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Vedolizumab, a monoclonal antibody to the gut homing α4β7 integrin, does not affect cerebrospinal fluid T-lymphocyte immunophenotype
Journal of Neuroimmunology 264 (2013) 123–126
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Vedolizumab, a monoclonal antibody to the gut homing α4β7 integrin, does not affect cerebrospinal fluid T-lymphocyte immunophenotype Catherine Milch a, Tim Wyant a, Jing Xu a, Asit Parikh b,⁎, Whitney Kent a, Irving Fox a, Joseph Berger c a b c
Millennium Pharmaceuticals, Inc., a Takeda Company, 35 Landsdowne St., Cambridge, MA 02139, USA Takeda Pharmaceuticals International Inc., 1 Takeda Pkwy, Deerfield, IL 60015, USA Department of Neurology, University of Kentucky College of Medicine, Room L-445, Kentucky Clinic, 740 S Limestone St, Lexington, KY 40536, USA
a r t i c l e
i n f o
Article history: Received 18 June 2013 Received in revised form 19 August 2013 Accepted 21 August 2013 Keywords: Vedolizumab Inflammatory bowel disease Cerebrospinal fluid Immune surveillance
a b s t r a c t Vedolizumab, a gut-homing α4β7 integrin antagonist, has demonstrated efficacy in ulcerative colitis and Crohn's disease. Development of progressive multifocal leukoencephalopathy, a serious brain infection associated with natalizumab (an α4β7 and α4β1 integrin antagonist), has raised concern that vedolizumab may convey a similar risk. Natalizumab is believed to impair central nervous system immune surveillance by affecting cerebrospinal fluid (CSF) lymphocyte counts and the CD4:CD8 ratio. To determine if vedolizumab elicits similar effects, we examined CSF of healthy volunteers by flow cytometry for T-lymphocyte surface markers 5 weeks after administration of intravenous vedolizumab 450 mg. No significant changes were observed in CSF T-lymphocyte populations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Vedolizumab, a gut-selective anti-inflammatory biologic agent in phase 3 development for the treatment of ulcerative colitis (UC) and Crohn's disease (CD), binds to the gut-homing integrin α4β7 and antagonizes its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (Soler et al., 2009), preventing migration of memory T lymphocytes into inflamed tissue. Vedolizumab is highly specific for a neoepitope formed by the heterodimerization of α4 integrin with β7 integrin and does not bind to the independent α4 and β7 monomers (Soler et al., 2009). Vedolizumab has shown benefit in UC and CD pivotal studies (Feagan et al., 2013; Sandborn et al., 2013). Concern has been raised about prolonged treatment with natalizumab and progressive multifocal leukoencephalopathy (PML), a demyelinating central nervous system (CNS) infection caused by reactivation of latent John Cunningham (JC) virus (Clifford et al., 2010; Tan and Koralnik, 2010). Natalizumab, approved in the United States for treatment of both multiple sclerosis (MS) and CD, binds to the α4 subunit of both α4β1 and α4β7 integrins, antagonizing their interaction with all known ligands (TYSABRI package insert, 2011). It is postulated that antagonizing the α4β1 integrin predisposes to PML by impairing CNS immune surveillance (Berger and Koralnik, 2005). Natalizumab reduces numbers of helper (CD4+) and cytotoxic (CD8+) T lymphocytes and the CD4+:
⁎ Corresponding author at: Takeda Pharmaceuticals International Inc., 1 Takeda Pkwy, Deerfield, IL 60015, USA. Tel.: +1 224 554 7573. E-mail address: [email protected] (A. Parikh). 0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.08.011
