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Accepted Manuscript

Association of Vedolizumab Level, Anti-drug Antibodies, and α4β7 Occupancy WithResponse in Patients With Inflammatory Bowel Diseases

Bella Ungar, Uri Kopylov, Miri Yavzori, Ella Fudim, Orit Picard, Adi Lahat, DanielCoscas, Matti Waterman, Ola Haj-Natour, Noam Orbach-Zingboim, Ren Mao,Minhu Chen, Yehuda Chowers, Rami Eliakim, Shomron Ben-Horin

PII: S1542-3565(17)31423-4DOI: 10.1016/j.cgh.2017.11.050Reference: YJCGH 55584

To appear in: Clinical Gastroenterology and HepatologyAccepted Date: 17 November 2017

Please cite this article as: Ungar B, Kopylov U, Yavzori M, Fudim E, Picard O, Lahat A, Coscas D,Waterman M, Haj-Natour O, Orbach-Zingboim N, Mao R, Chen M, Chowers Y, Eliakim R, Ben-HorinS, Association of Vedolizumab Level, Anti-drug Antibodies, and α4β7 Occupancy With Response inPatients With Inflammatory Bowel Diseases, Clinical Gastroenterology and Hepatology (2018), doi:10.1016/j.cgh.2017.11.050.

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https://doi.org/10.1016/j.cgh.2017.11.050

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Association of Vedolizumab Level, Anti-drug Antibodies, and α4β7 Occupancy With

Response in Patients With Inflammatory Bowel Diseases

Bella Ungar1, Uri Kopylov1, Miri Yavzori1, Ella Fudim1, Orit Picard1, Adi Lahat1,

Daniel Coscas1, Matti Waterman2, Ola Haj-Natour1, Noam Orbach-Zingboim1, Ren

Mao3, Minhu Chen3, Yehuda Chowers2, Rami Eliakim1, Shomron Ben-Horin1,3

Department of Gastroenterology, Sheba Medical Center Tel Hashomer, Sackler

School of Medicine, Tel-Aviv University, Tel Aviv1 ,Rambam Health Care Campus,

Bruce & Ruth Rappaport Faculty of Medicine, Technion - Israel Institute of

Technology, Haifa2, Israel, and The First Affiliated Hospital of Sun-Yatsen

University, Guangzhou, China3

Short title: Pharmacokinetics & pharmacodynamics of response to vedolizumab therapy

Abbreviations: AVA - anti-vedolizumab-antibodies; CD - Crohn's disease; CRP - C-

reactive protein; HBI - Harvey-Bradshaw index; IBD - inflammatory bowel disease;

SCCAI - Simple Clinical Colitis Activity Index; UC - ulcerative colitis.

Keywords: Vedolizumab; IBD; immunogenicity; clinical outcome.

Corresponding authors:

Bella Ungar, Department of Gastroenterology, Sheba Medical Center, Derech Sheba

2, Ramat-Gan, Israel, 5262100, [email protected], Tel. 972-3-530-2060, Fax

972-3-530-3160

Shomron Ben-Horin, Department of Gastroenterology, Sheba Medical Center, Derech

Sheba 2, Ramat-Gan, Israel, 5262100, [email protected], Tel. 972-3-

530-2060, Fax 972-3-530-3160

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Financial support: This work was supported in part by the 7th Dr. Pinchas

Borenstein Talpiot Medical Leadership Program, The Chaim Sheba Medical Center

(to BU), the 'Talpiot' Medical Leadership Program, The Chaim Sheba Medical Center

(to SBH), the Legacy heritage foundation, Rambam Health Care Center (to YC), and

ECCO-AOCC Visiting Grant (to MR), The Leona M. and Harry B. Helmsley

Charitable Trust (to SBH, RE and YC)

Potential competing interests: Shomron Ben-Horin received consulting and advisory

board fees and/or research support from AbbVie, MSD, Janssen, Takeda and

CellTrion. Uri Kopylov received speaker fees from Abbvie, Janssen and Takeda,

research support from Takeda and Janssen and consulting fees from Takeda and CTS.

Yehuda Chowers received grant support, speaker & consultant fees from AbbVie,

speaker & consultant fees from Janssen, grant support, speaker and consultant fees

from Takeda, speaker fees from Ferring and consultant fees from Medtronics. Rami

Eliakim received consultant and speaker fees from Janssen, Abbvie, Takeda and

Medtronic. Min-hu Chen received speaker fees from Janssen, Falk, Takeda and Ipson.

Matti Waterman received consultant and speaker fees from Janssen, Abbvie, Takeda

and Medtronic. Bella Ungar received consultation fees from Janssen and Abbvie.

None of the other authors have any conflicts to declare. Sheba Medical Center and

Rambam Health Care Campus have filed intellectual property requests on the assays

for vedolizumab and AVA levels.

Author contributions: SBH conceived the study and drafted the manuscript; BU was

involved in study conception, analysis and interpretation of data and manuscript

drafting; UK, MW, DC, AL, OHN and NOZ participated in acquisition of data; EF,

MY and OP took part in data analysis; RE, YC, MR and MC participated in data

interpretation and in critical revision of the manuscript for important intellectual

property. All authors have approved the final draft submitted.

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Background & Aims: There are few data available on the real-life pharmacokinetic

and pharmacodynamics features of vedolizumab, a monoclonal antibody against

integrin α4β7. We performed a prospective study of patients with inflammatory bowel

diseases (IBD) treated with vedolizumab to determine serum drug concentrations,

formation of anti-vedolizumab antibodies (AVAs), and integrin α4β7 saturation.

