Published OnlineFirst November 13, 2018; DOI: 10.1158/2326-6066.CIR-18-0245 Cancer Immunology Miniature Cancer Immunology Research Autoantibody Development under Treatment with Immune-Checkpoint Inhibitors Emma C. de Moel1, Elisa A. Rozeman2, Ellen H. Kapiteijn3, Els M.E. Verdegaal3, Annette Grummels4,JaapA.Bakker4, Tom W.J. Huizinga1, John B. Haanen2,3, Rene E.M. Toes1, and Diane van der Woude1 Abstract Immune-checkpoint inhibitors (ICIs) activate the and any irAEs [OR, 2.92; 95% confidence interval (CI) immune system to assault cancer cells in a manner that is 0.85–10.01]. Patients with antithyroid antibodies after ipi- not antigen specific. We hypothesized that tolerance may limumab had significantly more thyroid dysfunction under also be broken to autoantigens, resulting in autoantibody subsequent anti–PD-1 therapy: 7/11 (54.6%) patients with formation, which could be associated with immune-related antithyroid antibodies after ipilimumab developed thyroid adverse events (irAEs) and antitumor efficacy. Twenty-three dysfunction under anti–PD1 versus 7/49 (14.3%) patients common clinical autoantibodies in pre- and posttreatment without antibodies (OR, 9.96; 95% CI, 1.94–51.1). Patients sera from 133 ipilimumab-treated melanoma patients were who developed autoantibodies showed a trend for better determined, and their development linked to the occurrence survival (HR for all-cause death: 0.66; 95% CI, 0.34–1.26) of irAEs, best overall response, and survival. Autoantibodies and therapy response (OR, 2.64; 95% CI, 0.85–8.16). We developed in 19.2% (19/99) of patients who were autoan- conclude that autoantibodies develop under ipilimumab tibody-negative pretreatment. A nonsignificant association treatment and could be a potential marker of ICI toxicity was observed between development of any autoantibodies and efficacy. Introduction autoantibodies were formed with ICI treatment and investigated their association with irAEs and clinical outcome. Immune-checkpoint inhibitors (ICIs) have improved the pre- viously dismal prognosis of patients with various types of cancer, but at the cost of immune-related adverse events (irAEs), includ- Materials and Methods ing arthritis, colitis, hepatitis, and various endocrinopathies (1). Patients and serological measurements ICIs inhibit negative costimulatory signals to T cells, thereby For this analysis, we included 133 patients with late-stage enhancing antitumor T-cell responses (2). Because this mode of melanoma who were treated with ipilimumab, a CTLA-4 inhib- action is not antigen-specific, ICIs may also (re)activate otherwise itor, and for whom pre- and posttreatment serum or plasma dormant autoreactive T cells. This, in turn, might lead to a break in samples were available. Patients were treated with a maximum T-cell tolerance to not only tumor antigens but also autoantigens, of four cycles of ipilimumab 3 mg/kg in an expanded resulting in activation of autoreactive B cells and ultimately the access program or according to the label after approval at the formation of autoantibodies. If true, the occurrence of autoanti- Netherlands Cancer Institute or the Leiden University Medical bodies may be associated with more frequent irAEs. Production of Center. Patients were included if they were at least 18 years of autoantibodies may indicate enhanced global immunogenicity, age and had histologically or cytologically proven irresectable which may, in turn, be associated with better antitumor responses, stage IIIc or IV melanoma, with measurable metastatic lesions as has been reported for changes in the T-cell repertoire (3–5). according to the RECIST 1.1 criteria. Patients were treated with Therefore, we determined if autoimmune disease-associated four cycles of intravenous 3 mg/kg ipilimumab every 3 weeks. Sixty-six (49.6%) patients were treated with anti–PD-1 therapy following ipilimumab: either 2 mg/kg intravenous pembroli- 1Department of Rheumatology, Leiden University Medical Center, Leiden, the zumab every 3 weeks or 240 mg intravenous nivolumab every Netherlands. 2Netherlands Cancer Institute, Amsterdam, the Netherlands. 4 weeks. The study was conducted in accordance with the 3 Department of Medical Oncology, Leiden University Medical Center, Leiden, Declaration of Helsinki after approval by the institutional 4 the Netherlands. Department of Clinical Chemistry and Laboratory Medicine, review boards of both centers. All patients signed informed Leiden University Medical Center, Leiden, the Netherlands. consent for withdrawal of extra blood samples for biomarker Note: Supplementary data for this article are available at Cancer Immunology analysis. According to the study protocol, serum or plasma for Research Online (http://cancerimmunolres.aacrjournals.org/). autoantibody determination was collected before initiation of Corresponding Author: Emma C. de Moel, Department of Rheumatology, ipilimumab treatment and 12 weeks after. Pre- and posttreat- Leiden University Medical Centre, Albinusdreef 2, 2333ZA Leiden, the ment serum was snap-frozen and stored at À80Cuntilauto- Netherlands. Phone: 0031 (0)71 52 65652; E-mail: [email protected] antibody determination. Due to failed measurements, post- doi: 10.1158/2326-6066.CIR-18-0245 ipilimumab autoantibody status could not be determined in Ó2018 American Association for Cancer Research. 