CD8+ lymphocyte ratio in cerebrospinal fluid (CSF) (Stuve et al., 2006a, 2006b). To understand if selectively targeting the gut-tropic α4β7 integrin averts effects on CNS immune homeostasis, we evaluated the CSF CD4+:CD8+ lymphocyte ratio and cell counts before and 5 weeks after a single 450-mg intravenous vedolizumab dose. Based on pharmacologic modeling, this dose was expected to provide saturation of the α4β7 target in peripheral blood for ~ 16 weeks (Data on file, Millennium Pharmaceuticals Inc., a Takeda company). 2. Materials and methods Healthy subjects 18–45 years old with a suitable body habitus for standard lumbar puncture (LP) and CSF measures within the normal range were eligible. Key exclusion criteria were: any major medical disorder, infection, immunosuppression, neurological disease, and coagulopathy. Oral corticosteroid and immunosuppressant use was prohibited throughout the study, with use of any corticosteroids (e.g., topical, inhaled) prohibited through week 5. Subjects underwent LP before and 5 weeks after a single 450-mg intravenous vedolizumab dose (1.5x the 300-mg dose studied in phase 3 trials [Feagan et al., 2013; Sandborn et al., 2013]). Approximately 15 mL CSF was obtained from each subject; the first 2 mL were discarded (to reduce effects of possible peripheral blood contamination), and the remaining sample was aliquotted for evaluation of pharmacokinetics (PK), chemistry, culture, and cell counts. Samples were processed by the 1:1 addition of stabilization buffer (RPMI + 5% fetal calf serum [FCS]) and shipped within 2 h of LP (laboratory received samples within 75 ± 24 min [mean ± standard deviation]). Upon receipt at a specialty
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laboratory, CSF was immediately centrifuged and the cell pellet was washed once with phosphate-buffered saline (PBS) + 5% FCS. A cocktail of fluorescently labeled antibodies against CD45, CD3, CD4, and CD8 was added and incubated on ice. Cell count beads were added, followed by fixation with PBS + 1% formaldehyde. Samples were run on an FC500 flow cytometer (Beckman Coulter, Brea, CA) with run times optimized to ensure collection of at least 1000 CD3+ CD4+ events. Prerequisites for analysis included: ≤10 RBCs/μL (to minimize peripheral blood contamination), negative culture result, adequate T-lymphocyte numbers, and absence of detectable serum antibodies to vedolizumab. Initial gating strategy included a CD45 versus side scatter gate to identify cells with the appropriate scatter characteristics typical of healthy lymphocytes as a surrogate for viability given the low number of anticipated cells in the CSF. Analyses, including immunophenotyping, were performed on the day of collection. Peripheral blood samples were obtained during screening and at weeks 2, 5, and 16 to evaluate vedolizumab effect on peripheral cell populations. Vedolizumab concentration in serum was determined on day 1 (pre- and postdose) and at weeks 5 and 16, and in CSF at baseline and at week 5. The extent of α4β7 receptor saturation was determined by MAdCAM-1-Fc binding on day 1 (predose) and at week 5. Testing for anti-vedolizumab antibodies was done on day 1 (predose) and at weeks 5 and 16. Safety was assessed by monitoring adverse events (AEs), serious AEs (SAEs), vital signs, clinical laboratory tests, and physical and neurological examinations at each visit. Formal statistical inference was performed for primary and secondary endpoints. For the primary endpoint, the null hypothesis (a clinically significant reduction in mean CD4+:CD8+ ratio from baseline) was rejected if the lower bound of the 90% confidence interval for the mean ratio change, derived from a paired t-test, was ≥−1.67. The secondary endpoint (mean postdose CD4+:CD8+ ratio b 1) was tested using a 1-sample t-test at α = 0.05 (1-sided). Descriptive statistics were provided for safety and other assessments.