Methods: We performed a prospective study of 106 patients with IBD (67 with

Crohn’s disease and 39 with ulcerative colitis) treated with vedolizumab from

September 2014 through March 2017 at 2 tertiary medical centers in Israel. Clinical

data and serum samples were collected before and during induction and maintenance

therapy. Clinical remission was defined as Harvey-Bradshaw index scores below 5 or

as Simple Clinical Colitis Activity Index scores of 3 or less. We measured serum

levels of vedolizumab, AVAs, and markers of inflammation. Peripheral blood

mononuclear cells were obtained from some patients at designated trough timepoints

and CD3+ CD45RO+ T cells were isolated from 36 samples. Cells were incubated

with fluorescent-conjugated vedolizumab and flow cytometry was used to quantify

α4β7 integrin saturation. We also performed flow cytometry analyses of CD3+

CD45RO+ lamina propria T cells isolated from intestinal mucosa of patients without

IBD (non-IBD controls, n=6), patients with IBD not treated with vedolizumab

(untreated IBD controls, n=8) and patients with IBD treated with vedolizumab (n=15).

Results: Clinical remission was achieved by 48/106 patients (45%) by week 6 and

50/106 patients (48%) by week 14 of treatment. The median level of vedolizumab at

week 6 was higher in patients in clinical remission (40.2 µg/mL) than in patients with

active disease (29.7 µg/mL; P=.05). The median serum level of vedolizumab was

significantly higher in patients with normal level of c-reactive protein (21.8 µg/mL

vedolizumab) vs the level in those with a high level of c-reactive protein (11.9 µg/mL

vedolizumab) during maintenance treatment (P=.0006). The other clinical outcomes

measured were not associated with median serum level of vedolizumab at any time

point examined. AVAs were detected in 17% of patients during induction therapy and

3% of patients during maintenance therapy, but did not correlate with clinical

outcomes. Flow-cytometry analysis of peripheral blood memory T cells (n=36)

showed near-complete occupancy of α4β7 integrin at weeks 2 and 14 and during

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maintenance phase, regardless of response status or drug levels. Most intestinal

CD3+CD45RO+ memory T cells of healthy and IBD controls expressed α4β7 (72%;

inter-quartile range, 56%–81%). In contrast, free α4β7 was detectable on only 5.6% of

intestinal memory cells (inter-quartile range, 4.4%–11.2%) (P<.0001) from

vedolizumab-treated patients, regardless of response.

Conclusion: In a prospective study of real-life patients with IBD, we associated

vedolizumab drug levels with remission and level of a marker of inflammation.

Integrin α4β7 was blocked in almost all T cells from patients treated with

vedolizumab, regardless of serum level of the drug or response to treatment. These

findings indicate a need to explore alternative mechanisms that prevent response to

vedolizumab.

KEY WORDS: trough level; CD; UC; immunogenicity; clinical response

Introduction

The leukocyte α4β7 integrin facilitates leukocyte homing to the gut via binding of

MADCAM1 on intestinal endothelium1-3. Vedolizumab is a monoclonal antibody

(mAb) against α4β7 which is efficacious for patients with moderate to severe CD and

UC4, 5. Whereas higher levels of anti-TNFs, both infliximab and adalimumab, were

associated with clinical remission and mucosal healing in UC and CD6-9, data

regarding pharmacokinetics and pharmacodynamics of vedolizumab are still scarce.

The GEMINI trials have demonstrated that vedolizumab drug levels were positively

associated with clinical response at week 6. However, drug levels have not been

reported for later time-points1011, and it is still unknown whether therapeutic drug-

monitoring (TDM) of vedolizumab could improve patient management. The

regulatory submission of vedolizumab asserts its mechanism of action is mediated by

blockade of α4β7 on target cells, predominantly memory T-cells, thereby inhibiting

these cells from binding to MADCAM1 and migrating into inflamed intestinal

tissues12. In the GEMINI program, integrin receptor saturation on target effector cells

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was demonstrated with high (6mg/kg) and low (2mg/kg) dosing regimens of

vedolizumab, but correlation of target occupancy with individual patients’ response

status was not shown13. Moreover, there are hitherto no data on α4β7 receptor

occupancy in the gut. The present study therefore aimed to assess, in a large

prospective real-life cohort, the relationship between vedolizumab levels, clinico-

biologic end-points and α4β7 target occupancy in the blood and the intestine.

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Methods

Patient population

This was a prospective observational study of IBD patients receiving scheduled

vedolizumab therapy between September 2014 and March 2017 at two tertiary

medical centers in Israel: Sheba medical center and Rambam Health Care Campus.

All patients who started scheduled vedolizumab therapy during the study period were

enrolled after providing an informed consent. Patients' clinical characteristics were

recorded before drug initiation. Drug and AVA serum levels, as well as inflammatory

markers, were measured in pre-infusion sera collected prospectively throughout

induction and maintenance therapy, and clinical scores were determined before each

infusion. Baseline colonoscopy was performed by physician discretion and was not

universally employed before vedolizumab initiation. In a subset of patients, peripheral

blood mononuclear cells (PBMC), obtained at the designated trough timepoints, and

lamina propria lymphocytes (LPLs), obtained during lower endoscopy, were also

collected. The study was approved by the medical centers' ethics committees and all

patients gave a written informed consent.

