Anti-integrin v6 autoantibodies are a potential biomarker for ulcerative colitis-like immune checkpoint inhibitor … – Nature.com

Patients

This was a retrospective study targeting patients with ICI-induced colitis who had undergone colonoscopy at the onset of the disease at Kyoto University Hospital, affiliated hospitals, and Kindai University Faculty of Medicine between April 2018 and April 2023. All but one patient (Case 1) was treated with only ICI treatment. ICI-induced colitis was defined as diarrhea or bloody stools following ICI administration and/or with histological evaluation [5]. Patients with preexisting IBD and infectious enteritis caused by pathogenic microorganisms, such as Clostridioides difficile, Campylobacter jejuni, and Cytomegalovirus, were excluded. The severity of ICI-induced colitis was evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 [19]. The clinical characteristics of each patient with ICI-induced colitis such as age, sex, ICI medication, cancer type, time from ICI initiation to onset, CTCAE grade of diarrhea and colitis, treatment for irAE, comorbidities, other irAEs, and prognosis are shown in Supplementary TableS1 and summarized in Supplementary TableS2. Serum samples were collected from each patient at the time of diagnosis. Among them, serial blood samples were available in four patients with ICI-induced colitis (Case 1, 10, 14, and 20), and the disease activity of the four patients was evaluated using the full or partial Mayo score [20]. Serum samples were also collected from 39 patients with irAEs in other organs, 77 patients with cancer but without irAEs, and 41 healthy volunteers as controls (Supplementary TableS3). Sera from 12 patients with UC were used to compare autoantibody characteristics between ICI-induced colitis and UC (Supplementary TableS4). All serum samples were stored at 80C until assayed.

The study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (protocol number: R1004). All participants provided written informed consent.

Two experienced endoscopists (M.O. and H.K.) who were blinded to the anti-integrin v6 antibody titer evaluated endoscopic findings. Both endoscopists majored in IBD and had over 10 years of work experience. The endoscopic findings of the patients were classified into typical UC findings as described in Supplementary TableS5 [21]. The presence or absence of the endoscopic findings in each patient was scored as 1 or 0, respectively, and the total score was calculated (Supplementary TableS6). The average of the scores assigned by the two endoscopists was used for further analysis.

Recombinant human integrin heterodimers were purchased from ACROBiosystems (Newark, DE, USA), and recombinant human integrin 6 monomer was kindly provided by Medical and Biological Laboratories (Tokyo, Japan) (Supplementary TableS7). To detect serum immunoglobulin G (IgG) antibodies against integrins, we used an enzyme-linked immunosorbent assay (ELISA) Starter Accessory kit (E101, Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturers instructions. Briefly, microtiter plates were coated with 100L of the recombinant proteins (2g/mL) overnight at 4C, blocked, and incubated for 60min with 100L of diluted serum (1:100) at room temperature. After five washes with wash solution, the plates were incubated for 60min with 100L rabbit anti-human IgG antibody conjugated with horseradish peroxidase (HRP) (1:50,000; ab6759, Abcam, Cambridge, UK) at room temperature. After additional five washes with wash solution, the bound reactants were identified by incubating with 3,3,5,5-tetramethylbenzidine (TMB) for 7min at room temperature. The absorbance was measured at 450nm. ELISA was performed with MgCl2 and CaCl2 (1mM each) [12].

The subclasses of the autoantibodies were identified using anti-human IgG1, IgG2, IgG3, and IgG4 secondary antibodies conjugated with HRP (1:2,000; BS-AP006, BS-AP007, BS-AP008, and BS-AP009, respectively; The Binding Site, Birmingham, UK). In addition, the autoantibody isotypes were evaluated using anti-human IgA, IgM, and IgE secondary antibodies conjugated with HRP (1:50,000 A80-102P, 1:100,000 A80-100P, and 1:1,000 A80-108P, respectively; Bethyl Laboratories).

To investigate whether the Arg-Gly-Asp (RGD) peptide inhibited the binding of IgG obtained from patients with ICI-induced colitis with anti-integrin v6 autoantibodies against integrin v6, we added the Arg-Gly-Asp-Ser (RGDS) peptide (A9041, Sigma-Aldrich, St. Louis, MO, USA) or the control peptide Arg-Gly-Glu-Ser (RGES) (A5686, Sigma-Aldrich) to the serum at concentrations of 25 and 100g/mL before incubation.

IgG was isolated from the sera of patients and healthy volunteers using Ab-Rapid SPiN EX (P-014; ProteNova, Higashikagawa, Japan) and stored at 30C. In our previous study, the rate of IgG recovery from the sera was verified to be >90% [12, 22].

A solid-phase integrin v6 binding assay was performed following a previously described method with minor modifications [12, 23]. Briefly, a 96-well microtiter plate was coated with 100L/well integrin v6 (2g/mL) overnight at 4C, blocked, and incubated with 100L of diluted patient or control IgG (1:10) for 60min at room temperature. After five washes with wash solution, the plates were incubated with 100L fibronectin (2g/mL; FC010, Millipore Sigma, Burlington, MA, USA) for 60min at room temperature. After five washes with wash solution, an anti-fibronectin antibody (1:5,000; ab2413, Abcam) was added, followed by incubation for 60min at room temperature. Afterward, the plates were washed with wash solution five times, and an anti-rabbit IgG HRP-conjugated secondary antibody (1:10,000; A27036, Thermo Fisher Scientific, MA, USA) was added, followed by incubation for 60min at room temperature. The plates were washed (five times with wash solution), incubated with TMB for 10min at room temperature, and bound reactants were identified. The absorbance was measured at 450nm. A solid-phase integrin v6 binding assay was performed in the presence of MgCl2 and CaCl2 (1mM each).

The inhibition rate was calculated as follows: [(control optical density (OD) sample OD)/control OD]. The control OD was measured by coating the control wells with integrin v6 and incubating with fibronectin in the absence of patient or control IgG.

The immunohistochemical analysis was performed according to standard procedures for human tissue sections. Because integrin 6 forms a dimer only with integrin v, whereas, v can dimerize with other subunits, including 1, 3, 5, and 8 [24], antibodies against integrin 6 were used to detect integrin v6 expression. Antigen retrieval was performed on sections by incubating them in citrate buffer (pH 6.0) for 20min at 121C in an autoclave before incubating overnight at 4C with the antibodies against integrin 6 (1:500; HPA023626, Sigma-Aldrich). Liquid DAB+ Substrate Chromogen System (K3468, Dako, Santa Clara, CA, USA) was used for staining. Detection times were equally standardized for all sections. Staining intensity of integrin 6 was graded as either 0, 1+, 2+, or 3+. The H-score was calculated using the following formula: [1 (% cells 1+) + 2 (% cells 2+) + 3 (% cells 3+)].

Fishers exact test was performed to evaluate categorical variables. Continuous variables were compared using MannWhitney U tests. Intraclass correlation coefficient (ICC) was used to determine the reliability of the endoscopic scores determined by the two endoscopists. GraphPad Prism Version 9 (GraphPad Software, San Diego, CA, USA) and Stata 18 (StataCorp, College Station, TX, USA) were used for statistical analysis. Two-tailed P<0.05 was considered statistically significant.

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