3. Results Fourteen subjects were enrolled and received vedolizumab on day 1; 10 (71%) were male; mean age was 27 years (range, 19–41 years); mean body mass index was 26 kg/m2. One subject tested positive for antivedolizumab antibodies and was therefore excluded from lymphocyte analyses, but was evaluated for safety. The mean serum vedolizumab concentration at week 5, 34.7 ± 7.54 μg/mL, was associated with N90% α4β7 receptor saturation as defined by inhibition of MAdCAM-1 binding. Vedolizumab was not detected in any CSF sample (detection limit, 0.125 μg/mL). The mean CSF CD4+:CD8+ ratios before vedolizumab administration and after 5 weeks were 3.592 and 3.605, respectively (Table 1; Fig. 1). In addition, there were no clinically relevant changes in mean or median absolute cell counts from baseline to week 5, or in mean percentage of CD4+- and CD8+-expressing T lymphocytes in CSF (Table 1; Fig. 2). No clinically meaningful changes were noted in analogous populations in peripheral blood (mean changes of 0.87% [CD4+] and −1.05% [CD8+]), or in memory (CD45RO+) subpopulations (mean changes of 0.7% and 0.4%, respectively [Table 2]). Thus the absence of CSF findings was not due to changes in peripheral blood lymphocyte populations (eg, reversal of CD4:CD8 ratio). Ten of the 14 subjects (71%) experienced at least 1 treatmentemergent AE, all mild or moderate. AEs reported by more than 1 subject were headache (6 subjects, 43%) and dizziness (2 subjects, 14%). Vasovagal syncope, nausea, and back pain were also reported. There were no reported SAEs, deaths, or AEs resulting in discontinuation and no clinically relevant changes in hematology or clinical chemistry.
Table 1 Effect of vedolizumab treatment on CSF lymphocyte CD4+:CD8+ ratio and count (n = 13).
CD4+:CD8+ ratio Mean (SE) Range 90% 2-sided CI for ratio CD4+ cells/μL, mean (SD) CD8+ cells/μL, mean (SD) CD4+ as % of lymphocytes, mean (SD) CD8+ as % of lymphocytes, mean (SD)
Baseline
Week 5
Change from baselinea
3.592 (0.9852) 1.53–5.67 3.105, 4.079 108.2 (94.93) 35.3 (35.72) 75.16 (7.383)
3.605 (0.9560)⁎ 1.42–5.15 3.132, 4.077 122.5 (90.20) 37.1 (25.85) 74.22 (6.373)
0.01 (0.197)+ −0.337, 0.363 14.4 (57.72) 1.8 (19.59) −0.94 (4.037)
22.27 (5.432)
22.01 (6.162)
−0.27 (2.911)
CI = confidence interval. a Difference is defined as week 5 CD4+:CD8+ ratio minus baseline CD4+:CD8+ ratio. ⁎ For the secondary endpoint, p b 0.0001 (1-sided, 1-sample t-test for H0: μ b1 vs H1: μ ≥ 1).
4. Discussion In this investigation, a single 450-mg vedolizumab dose did not alter the mean CSF CD4+:CD8+ ratio or affect mean CSF CD4+ and CD8+ cell counts in healthy volunteers. Serum vedolizumab concentrations at week 5 corresponding to postdose CSF assessments exceeded mean trough concentrations observed in a maintenance dosing regimen associated with efficacy in UC and CD and resulted in continuous target saturation (Feagan et al., 2013; Sandborn et al., 2013). Therefore, the absence of CSF changes cannot be attributed to insufficient exposure or target saturation. As prespecified, the subject who developed antivedolizumab antibodies was excluded to minimize confounding by altered PK resulting from drug sensitization. There were no drug-related AEs. These human CSF data are consistent with results in healthy monkeys following prolonged exposure to high-dose vedolizumab (Fedyk et al., 2012). Contrasting effects of vedolizumab and natalizumab on T-lymphocyte function have been further elucidated in monkeys with experimental autoimmune encephalitis (EAE), wherein natalizumab inhibited the formation of EAE while vedolizumab did not (Haanstra et al., 2013). Clinical studies in patients with MS treated with natalizumab have shown significant changes in the CSF CD4+:CD8+ ratio (as defined by ratio b1) (Stuve et al., 2006a, 2006b); no corresponding ratio change was seen with vedolizumab. Natalizumab has also been shown to cause peripheral lymphocytosis, stimulating B-cell release from the bone marrow, which represents a potential reservoir of neurotropic strains of JC virus (Major, 2011). In contrast, vedolizumab does not cause lymphocytosis (Parikh et al., 2010) or obvious changes in circulating B-cell populations (Data on file, Millennium Pharmaceuticals Inc., a Takeda company).