Clinical Scores

Clinical status was determined before each infusion prospectively by HBI (Harvey-

Bradshaw index) for CD and by SCCAI (Simple Clinical Colitis Activity Index) for

UC patients15,16. Clinical remission was defined as HBI <5 or as SCCAI≤ 3,

respectively. Clinical response was defined as drop of ≥3 points of the HBI and

SCCAI scores17. Primary non-response was defined as cessation of vedolizumab

therapy by week 14, due to lack of clinical response as defined above18. Secondary

loss of response was defined as clinical worsening necessitating drug-discontinuation

or additional intervention during maintenance period31.

Development of assay for measurement of vedolizumab concentration

Integrin alfa4-beta7 (2µg/ml, R&D, MN, USA) was added to pre-plated anti-His-tag

(4µg/ml, R&D, MN, USA) wells of ELISA plates (Nunc, Roskilde, Denmark). After

drying, 100µl of 1:1000 diluted serum was added and incubated for 60 min at room

temperature. Plates were then washed and goat anti-human κ chain HRP-labeled

antibody (Serotec, Oxford, UK) was added at a concentration of 66 ng/ml for 40 min.

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The results were read by an ELISA reader EL-800 (Biotek Instruments, Winooski,

USA) and expressed as µg/ml. The Level of detection (LoD) of the assay was 3ng/ml

and level of quantitation (LoQ) was 3μg/ml. For assay validation, graded

vedolizumab concentrations were added to vedolizumab-negative sera, demonstrating

significant correlation of signal optical density (OD) with the concentrations added

(rho=1, p<0.0001, Supplementary figure 1).

Determination of anti-vedolizumab antibodies (AVA) concentration

A drug-tolerant ELISA assay using an anti-human λ chain-conjugated detector

antibody was developed. Briefly, 100µl of 1:500 diluted serum was added to pre-

plated 5µg/ml vedolizumab (Takeda Pharma, Denmark) and incubated for 60 min.

Following washing, horseradish peroxidase (HRP) labelled goat anti-human λ-chain

antibody (MP Biomedical, Solon, Ohio, USA) at a concentration of 33 ng/ml was

added for 40 min and reacted with tetramethylbenzidine (TMB) substrate. The results

were read by an ELISA reader EL-800 (Biotek Instruments, Winooski, USA) and

expressed as µg/ml-equivalent (µg/ml-eq) after normalization versus graded

concentrations of 9-1200ng/ml of goat anti-human F(ab′)2 fragment antibody (MP

Biomedicals, USA). AVA levels above 30μg/ml-eq cut-off were considered positive,

as determined by three standard deviations above the mean of 30 unexposed controls.

Fluorescence-activated cell sorting (FACS) analysis of α4β7 occupancy on T-cells

Freshly obtained PBMC and lamina propria (LP) lymphocytes were isolated and

prepared as previously described19. Vedolizumab was biotinylated (EZ-LinkTM

NHS-PEO solid Phase biotinylation kit, Thermo Scientific, USA). Cells were co-

stained with CD3 T-cell marker, CD45RO as a marker of previously-activated cells

(surrogate for memory cells) and with 30mcg/ml of biotinylated-vedolizumab-

strepavidine-PE or a biotinylated-IgG control antibody. The expression of α4β7

(vedolizumab-positive cells) on gated PB or LP CD3+CD45RO+ memory T-cells was

determined by FACS analysis (Navios Flow cytometer, Beckman Coulter, USA) and

results analyzed by Kaluza software (Beckman Coulter, USA). To examine the drug

concentration that completely block all free membranal α4β7, graded concentrations

of vedolizumab were added to PBMC, cultured 5 days in media alone or after

polyclonal stimulation with 5mcg/ml Muromonab-CD3 (OKT3).

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Statistical analysis

Continuous variables were compared by Mann-Whitney test and Fischer's exact test

was used for categorical data. Wilcoxon test was used to compare paired samples. A

receiver-operating characteristic (ROC) analysis was performed for analysis of

vedolizumab levels discriminatory accuracy for categorical variables. Correlations

were analyzed by the Spearman rank test. One-way analysis of variance (ANOVA)

and Cochran's Q test were used to test the differences between quartiles. Multivariable

analysis was performed using backward logistic regression. All P-values were 2-

sided, and a P-value <0.05 was considered statistically significant. All statistics were

performed with MedCalc software (version 12.2.1.0, Mariakerke, Belgium).

Results

Demography and clinical outcomes

124 patients started vedolizumab treatment during the study period; 18 patients were

excluded due to missing sera, non-consent, pregnancy, or cessation of vedolizumab

before the third infusion due to reasons unrelated to clinical non-response. Thus, the

study cohort consisted of 106 IBD (67 CD, 39 UC) patients. The median follow-up

for the study cohort was 30 weeks (IQR 14-54 weeks). The patients' clinical and

demographic characteristics are depicted in table 1. Clinical remission by weeks 6 and

14 were achieved by 48/106 patients (45%) and 50/106 (48%), respectively. Nine

patients (8.4%) experienced primary non-response to induction and 17 (18%) lost

response and discontinued vedolizumab therapy during maintenance; 12 patients

(11.3%) underwent interval shortening during induction (week 10) and 11 patients

(11.5%) during maintenance. Fifty-four patients completed one year of therapy, 17 of

whom (32%) were in clinical remission.

