanomolar affinity, and inhibits CD73 enzymatic activity in a manner that is non-competitive for AMP binding. In functional studies, INCA00186 restored effector T cell proliferation in the presence of high concentrations of AMP. In the human A375 melanoma tumor model in CD34+ humanized NSG mice, INCA00186 decreased levels of cell surface CD73, displayed high receptor occupancy on tumor cells, and decreased CD73 activity in ex vivo assays of tumor homogenates. In the human MDA-MB-231 breast tumor model in CD34+ humanized NSG mice, INCA00186 decreased intratumoral adenosine concentrations. In combination with the novel A2A/A2B adenosine receptor antagonist INCB106385, INCA00186 synergistically restored effector T cell activity as measured by interferon gamma (IFNγ) production in the presence of high concentrations of AMP. In the human MDA-MB-231 breast tumor model in CD34+ humanized NSG mice, combination treatment with INCA00186 and INCB106385 controlled tumor growth significantly better than monotherapies. In summary, the data presented in this study demonstrates that INCA00186 is a potent CD73 antagonist and effectively attenuates adenosine induced immunosuppression in the tumor microenvironment. INCA00186 demonstrated anti-tumor immunity alone and, to a greater extent, in combination with the A2A/A2B adenosine receptor antagonist INCB106385. Citation Format: Shaun Stewart, Rebecca Buonpane, Jing Zhou, Michael Hansbury, Michael Smith, Hui Wang, Lu, Bin Su, Rahel Awdew, Cheng-Yen Huang, Ashwini Kulkarni, Shane Harvey, Arpita Mondal, Steve Wang, Christina Stevens, Michael Pratta, Elham Behshad, Alexandra Fanuka, Xiaodi Ren, Holly Koblish, Horacio Nastri, Patrick Mayes. Discovery and preclinical characterization of INCA00186, a humanized monoclonal antibody antagonist of CD73, as a cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB174.

Acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic cell transplantation (HCT) is a primary cause of nonrelapse mortality and a major barrier to successful transplant outcomes. Itacitinib is a Janus kinase (JAK)1-selective inhibitor that has demonstrated efficacy in preclinical models of aGVHD. We report results from the first registered study of a JAK inhibitor in patients with aGVHD. This was an open-label phase 1 study enrolling patients aged ≥18 years with first HCT from any source who developed grade IIB to IVD aGVHD. Patients with steroid-naive or steroid-refractory aGVHD were randomized 1:1 to itacitinib 200 mg or 300 mg once daily plus corticosteroids. The primary endpoint was safety and tolerability; day 28 overall response rate (ORR) was the main secondary endpoint. Twenty-nine patients (200 mg, n = 14; 300 mg, n = 15) received ≥1 dose of itacitinib and were included in safety and efficacy assessments. One dose-limiting toxicity was reported (grade 3 thrombocytopenia attributed to GVHD progression in a patient receiving 300 mg itacitinib with preexisting thrombocytopenia). The most common nonhematologic treatment-emergent adverse event was diarrhea (48.3%, n = 14); anemia occurred in 11 patients (38%). ORR on day 28 for all patients in the 200-mg and 300-mg groups was 78.6% and 66.7%, respectively. Day 28 ORR was 75.0% for patients with treatment-naive aGVHD and 70.6% in those with steroid-refractory aGVHD. All patients receiving itacitinib decreased corticosteroid use over time. In summary, itacitinib was well tolerated and demonstrated encouraging efficacy in patients with steroid-naive or steroid-refractory aGVHD, warranting continued clinical investigations. This trial was registered at www.clinicaltrials.gov as #NCT02614612.

116 RNAScope is a sensitive, specific platform to detect IDO1 expression in tumor tissue sections. M Pratta, M Rupar, P Waeltz, T Burn, G Hollis, M Covington, M Smith, and R Newton. Incyte Corp. Wilm. DE. Background: Indoleamine 2,3-Dioxygenase 1 (IDO1) catalyzes the primary and rate-limiting step in tryptophan catabolism to generate N-formyl-kynurenine (Kyn). Through a combination of local depletion of tryptophan and an increase in Kyn concentrations, IDO-1 activity can result in the suppression of antitumor immune responses. Because IDO-1 inhibitors are now in the clinic for treatment of multiple tumor types, immunohistological approaches are employed to demonstrate IDO1 expression in tumor biopsies. However, using a commercially available antibody to detect IDO1 by immunohistochemistry (IHC), the level of sensitivity was inadequate. Methods: In order to improve the sensitivity of IDO1 detection, we evaluated in situ hybridization (ISH) using RNAScope technology and digital quantitation by HALO analysis in collaboration with Advanced Cell Diagnostics (ACD). The technology was cross-validated using IDO1 qRT-PCR, Western blot, and activity analysis and compared with standard IHC. We initially evaluated IDO1 expression in HeLa cells stimulated with various concentrations of IFNγ, and then extended the observations using tissue sections from multiple tumor types. Results: In the HeLa cell model, IFNγ induced a time- and concentration-dependent increase of IDO1 at the mRNA, protein, and activity level. Although IDO1 was successfully detected in the HeLa cell samples by IHC, comparison of the platforms indicated IFNγ EC50 values were in strong agreement between RNAScope (193.8 pg/ml) and Western blot analysis (170.8 pg/ml), but was much higher by IHC analysis (2206 pg/ml). A strong positive correlation (*p < 0.0001) between RNAScope and Western blot analysis was observed, suggesting a highly coordinated induction of IDO1 by IFNγ at both the mRNA and protein levels. FFPE tumor tissue from melanoma, HNSCC, bladder, renal, ovarian, and lung cancers visualized by RNAScope all show varying levels of IDO1 expression. Conclusions: These data support the use of RNAScope for the analysis of IDO1 expression in clinical trials.