Fig. 1. CD4+:CD8+ ratio of individual subjects before and after vedolizumab exposure (n = 13). Each dot represents the CD4+:CD8+ ratio of an individual subject; blue lines connect the CD4+:CD8+ ratios of a subject before and after vedolizumab exposure; solid black line represents the mean ratios; dotted line corresponds to a ratio of 1.
C. Milch et al. / Journal of Neuroimmunology 264 (2013) 123–126
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Fig. 2. Median and interquartile range (IQ) CSF lymphocyte counts at baseline and week 5 (n = 13). Center horizontal line in each box corresponds to median; upper and lower box margins correspond to 75th and 25th percentiles. The lower bar indicates the data point that is the lower quartile minus 1.5 times the IQ; the upper bar indicates the data point that is the upper quartile plus 1.5 times the IQ. The isolated data points (+) indicate values that are beyond these thresholds.
These data suggest a link between alterations in CNS lymphocyte homeostasis and impaired CNS immune surveillance, creating a permissive environment for PML. The absence of detectable effects on CNS T lymphocytes by selective targeting of α4β7 integrin provides a plausible mechanistic basis for a lower or absent PML risk with vedolizumab (Sathish et al., 2013). Consistent with this, no PML cases were reported during the vedolizumab development program, which comprised ~3000 subjects, many with prolonged exposure. If the premise of gutselective targeting as a means of achieving disease control while averting PML holds, it could translate to a meaningful safety advantage. Limitations of the current research include the choice of healthy volunteers. This differs from natalizumab studies in that the MS population displays an extensive T-lymphocyte infiltrate in CSF at baseline (Stuve et al., 2006a). Healthy volunteers were considered appropriate for this study because inflammatory bowel disease (IBD) patients do not exhibit CSF pleocytosis and to minimize confounding by concomitant immunosuppressants required to manage (IBD). Another is the use of a single dose and 5-week endpoint. Although pharmacologically relevant drug concentrations were present throughout the investigational period, it is possible that vedolizumab could require longer exposure to affect CSF lymphocytes. However, this is unlikely for several reasons. Hickey (1999) has shown mature lymphocytes readily cross into the CSF; without antigenic stimulation, these cells exit after 24 h. This constant cellular turnover results in low levels of CSF lymphocytes and, on average, a CD4+:CD8+ ratio of ~3 in healthy individuals. Moreover, the effect of natalizumab can occur within a day of administration of a single dose (Stuve et al., 2006a). Thus, a potential effect of vedolizumab on CSF lymphocyte populations should be rapid if trafficking into this compartment is dependent on the α4β7 integrin.
Table 2 Peripheral blood memory T-lymphocyte (RO+) percentages (n = 13).