Baseline factors association with pharmacokinetics

Baseline albumin and CRP values significantly correlated with weeks 2, 6 and 14

vedolizumab trough levels (supplementary table 1) and albumin level quartiles were

associated with increased week 14 vedolizumab trough levels (p<0.001, Figure 1). In

addition, female gender, lower baseline weight and younger age were associated with

higher levels at week 2. Female gender was significantly associated with week 6

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levels as well. On multivariate analysis, only baseline albumin remained significantly

correlated with both week 6 and 14 levels (supplementary table 1).

Association of induction period drug levels and clinical and biomarker response

Median week 6 vedolizumab levels were higher in week 6 clinical remission versus

non-remitters patients (40.2µg/mL, IQR 18.3-57.2, versus 29.7µg/mL, IQR 17.5-41.8,

respectively, p=0.05, figure 2a). On quartile analysis, a statistically significant

difference in remission rate was noted between week 6 drug-level-quartiles 2, 3 and 2,

4 (p=0.02, 0.006, figure 2b). However, drug levels were not associated with remission

status at other time-points (supplementary table 2). Week 6 drug levels in partially-

responding patients requiring interval-shortening at week 10 (45.4µg/mL, IQR 19.6-

84.2, n=12) were similar to levels in patients whose intervals were not shortened

(33.9µg/mL, IQR 18.3-48, p=0.28). A sub-analysis including only patients who did

not receive steroids during induction (n=71) similarly showed significant association

of vedolizumab levels with clinical remission only for week 6 (median 41.3µg/mL,

IQR 19.8-40.5, versus 29.8µg/mL, IQR 24.6-53.4, p=0.04).

In an analysis of biomarker response, week 2 vedolizumab levels demonstrated a

significant, albeit weak, negative correlation with week 14 CRP values (rho=-0.25,

p=0.02) and week 6 drug levels were weakly associated with week 6 CRP values

(rho=-0.24, p=0.04, figure 2c). No association was detected between drug levels and

CRP normalization for other time-points (supplementary table 3), and clinical

remission was not predicted by any of the other clinical/demographic factors

(supplementary table 5). A separate sub-group analysis for PK-response correlation

for CD (n=67) and UC patients (n=39) showed similar findings to those of the entire

cohort (supplementary document 1).

Maintenance period drug levels association with clinical-biomarker response

Analysis of 140 serial sera obtained from 60 consecutive patients during vedolizumab

maintenance therapy (week 22 onward) exhibited significantly higher median

vedolizumab levels at time-points with normal versus elevated CRP (21.8µg/mL, IQR

12.9-40.5, versus 11.9µg/mL, IQR 5-26.5, respectively, p=0.0006, figure 3). In

contrast, median levels in clinical responders did not differ from levels in non-

responders (15.9µg/mL, IQR 7.7-25 versus 14µg/mL, IQR 5.3-31.9, respectively,

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p=0.57). Drug levels in patients before interval-shortening were similar to levels in

patients not requiring this intervention, matched for the same time-point (27.8µg/mL,

IQR 11.4-45, versus 15.4µg/mL, IQR 7.1-26.2, p=0.09), and intra-individual temporal

variations in vedolizumab levels appeared similar in responders versus those with

loss-of-response (supplementary figures 3a, 3b). Furthermore, week 6 levels among

patients who went on to have sustained response during maintenance were not

different than levels in those with subsequent loss-of-response (35.7µg/mL, IQR 19-

48.5 vs. 31.5µg/mL, IQR 16.5-53, respectively, p=0.98).

Immunogenicity

Anti-vedolizumab-antibodies (AVA) were measured in 180 sera. Of these, 120 were

consecutive (week 2-6-14) samples of 41 patients during induction (21 with week 14

clinical remission , 20 clinically active). Additional 60 sera obtained from 60

consecutive patients during maintenance therapy were also analyzed (30 of whom

were clinically active at the time of sampling). Positive antibodies at weeks 2 or 6

were detected in 7/41 patients (17%): 3/21 (14.3%) responders and 4/20 (20%) of the

non-responders (p=0.63, OR=1.5, 95% CI 0.3-7.7). AVA were detected with zero

drug levels in one patient and with measurable drug levels (i.e. drug-positive/AVA-

positive) in the other six, with the use of the described drug-tolerant assay. Only 2/60

(3%) patients during maintenance therapy were found AVA positive (One AVA-

positive/drug-positive in a patient in remission, one AVA-positive/drug-negative in a

patient with loss of response).

Intestinal and peripheral blood α4β7 integrin target occupancy in relation to

drug levels and clinical outcome

Peripheral blood lymphocytes (PBL) were prospectively collected at weeks 0, 2 and14

in 13 patients starting vedolizumab therapy (six responders, seven non-responders to

induction). The percentage of free (unbound) α4β7 on CD3+CD45RO+ memory T-

cells was determined by FACS analysis. Median free α4β7 (positive α4β7 CD45RO+

cells) at week 0 was 27% (IQR 18%-31%), and sharply declined to near-complete

target occupancy at week 2 (0.09%, IQR 0.03-0.59%) and week 14 (2%, IQR 0.77%-

3.4%, P<0.0001 for both comparisons, figures 4a, 4b). Near-complete target

occupancy at weeks 2 and 14 was universal and unrelated to response/non-response to

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induction or to drug levels at week 14 (Figure 4b). The percentage of positive α4β7 T-

cells before vedolizumab initiation (day 0), did not predict response to induction in

the present cohort (n=13), nor in an additional exploratory cohort (n=26, data not

shown). Similar to the findings during induction, near-complete α4β7 occupancy was

also found on T-cells obtained during maintenance phase (n=23), regardless of

whether patients were in clinical response (n=12) or loss-of-response (n=11, figure

4b). Similar near-complete blockade of α4β7 was also found for naïve T-cells

(CD3+CD45RO-, data not shown). Given these results, the minimal concentration of

vedolizumab able to block PB T-cells α4β7, in vitro, was explored (see Methods

section). A near-complete blockade of α4β7 (<2% free α4β7) was apparent already

with concentrations of 3μg/ml of vedolizumab in unstimulated resting cells, whereas

stimulated cells had higher baseline expression of α4β7 and required somewhat

higher vedolizumab concentrations to achieve comparable target occupancy (figure

4c).

