In vivo imaging using fluorescent antibodies to TNF predicts therapeutic response in Crohn s disease

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1 In vivo imaging using fluorescent antibodies to TNF predicts therapeutic response in Crohn s disease Raja Atreya, Helmut Neumann, Clemens Neufert, Maximilian J. Waldner, Ulrike Billmeier, Yurdagül Zopf, Marcus Willma, Christine App, Tino Münster, Hermann Kessler, Stefanie Maas, Bernd Gebhardt, Ralph Heimke-Brinck, Eva Reuter, Frank Dörje, Tilman T. Rau, Wolfgang Uter, Thomas D. Wang, Ralf Kiesslich, Michael Vieth, Ewald Hannappel & Markus F. Neurath Supplementary Figure 1 Supplementary Figure 1 Analysis of fluorescent adalimumab (a) SDS-PAGE gel electrophoresis of fluorescein isothiocyanate-adalimumab, which was labeled with a 14-fold excess of fluorescein isothiocyanate (HF1) and adalimumab (H) and fluorescein isothiocyanate-adalimumab after removing the excess fluorescent dye with a dye removal column according to the study protocol (HF2). The left panel depicts the fluorescence when the gel was exposed to UV light. The right panel shows the gel after Coomassie staining. All lanes contained 1 µg of the protein. (b) Hypothetical model of fluorescent adalimumab based on the above analysis. 1

2 Supplementary Figure 2 Supplementary Figure 2 In vivo staining with labeled adalimumab. (a) In vivo molecular imaging with fluorescent adalimumab led to the identification of mtnfexpressing mucosal immune cells. Scale bar represents 25 µm. (b) The same confocal image was contrast-enhanced by Fiji imaging technique to augment the contrast between mtnf-positive mucosal cells and the background. Scale bar represents 25 µm. (c) Autofluorescence background of the mucosa in a patient with Crohn s disease before staining was performed with fluorescent adalimumab. Scale bar represents 25 µm. (d) Receiver operator characteristics (ROC) analysis of the mean number of in vivo mtnf expressing cells patient -1 during the molecular imaging procedure. The ROC analysis identified values of , rounded to 20, as an optimum cut-point (arrow) to differentiate between patients in the high and low mtnf groups, with a sensitivity of 84.6% and a specificity of 91.7% for the prediction of response to adalimumab therapy. 2

3 Supplementary Figure 3 Supplementary Figure 3 Ex vivo staining of histological gut sections from the mucosal area adjacent to the site where molecular imaging was performed using fluorescent adalimumab. (a b) Patients with low and high numbers of mtnf expressing mucosal immune cells could be differentiated by ex vivo staining. Scale bar represents 25 µm. (c) Quantitative analysis demonstrated that patients with Crohn s disease and clinical response to adalimumab therapy after 12 weeks had a significantly higher number of mtnf expressing immune cells than patients lacking clinical response to adalimumab therapy. Data represent mean values ±s.e.m.; *P = 0.02 (t-test; two-sided). (d) Representative immunofluorescence staining for mtnf with FITC labeled adalimumab (green) and antibodies against CD4 or CD14 (all red) in gut cryosections from patients with Crohn s disease. Nuclei were counterstained with DAPI (blue). Double positive cells are shown in yellow (enlarged image). Representative images of one out of 11 patients are shown. Scale bars represent 10 µm. (e) Higher percentage of mtnf positive cells among intestinal CD14+ macrophages compared to CD4+ T-cells in patients with Crohn s disease (n = 11). Data represent mean values ±s.e.m.; ***P = (ttest; two-sided). 3

4 Supplementary Figure 4 * * Supplementary Figure 4 Clinical findings upon adalimumab therapy. (a) Clinical outcome analysis showed that patients with a higher number of mtnf positive intestinal cells had a statistically significant reduction of their CDAI levels after 4 and 12 weeks of adalimumab treatment in comparison to the baseline CDAI before initiation of adalimumab therapy. Patients were subsequently followed over a period of 52 weeks. In the follow up of the patients with high mtnf expression it could be shown that this group has a sustained significant reduction of the CDAI score even one year after the initiation of the adalimumab treatment. In contrast, patients with low numbers of mtnf positive cells did not show any significant reduction in CDAI scores. Data represent mean values ±s.e.m.; *P = 0.04; **P = ***P = (t-test; two-sided). (b) Patients with high numbers of mtnf expressing cells had a statistically significant reduction of their corticosteroid use after 4 and 12 weeks of adalimumab treatment in comparison to patients with low numbers of mtnf expressing cells. Data represent mean values ±s.e.m.; *P = 0.04 (t-test; two-sided). 4

5 Supplementary Figure 5 Supplementary Figure 5 Ulcerations and mucosal healing rate during follow-up endoscopy in the high mtnf group. Endoscopic analysis at the beginning of the study showed that 100% of the patients in the low and high mtnf groups had mucosal ulcerations prior to the induction of the treatment with the antibody to TNF adalimumab. In 11 out of 12 patients with high mtnf expression, a follow up white light endoscopic examination could be performed at a mean of 17 months (range months) after initiation of adalimumab therapy. 27% of these patients still had mucosal ulcerations during the follow-up indicating that 73% of the patients with high mtnf expression had mucosal healing. Mucosal healing was defined as the absence of mucosal ulcerations during the follow-up endoscopy. 5

6 Supplementary Table 1 Clinical characteristics of responders and nonresponders Characteristic Responder Nonresponder P value Female, n (%) 5 (38.5) 4 (33.3) 1 Age in years, mean (s.d.) 43.5 (17.3) 39.3 (11.3) 0.57 Body weight at V1 in kilograms, mean (s.d.) Disease duration in years, mean (s.d.) Involved intestinal area, n (%): Colonic Ileal Gastroduodenal 75.5 (10.3) 72.4 (13.4) (10.2) 11.3 (5.8) (53.8) 8 (61.5) 3 (23.1) 8 (66.7) 9 (75) 1 (7.7) Previous anti-tnf exposure, n (%) 1 (7.7) 1 (8.3) 1 Concomitant medication, n (%): Aminosalicylates Corticosteroids Azathioprine Antibiotics 6 (46.2) 6 (46.2) 5 (38.5) 0 (0) 1 (8.3) 7 (58.3) 4 (33.3) 1 (8.3) Current smoker, n (%) 4 (30.8) 0 (0) 0.10 CRP at V3, mean ( s.d.) 8.8 (16.9) 18.1 (22.3) 0.19 Stenosis, n (%) 4 (30.8) 8 (66.7) 0.16 Comparison of patient characteristics between responders and nonresponders to adalimumab therapy. Results of statistical tests yielded no evidence of significant differences between the two groups. V1, visit 1; V3, visit 3.