4 patients (Supplementary Fig. S1). 6 Cancer Immunol Res; 7(1) January 2019 Downloaded from cancerimmunolres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst November 13, 2018; DOI: 10.1158/2326-6066.CIR-18-0245 Autoantibodies under Immune-Checkpoint Inhibitors Indirect immunofluorescence assays adamantyl dioxetane phosphate tracer and were performed using Autoimmune hepatitis and primary biliary cirrhosis-associated reagents provided by the manufacturer according to instructions antismooth muscle, antimitochondrial, and anti-liver/kidney in the package insert. microsome (LKM) antibodies were measured by indirect immu- nofluorescence assay (IFA) using mouse liver/kidney/stomach Clinical data substrate (Aesku). Antinuclear antibodies (ANA) were deter- Information about demographics, treatment response, survival mined in all patients by IFA using the HEp-2000 ANA Test System status, and the occurrence of irAEs was obtained from retrospec- which uses human epithelioid cells stably transfected with the tive review of medical records. irAEs were recorded starting from SSA/Ro autoantigen, cultured, and fixed directly on the test wells the first ipilimumab treatment until one year later, death, or the (Immuno Concepts). Patient serum samples at a dilution of 1:40 start of different therapy (whichever occurred first), using Com- were incubated with antigen substrate for 30 minutes at room mon Terminology Criteria for Adverse Events version 4.03: any temperature to allow specific binding of autoantibodies to cell grade arthralgia/arthritis, colitis, hypophysitis, primary adrenal nuclei. After washing with phosphate-buffered saline to remove insufficiency, primary thyroid dysfunction, dermatitis (rash, vit- nonspecifically bound antibodies, the substrate was incubated iligo, or psoriasis), uveitis, or grade 3–4 hepatitis. Primary thyroid with an anti-human antibody conjugated to fluorescein. After dysfunction as an irAE during anti–PD-1 treatment (nivolumab or another washing step, the nuclear staining pattern was read using pembrolizumab) following ipilimumab treatment was deter- the international consensus on antinuclear antibody pattern mined in the same manner. Hematologic and serum parameters (ICAP; ref. 6) by two experienced, independent readers trained necessary for making the above diagnoses were determined at in ANA-pattern reporting and blinded to time order and patient baseline, every 3 months during follow-up, at progressive disease, data of samples. In the case of lack of consensus, a third reader and according to the treating oncologist's clinical judgment. Three functioned as tiebreaker. All system reagents, conjugates, calibra- patients had preexisting hypothyroidism. Thyroid dysfunction tors, and positive and negative controls were provided by and was registered only as an irAE in these patients if symptoms were used according to instructions of the manufacturer (Immuno aggravated and a new medical intervention was indicated. Two of Concepts). All steps of the IFA were conducted using a Helmed the four cases of arthralgia/arthritis constituted a flare of preexist- fully automated IFA slide processor (Aesku). ing rheumatoid arthritis (RA). Survival was defined as time from start of ipilimumab to death of any cause, recorded between start Fluorescence enzyme immunoassays of first ipilimumab treatment until January 2018, for a median Anticyclic citrullinated peptide 2 (CCP2) IgG, rheumatoid follow-up time of 20.4 months (IQR: 8.8–40.8). Radiologic factor (RF) IgM, antigliadin IgG, and (if ANA was positive by evaluation (CT or PET/CT scanning) was performed at baseline, IFA) antibodies to extractable nuclear antigens (ENA) were deter- week 12, and subsequently every 3 months until progression. mined by EliA technique on a Phadia ImmunoCap 250 instru- Response was scored according to RECIST 1.1 criteria. Best overall ment (Thermo Fisher Scientific). This is a fully automated and response was defined as the best response recorded from start of high-throughput fluorescence enzyme immunoassay system used ipilimumab until date of progression, death, or the start of a for routine diagnostic laboratory testing. The fluorescence