Finally, mucosal samples were obtained during colonoscopies in control healthy

individuals, IBD controls (not treated with vedolizumab) and IBD patients treated

with vedolizumab (n=29), and LP T-cells were isolated and stained as above.

CD3+CD45RO+ LP-T cells in both healthy and IBD control individuals were

predominantly α4β7positive, regardless of controls’ disease activity state (median

71%, IQR 55.6-81%, n=14). In contrast, in vedolizumab-treated patients, free α4β7 on

intestinal memory cells was significantly lower (median CD3+CD45RO+α4β7+

5.6%, IQR 4.4-11.2%, n=15, p<0.0001, figures 4d, 4e). Intestinal α4β7 occupancy in

vedolizumab-treated patients was similar in inflamed and non-inflamed mucosa

(5.6%, IQR 4.4-11.2, n=15, versus 4.8%, IQR 3.8-11.8, n=9, respectively, p=0.7,

supplementary table 6). There was also no difference in the rate of free α4β7+ on T-

cells among responders and non-responders to therapy (data not shown).

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Discussion

Vedolizumab is efficacious for moderate-to-severe CD and UC4, 20-22. Nevertheless,

scarce data exist pertaining to its pharmacokinetics/pharmacodynamics and data on its

target occupancy in the gut are completely absent. This study showed a modest

association of vedolizumab drug levels with some outcomes of clinical and biomarker

response to therapy. It further showed a nearly-full target α4β7 receptor occupancy on

both peripheral blood and intestinal memory T-cells, within a wide range of drug

concentrations and regardless of response to therapy or not.

Similar to the GEMINI trials23, we also found body weight and albumin to correlate

with induction drug concentration and identified female gender as additional predictor

– possibly due to the lower average weight of females. However, the only predictor

retained on multi-variable analysis was albumin, whose correlation with drug levels

was not only found for extreme albumin values, unlike the GEMINI analysis. This

may be related to differences in FcRn salvage24 and/or in fecal loss of albumin/IgG,

which may be different for the present population compared to the GEMINI trial

population.

In the GEMINI 1 & 2 studies, vedolizumab drug level quartiles at week 6 were

positively associated with week 6 clinical response10 11. In the present cohort, week 6

levels were similarly higher in week 6 responders compared to non-responders,

regardless of concomitant steroid therapy. However, clinical outcomes at other time-

points were not predicted by week 2 or 6 levels. This is echoed in a recently published

analysis of GEMINI trial, in which no definite vedolizumab threshold level could be

determined for discriminating remitters from non-remitters after consideration of co-

variates25. This could suggest some detected drug-response associations may arise

from multiple-testing, implying further corroborating studies are imperative.

Interestingly, a recent real-life study similarly did not find different vedolizumab

levels between responders and non-responders, although week 6 levels<19µg/mL

modestly associated with subsequent need for interval-shortening26. We could not

reproduce such a correlation, possibly because of different cohort characteristics or

size (106 patients in the present cohort versus 47 in the previously reported one), or

due to variations in assay techniques or outcome definitions.

Vedolizumab levels were associated with biomarker response (CRP normalization),

especially during maintenance treatment. Data on maintenance-phase drug levels and

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outcomes are still scant, but the present observations are in line with recent data from

another real-world cohort, presented in an abstract form27. Immunogenicity rates of

vedolizumab were 17% during induction and 3% during maintenance, and were in

agreement with reported low incidence of AVA and with their limited clinical

impact27, 28.

As concluded by FDA and EMA, the mechanism of action of vedolizumab is

blockade of α4β7, predominantly on memory T-cells, to inhibit their MADCAM1-

binding mediated migration into intestinal tissue12. Earlier work showed that even

2mg/kg vedolizumab dose provided near-complete saturation of the integrin-

receptor29, thereby suggesting the need to elucidate the pharmacodynamics impact of

fluctuations of vedolizumab exposure within the current dosing scheme. Recently,

higher baseline mRNA expression of integrin αΕβ7, and its decline after therapy,

were correlated with response to etrolizumab, an anti-β7 integrin antibody30.

However, the rate of α4β7 occupancy with this agent was not reported. The present

findings show target membranal α4β7-integrin on peripheral blood memory T-cells to

be nearly-fully occupied during both induction and maintenance with vedolizumab

and regardless of clinical response status. Whether these results indicate blocking of

α4β7 by vedolizumab or post-binding internalization of α4β7-vedolizumab complex is

still unclear, but even the later implies a near-complete binding of α4β7 by

vedolizumab before putative internalization. Similar to findings in blood, near-full

receptor saturation was demonstrated - for the first time - also on activated T-cells in

the intestinal mucosa, indicating in-situ blockade of T-cells by vedolizumab and/or

the effective prevention of these cells from migration to the gut. Full receptor

occupancy was evident throughout a wide-range of drug concentrations and was

unrelated to response status, thereby arguing against sub-dosed vedolizumab (being

unable to block its target in full) as the cause for suboptimal response in non-

responders. Conversely, however, these data may hint towards other mechanisms

underlying the biologic effect of vedolizumab or its lack-of, such as receptor

occupancy on other T-cell subsets32, which may hypothetically require higher drug

concentration for optimized effect.