Vedolizumab exposure did not affect the CSF CD4+:CD8+ ratio or cell counts in healthy volunteers. These results support the concept that the α4β7 integrin is unlikely to affect CNS immune surveillance, making PML an unlikely consequence of pharmacologic modulation of this pathway. Disclosures Catherine Milch, Tim Wyant, Jing Xu, Whitney Kent, and Irving Fox are employees of Millennium Pharmaceuticals Inc., a Takeda company. Asit Parikh is an employee of Takeda Pharmaceuticals International Inc. Dr. Berger serves on PML Adjudication Committees for Millennium, Johnson and Johnson, and Amgen; has served on scientific advisory boards or as a consultant for Amgen, Bayer, Biogen Idec, Eisai, Genzyme, GlaxoSmithKline, Genentech, Novartis, and Pfizer; has received speaking fees from CMSC, AAN, Bayer, and Biogen Idec; serves as an associate editor of the Journal of Neurovirology and serves on the editorial boards of ISRN Education, Multiple Sclerosis and Related Disorders, Neuroscience, World Journal of Rheumatology; and receives research support from the PML Consortium. This study was supported by Millennium Pharmaceuticals Inc., a Takeda company. Medical writing assistance was provided by Ms. Dorothy Scott, and editorial assistance was provided by Audrey Suh, PharmD, of Takeda Pharmaceuticals International Inc. Acknowledgments The authors wish to acknowledge critical review of this manuscript by Dr. Brian G. Feagan (Western University, London, Ont, Canada) and Dr. Eric Fedyk (Takeda Pharmaceuticals International Inc., Deerfield, IL). Medical writing assistance was provided by Ms. Dorothy Scott, Takeda Pharmaceuticals International, Inc., Deerfield, IL. References
CD4+CD45RO+ (%) Mean (SD) Median CD8+CD45RO+ (%) Mean (SD) Median
Baseline
Week 5
Mean (SD)
Mean (SD)
27.8 (4.98) 28.6
27.1 (5.02) 26.2
11.2 (3.40) 12.5
10.8 (2.98) 10.7
Berger, J.R., Koralnik, I.L., 2005. Progressive multifocal leukoencephalopathy and natalizumab— unforeseen consequences. N. Engl. J. Med. 353, 414–416. Clifford, D.B., De Luca, A., Simpson, D.M., Arendt, G., Giovannoni, G., Nath, A., 2010. Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases. Lancet Neurol. 9, 438–446. Feagan, B.G., Rutgeerts, P., Sands, B.E., Hanauer, S., Colombel, J.-F., Sandborn, W.J., Van Assche, G., Axler, J., Kim, H.-J., Danese, S., Fox, I., Milch, C., Sankoh, S., Wyant, T., Xu, J., Parikh, A., GEMINI 1 Study Group, 2013. Vedolizumab as induction and maintenance therapy for ulcerative colitis. N. Engl. J. Med. 369, 699–710.
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Fedyk, E.R., Wyant, T., Yang, L., Csizmadia, V., Burke, K., Yang, H., Kadambi, V.J., 2012. Exclusive antagonism of the alpha4beta7 integrin by vedolizumab confirms the gut-selectivity of this pathway in primates. Inflamm. Bowel Dis. 18, 2107–2119. Haanstra, K.G., Hofman, S.O., Lopes Estevao, E.S., 2013. Antagonizing the alpha4beta1 integrin but not alpha4beta7 inhibits leukocytic infiltration of the CNS in rhesus EAE. J. Immunol. 190, 1961–1973. Hickey, W.F., 1999. Leukocyte traffic in the central nervous system: the participants and their roles. Semin. Immunol. 11, 125–137. Major, E.O., 2011. History and current concepts in the pathogenesis of PML. Cleve. Clin. J. Med. 78 (Suppl. 2), S3–S7. Parikh, A., Wyant, T., Clifford, D.B., Berger, J.R., Feagan, B.G., Fox, I.H., Milch, C., 2010. No increase in JC viremia, lymphocyte count, or circulating CD34+ hematopoietic progenitor cells after treatment with vedolizumab, a humanized monoclonal antibody to α4β7 integrin (Digestive Disease Week 2010 abstract 1008). Gastroenterology 138 (5 Suppl. 1), S145–S146. Sandborn, W.J., Feagan, B.G., Rutgeerts, P., Hanauer, S., Colombel, J.-F., Sands, B.E., Lukas, M., Fedorak, R.N., Lee, S., Bressler, B., Fox, I., Rosario, M., Sankoh, S., Xu, J., Stephens, K., Milch, C., Parikh, A., GEMINI 2 Study Group, 2013. Vedolizumab as induction and maintenance therapy for Crohn's disease. N. Engl. J. Med. 369, 711–721.
Sathish, J.G., Sethu, S., Bielsky, M.C., de Haan, L., French, N.S., Govindappa, K., Green, J., Griffiths, C.E., Holgate, S., Jones, D., Kimber, I., Moggs, J., Naisbitt, D.J., Pirmohamed, M., Reichmann, G., Sims, J., Subramanyam, M., Todd, M.D., Van Der Laan, J.W., Weaver, R.J., Park, B.K., 2013. Challenges and approaches for the development of safer immunomodulatory biologics. Nat. Rev. Drug Discov. 12, 306–324. Soler, D., Chapman, T., Yang, L.L., Wyant, T., Egan, R., Fedyk, E.R., 2009. The binding specificity and selective antagonism of vedolizumab, an anti-alpha4beta7 integrin therapeutic antibody in development for inflammatory bowel diseases. J. Pharmacol. Exp. Ther. 330, 864–875. Stuve, O., Marra, C.M., Bar-Or, A., Niino, M., Cravens, P.D., Cepok, S., Frohman, E.M., Phillips, J.T., Arendt, G., Jerome, K.R., Cook, L., Grand'Maison, F., Hemmer, B., Monson, N.L., Racke, M.K., 2006a. Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis. Arch. Neurol. 63, 1383–1387. Stuve, O., Marra, C.M., Jerome, K.R., Cook, L., Cravens, P.D., Cepok, S., Frohman, E.M., Phillips, J.T., Arendt, G., Hemmer, B., Monson, N.L., Racke, M.K., 2006b. Immune surveillance in multiple sclerosis patients treated with natalizumab. Ann. Neurol. 59, 743–747. Tan, C.S., Koralnik, I.J., 2010. Progressive multifocal leukoencephalopathy and other disorders caused by JC virus: clinical features and pathogenesis. Lancet Neurol. 9, 425–437. TYSABRI® (natalizumab) package insert. Biogen Idec Inc., Cambridge, MA; 2011.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Vedolizumab, a monoclonal antibody to the gut homing α4β7 integrin, does not affect cerebrospinal fluid T-lymphocyte immunophenotype Catherine Milch a, Tim Wyant a, Jing Xu a, Asit Parikh b,⁎, Whitney Kent a, Irving Fox a, Joseph Berger c a b c
Millennium Pharmaceuticals, Inc., a Takeda Company, 35 Landsdowne St., Cambridge, MA 02139, USA Takeda Pharmaceuticals International Inc., 1 Takeda Pkwy, Deerfield, IL 60015, USA Department of Neurology, University of Kentucky College of Medicine, Room L-445, Kentucky Clinic, 740 S Limestone St, Lexington, KY 40536, USA
a r t i c l e
i n f o
Article history: Received 18 June 2013 Received in revised form 19 August 2013 Accepted 21 August 2013 Keywords: Vedolizumab Inflammatory bowel disease Cerebrospinal fluid Immune surveillance
a b s t r a c t Vedolizumab, a gut-homing α4β7 integrin antagonist, has demonstrated efficacy in ulcerative colitis and Crohn's disease. Development of progressive multifocal leukoencephalopathy, a serious brain infection associated with natalizumab (an α4β7 and α4β1 integrin antagonist), has raised concern that vedolizumab may convey a similar risk. Natalizumab is believed to impair central nervous system immune surveillance by affecting cerebrospinal fluid (CSF) lymphocyte counts and the CD4:CD8 ratio. To determine if vedolizumab elicits similar effects, we examined CSF of healthy volunteers by flow cytometry for T-lymphocyte surface markers 5 weeks after administration of intravenous vedolizumab 450 mg. No significant changes were observed in CSF T-lymphocyte populations. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Vedolizumab, a gut-selective anti-inflammatory biologic agent in phase 3 development for the treatment of ulcerative colitis (UC) and Crohn's disease (CD), binds to the gut-homing integrin α4β7 and antagonizes its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (Soler et al., 2009), preventing migration of memory T lymphocytes into inflamed tissue. Vedolizumab is highly specific for a neoepitope formed by the heterodimerization of α4 integrin with β7 integrin and does not bind to the independent α4 and β7 monomers (Soler et al., 2009). Vedolizumab has shown benefit in UC and CD pivotal studies (Feagan et al., 2013; Sandborn et al., 2013). Concern has been raised about prolonged treatment with natalizumab and progressive multifocal leukoencephalopathy (PML), a demyelinating central nervous system (CNS) infection caused by reactivation of latent John Cunningham (JC) virus (Clifford et al., 2010; Tan and Koralnik, 2010). Natalizumab, approved in the United States for treatment of both multiple sclerosis (MS) and CD, binds to the α4 subunit of both α4β1 and α4β7 integrins, antagonizing their interaction with all known ligands (TYSABRI package insert, 2011). It is postulated that antagonizing the α4β1 integrin predisposes to PML by impairing CNS immune surveillance (Berger and Koralnik, 2005). Natalizumab reduces numbers of helper (CD4+) and cytotoxic (CD8+) T lymphocytes and the CD4+:
⁎ Corresponding author at: Takeda Pharmaceuticals International Inc., 1 Takeda Pkwy, Deerfield, IL 60015, USA. Tel.: +1 224 554 7573. E-mail address: [email protected] (A. Parikh). 0165-5728/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jneuroim.2013.08.011
CD8+ lymphocyte ratio in cerebrospinal fluid (CSF) (Stuve et al., 2006a, 2006b). To understand if selectively targeting the gut-tropic α4β7 integrin averts effects on CNS immune homeostasis, we evaluated the CSF CD4+:CD8+ lymphocyte ratio and cell counts before and 5 weeks after a single 450-mg intravenous vedolizumab dose. Based on pharmacologic modeling, this dose was expected to provide saturation of the α4β7 target in peripheral blood for ~ 16 weeks (Data on file, Millennium Pharmaceuticals Inc., a Takeda company). 2. Materials and methods Healthy subjects 18–45 years old with a suitable body habitus for standard lumbar puncture (LP) and CSF measures within the normal range were eligible. Key exclusion criteria were: any major medical disorder, infection, immunosuppression, neurological disease, and coagulopathy. Oral corticosteroid and immunosuppressant use was prohibited throughout the study, with use of any corticosteroids (e.g., topical, inhaled) prohibited through week 5. Subjects underwent LP before and 5 weeks after a single 450-mg intravenous vedolizumab dose (1.5x the 300-mg dose studied in phase 3 trials [Feagan et al., 2013; Sandborn et al., 2013]). Approximately 15 mL CSF was obtained from each subject; the first 2 mL were discarded (to reduce effects of possible peripheral blood contamination), and the remaining sample was aliquotted for evaluation of pharmacokinetics (PK), chemistry, culture, and cell counts. Samples were processed by the 1:1 addition of stabilization buffer (RPMI + 5% fetal calf serum [FCS]) and shipped within 2 h of LP (laboratory received samples within 75 ± 24 min [mean ± standard deviation]). Upon receipt at a specialty
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laboratory, CSF was immediately centrifuged and the cell pellet was washed once with phosphate-buffered saline (PBS) + 5% FCS. A cocktail of fluorescently labeled antibodies against CD45, CD3, CD4, and CD8 was added and incubated on ice. Cell count beads were added, followed by fixation with PBS + 1% formaldehyde. Samples were run on an FC500 flow cytometer (Beckman Coulter, Brea, CA) with run times optimized to ensure collection of at least 1000 CD3+ CD4+ events. Prerequisites for analysis included: ≤10 RBCs/μL (to minimize peripheral blood contamination), negative culture result, adequate T-lymphocyte