There are several limitations to our study. Vedolizumab levels were measured using

an in-house novel validated ELISA-based assay; Thus, corroborating studies using

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other assays are pertinent. Nonetheless, the vedolizumab concentrations herein are in

agreement with those reported in the GEMINI trials, and a high accuracy for detecting

exogenous vedolizumab was shown (supplementary figure 1), thus supporting the

validity of the observations. Secondly, various clinical-biomarker end-points in

relation to drug levels were assessed, but endoscopic outcomes were not evaluated.

In conclusion, in this real-life prospective pharmacokinetic cohort of vedolizumab-

treated IBD patients, drug levels were associated with some – albeit not all - clinical

outcomes of induction and with CRP normalization during maintenance. Peripheral

blood and intestinal target α4β7-integrin is nearly-fully occupied by vedolizumab

throughout wide-range of drug concentrations and regardless of therapy. These

pharmacokinetic-pharmacodynamic data suggest that within current dosing schemes,

drug levels in most non-responders are still sufficient to block the α4β7 target

molecule, indicating the need to explore additional modes-of-action of vedolizumab

and to elucidate other mechanisms underlying non-response to the drug.

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Table 1 - Patients' Demographic and Clinical Characteristics

N 106 Age, years (median, IQR) 37 (29.3-46.8) Disease duration, years (median, IQR) 10.5 (6-17) Male / Female ratio 1.2 Smoking at induction, n (%) 15 (14) Jewish Ashkenazi ethnicity, n (%) 41 (39) Previous surgery, n(%) 35 (33) Concomitant medical condition, n (%) 23 (21) BMI (median, IQR) 22 (19.6-25.3) Extra-intestinal manifestations, n (%) 36 (34) Concomitant immunomodulator therapy, n (%) 15 (14) Concomitant steroids at baseline 33 (31) CD, n (%) 67 (63) UC, n (%) 39 (37) CD behavior Inflammatory (%) 27 (45)

Stricturing (%) 18 (30) Penetrating (%) 15 (25)

CD location Ileal (%) 34 (50) Ileo-colonic (%) 22 (33) Colonic (%) 11 (17)

Perianal CD (%) 20 (33.3)

UC location* Left sided colitis (%) 18 (47)

Pancolitis (%) 20 (52) Failure of anti-TNFα therapy, n (%) 91 (86) Baseline albumin level (g/dl, median, IQR) 3.9 (3.6- 4.3) Baseline CRP level (mg/l, median, IQR) 9 (4.3 - 23.7) Vedolizumab week 2 trough serum level, µg/mL (median, IQR)

42.7 (28.9 - 60.7)

Vedolizumab week 6 trough serum level, µg/mL (median, IQR)

33.8 (17.9 - 48)


11.4 (4.6 - 20.4)


16.9 (7.5-26.4)


15 (8.4-26.2)

IBD -Inflammatory bowel disease, CD - Crohn's disease, UC - ulcerative colitis, TNF - Tumor necrosis factor, IQR - interquartile range, CRP - C-reactive protein * One UC patient received vedolizumab induction therapy after proctocolectomy and resultant pouchitis.

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Figure Legends

Figure 1

Association between baseline albumin level quartiles and week 14 vedolizumab trough levels (n=106).

Figure 2a

Week 6 vedolizumab levels among patients in clinical remission versus clinically active patients at week 6.

Figure 2b

Vedolizumab trough level quartiles at week 6 in relation to clinical remission rates at the same time-point.

Figure 2c

Correlation between week 6 vedolizumab levels and CRP values. CRP - C-reactive protein

Figure 3

Maintenance-phase vdolizumab levels among 60 patients with normal versus elevated CRP values. CRP - C-reactive protein

Figure 4a

FACS analysis of free α4β7-integrin on peripheral blood memory T-cells

(α4β7+CD45RO+CD3+ cells, out of total CD45RO+CD3+ T-cells), detected by staining with conjugated-vedolizumab at weeks 0-2-14. Exemplary four patients are depicted with their respective induction’s clinical outcome and drug levels. FACS - flow cytometry and fluorescence-activated cell sorting

Figure 4b

Graphic summary of the percentage of free α4β7 on memory T-cells (α4β7+ CD45RO+CD3+) in peripheral blood of vedolizumab-treated patients (separately presented for responders and non-responders). Induction and maintenance period observations are depicted. Each point denotes a single patient, except during induction when two points connected with a line represent a single patient at the two designated timepoints. The color of the connecting line denotes if a patient’s week 14 level vedolizumab was low (‘Low vedo’ - defined as level below the median value at week 14 of entire 106 patients’ cohort, red line) or high (‘high vedo’ - above median, blue line). vedo – vedolizumab. W- week.

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Figure 4c

Results of experiments (n=3) of drug concentration- α4β7 blockade titration curve. Unstimulated peripheral blood T-cells were cultured with graded concentrations of un-conjugated vedolizumab in media alone or after polyclonal stimulation by OKT3. Staining with conjugated vedolizumab was performed after five days in culture, to delineate the drug concentration that confers complete receptor occupancy. FACS - flow cytometry and fluorescence-activated cell sorting

Figure 4d

FACS analysis of intestinal memory LP T-cells free α4β7 integrin

(α4β7+CD45RO+CD3+ cells, out of total CD45RO+CD3+ T-cells), determined by staining with conjugated-vedolizumab. Two controls were employed: An IgG-isotype control and a pre-incubation with un-conjugated 50mcg/ml vedolizumab. Exemplary three experiments are shown.

Figure 4e

Box-and-Whisker plot depicting the percentage of α4β7+ on LP-memory T-cells

(α4β7+CD45RO+CD3+ cells, out of total CD45RO+CD3+ T-cells) in intestinal biopsies of control (n=14) or vedolizumab-treated patients (n=15).

Supplementary figure 1

Correlation between graded concentrations of exogenously-added vedolizumab to unexposed serum and ELISA reading by optical density (OD).

Supplementary figure 2a

ROC analysis of vedolizumab levels among CD patients, with clinical remission as a classification variable. CD - Crohn's disease, ROC - receiver-operating characteristic

Supplementary figure 2b

ROC analysis of vedolizumab levels among UC patients, with clinical remission as a classification variable. UC - ulcerative colitis, ROC - receiver-operating characteristic

Supplementary figure 3a

Intra-individual vedolizumab levels during maintenance therapy among responders.

Supplementary figure 3b

Intra-individual vedolizumab levels among patients who lost response to maintenance therapy.

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Supplement 1, Page S39

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Supplementary document 1 - Induction period drug levels and clinical and

biomarker response- subanalyses among CD and UC patients

Among CD patients (n=67), similar to the general study population, week 6

vedolizumab levels associated with clinical remission at week 6 (median levels 47.7

µg/mL, IQR 18.4-61 versus 33 µg/mL, IQR 21.2-42.5, p=0.037 in those in remission

versus clinically active patients, respectively). However, ROC analysis demonstrated

only modest discriminatory accuracy (AUC=0.66, 73% sensitivity, 70.3% specificity

for drug level>37.8 µg/mL, p=0.055. Odds-ratio 5.5 for remission with levels over

37.8 µg/mL, p<0.01, supplementary figure 2a). All other associations were

insignificant among CD patients (supplementary table 7). A separate analysis of week

2, 6 levels in relation to HBI values yielded no significant association either (data not

shown).

Among UC patients (n=39), week 2 median vedolizumab levels were significantly

higher - 55.8 µg/mL (IQR 35.4-80.8) in patients who went on to have clinical

remission at week 6, compared to 29.9 µg/mL (IQR 24-47.7) in patients without

clinical remission at week 6 (p=0.02). ROC analysis demonstrated that vedolizumab

week 2 levels above 32.7 µg/mL had a 90% sensitivity and a 61.1% specificity for

clinical remission at week 6 among UC patients (AUC=0.73, p=0.01, odds-ratio=8 for

remission with a drug level>32.7mcg/ml, p=0.019, supplementary figure 2b).

Nevertheless, sub-analyses of vedolizumab levels among UC patients at week 6 vis-a-

vis clinical remission and clinical score (SCCAI) at weeks 6 or 14 did not yield

statistically significant correlations (supplementary table 8).

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Supplementary table 7 - Association between week 2 and 6 vedolizumab trough levels and clinical remission by the end of induction among CD patients (n=67)

Week of trough level measurement

Patients in remission - week 6

Patients clinically active -week 6

P value



P value

Week 2 drug level (median, IQR)

41.2, 28.2-60.5

46.8, 34.3-54.5

0.53 44.4, 31-60.3

46.5, 30.7-58.8

0.87


47.7, 18.4-61

33, 21.2-42.5

0.037 40.2, 25.5-57.2

33.2, 20.7-47.5

0.23

CD - Crohn's disease, IQR - interquartile range

Supplementary table 8 - Association between week 2 and 6 vedolizumab trough levels and clinical remission by the end of induction among UC patients (n=39)




P value



P value


55.8, 35.4-80.8

29.9, 24-47.7

0.02 38.4, 28.4-61.9

40.2, 24.6-72.5

0.89


35.7, 18.1-48.5

23, 12-42.8

0.31 24.4, 16.4-37.6

36.6, 13.5-54.3

0.2

UC - ulcerative colitis, IQR - interquartile range

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Supplementary Table 1 - Analysis of pharmacokinetic parameters associated with week 2, 6, 14 vedolizumab levels (n=98)

CRP - C reactive protein, CD - Crohn's disease

Supplementary Table 2 - Comparison of clinical remission rates between quartiles of vedolizumab trough levels

Quartiles compared P value Odd Ratio Confidence interval 1, 2 0.09 3.2 0.8-12.1 1, 3 0.4 0.6 0.2-2 1, 4 0.2 0.5 0.15-1.5 2, 4 0.006 0.15 0.04-0.6 2, 3 0.02 0.2 0.05-0.7 3, 4 0.7 0.8 0.25-25

Variable Week 2 univariate

analysis P Value

Week 2 multivariate

analysis P Value

Week 6 univariate

analysis P Value

Week 6 multivariate

analysis P Value

Week 14 univariate

analysis P value

Week 14 multivariate

analysis P Value

Baseline albumin (g/dl)

0.009 0.34 0.002 0.05 <0.0001 0.004

Baseline CRP (mg/l)

0.02 0.53 0.007 0.25 0.01 0.77

Baseline weight (kg)

0.007 0.02 0.28 0.25

Gender 0.02 0.07 0.05 0.99 Concomitant steroidal therapy

0.24 0.83 0.55

Concomitant immunomodulator therapy

0.67 0.28 0.81

IBD type (CD) 0.67 0.17 0.92 Age 0.02 0.74 0.37 0.25 Prior anti-TNFα therapy

0.53 0.9 0.5

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Supplementary Table 3 - Association between week 2, 6 vedolizumab trough levels and CRP normalization by the end of induction (weeks 6, 14, n=95)


Patients with elevated CRP - week 6

Patients whose CRP normalized - week 6

P value

Patients with elevated CRP - week 14

Patients whose CRP normalized - week 14

P value

Week 2, µg/mL (median, IQR)

40, 31.4-48

35.4, 28-50.6

0.96 37 24.7-47.7

46.4, 28.7-50.9

0.3

Week 6, µg/mL (median, IQR)

22.4, 13.1-36.9

29.4, 17.4-50.3

0.19 24.9, 15-37.2

29.4, 17-45

0.6

CRP - C-reactive protein, IQR - interquartile range

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Supplementary Table 4 - Univariable analysis of pharmacokinetic parameters associated with week 6 and 14 clinical remission (n=106)

CRP - C-reactive protein, CD - Crohn's disease, OR - odds ratio, CI - confidence interval

Supplementary Table 5 - Multivariable analysis of pharmacokinetic parameters associated with week 6 clinical remission

Variable Week 6 remission, p value

Week 6 vedolizumab level, µg/mL 0.34

Baseline CRP, mg/l 0.35 Prior anti-TNFα therapy 0.37 CRP - C-reactive protein. *Only parameters with significant/borderline association with week 6 remission were incorporated into the analysis. Only one parameter was borderline for week 14 remission, hence multivariate analysis was not performed.

Variable Week 6 remission

Week 14 remission

Median - patients in

clinical remission

Median - clinically

active patients

P value

Median - patients in

clinical remission

Median - clinically

active patients

P value

Week 2 vedolizumab level, µg/mL

47.7 43.5 0.3 41.2 46.5 0.88

Week 6 vedolizumab level, µg/mL

40.2 29.7 0.05 35.6 33.1 0.96

Baseline albumin, g/dl 3.9 3.9 0.89 3.9 3.9 0.97 Baseline CRP, mg/l 7.5 12.4 0.09 9.5 8.7 0.8 Baseline weight, kg 60.5 65.4 0.67 70 60.5 0.09 Age, years 37 37 0.44 37 37 0.96 Disease duration, years 10.5 11 0.94 12.5 10 0.63

Variable OR CI P value

OR CI P value

Concomitant immunomodulator therapy

0.5 0.16-1.5 0.22 1.1 0.4-3.3 0.85

Concomitant steroidal therapy

0.54 0.24-1.3 0.15 1.5 0.6-3.4 0.36

IBD type (CD) 1.5 0.7-3.24 0.35 1.6 0.7-3.5 0.25 Prior anti-TNFα therapy 2.5 0.8-8 0.09 1.5 0.5-4.8 0.45 Gender (female) 0.6 0.3-1.3 0.2 1.5 0.7-3.2 0.32

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Supplementary Table 6 - Clinical, endoscopic and histological characteristics of 15 patients whose lamina propria biopsies were analyzed

Patient IBD type

Endoscopy Histology Biopsy location

Days since last

infusion 1 UC Hyperemic mucosa in left

colon, MAYO 1 Mild chronice active

inflammation sigmoid colon

14

2 UC Inflammation with exudates and hemorrhage, MAYO 3

Moderately to severe chronic active colitis with ulceration and granulation

recto-sigmoid

9

3 UC Ulcerated hyperemic mucosa MAYO 1-2

Moderate chronic active colitis with crypt abscesses

transverse - sigmoid

colon

22

4 UC Severely inflamed colonic mucosa

Chronic colitis sigmoid colon

14

5 UC Moderate inflammation with ulcerations

Mild to moderate chronic active colitis

sigmoid colon

14

6 CD Edematous inflamed ulcerated mucosa

Chronic colitis with moderate activity including

cryptitis and granulation tissue formation.

sigmoid colon

12

7 UC Moderate inflammation Colonic mucosa with mild to moderate glandular

architectural distortion and reactive lymphoid

hyperplasia

transverse colon

14

8 Severe inflammation with ulcerations MAYO 3

Colonic mucosa with severe active chronic

colitis.

transverse - sigmoid

colon

21

9 CD Severe inflammation with spontaneous bleeding

Active chronic colitis with ulceration and granulation and regenerative epithelial

changes

sigmoid colon

14

10 CD Hyperemic mucosa at anastomosis

Minimally active colitis with hyperplastic crypts

ileo- colonic

anastomosis

20

11 CD Inflammation with ulceration at anastomosis -

Rutgreets 2

Minimally active colitis with lymphoid follicle formation

sigmoid colon

5

12 UC Inflamed mucosa MAYO 2 Mild to moderate chronic active colitis

sigmoid colon

6

13 UC Mild - moderate inflammation

Active chronic colitis sigmoid colon

18

14 UC Severe inflammation with exudates MAYO 2-3

Moderate to severe active chronic colitis with crypt abscess and granulation

tissue

recto-sigmoid

14

15 UC Erythematous mucosa mildly active chronic colitis with focal cryptitis

rectum 0

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