numbers, and absence of detectable serum antibodies to vedolizumab. Initial gating strategy included a CD45 versus side scatter gate to identify cells with the appropriate scatter characteristics typical of healthy lymphocytes as a surrogate for viability given the low number of anticipated cells in the CSF. Analyses, including immunophenotyping, were performed on the day of collection. Peripheral blood samples were obtained during screening and at weeks 2, 5, and 16 to evaluate vedolizumab effect on peripheral cell populations. Vedolizumab concentration in serum was determined on day 1 (pre- and postdose) and at weeks 5 and 16, and in CSF at baseline and at week 5. The extent of α4β7 receptor saturation was determined by MAdCAM-1-Fc binding on day 1 (predose) and at week 5. Testing for anti-vedolizumab antibodies was done on day 1 (predose) and at weeks 5 and 16. Safety was assessed by monitoring adverse events (AEs), serious AEs (SAEs), vital signs, clinical laboratory tests, and physical and neurological examinations at each visit. Formal statistical inference was performed for primary and secondary endpoints. For the primary endpoint, the null hypothesis (a clinically significant reduction in mean CD4+:CD8+ ratio from baseline) was rejected if the lower bound of the 90% confidence interval for the mean ratio change, derived from a paired t-test, was ≥−1.67. The secondary endpoint (mean postdose CD4+:CD8+ ratio b 1) was tested using a 1-sample t-test at α = 0.05 (1-sided). Descriptive statistics were provided for safety and other assessments.
3. Results Fourteen subjects were enrolled and received vedolizumab on day 1; 10 (71%) were male; mean age was 27 years (range, 19–41 years); mean body mass index was 26 kg/m2. One subject tested positive for antivedolizumab antibodies and was therefore excluded from lymphocyte analyses, but was evaluated for safety. The mean serum vedolizumab concentration at week 5, 34.7 ± 7.54 μg/mL, was associated with N90% α4β7 receptor saturation as defined by inhibition of MAdCAM-1 binding. Vedolizumab was not detected in any CSF sample (detection limit, 0.125 μg/mL). The mean CSF CD4+:CD8+ ratios before vedolizumab administration and after 5 weeks were 3.592 and 3.605, respectively (Table 1; Fig. 1). In addition, there were no clinically relevant changes in mean or median absolute cell counts from baseline to week 5, or in mean percentage of CD4+- and CD8+-expressing T lymphocytes in CSF (Table 1; Fig. 2). No clinically meaningful changes were noted in analogous populations in peripheral blood (mean changes of 0.87% [CD4+] and −1.05% [CD8+]), or in memory (CD45RO+) subpopulations (mean changes of 0.7% and 0.4%, respectively [Table 2]). Thus the absence of CSF findings was not due to changes in peripheral blood lymphocyte populations (eg, reversal of CD4:CD8 ratio). Ten of the 14 subjects (71%) experienced at least 1 treatmentemergent AE, all mild or moderate. AEs reported by more than 1 subject were headache (6 subjects, 43%) and dizziness (2 subjects, 14%). Vasovagal syncope, nausea, and back pain were also reported. There were no reported SAEs, deaths, or AEs resulting in discontinuation and no clinically relevant changes in hematology or clinical chemistry.
Table 1 Effect of vedolizumab treatment on CSF lymphocyte CD4+:CD8+ ratio and count (n = 13).
CD4+:CD8+ ratio Mean (SE) Range 90% 2-sided CI for ratio CD4+ cells/μL, mean (SD) CD8+ cells/μL, mean (SD) CD4+ as % of lymphocytes, mean (SD) CD8+ as % of lymphocytes, mean (SD)
Baseline
Week 5
Change from baselinea
3.592 (0.9852) 1.53–5.67 3.105, 4.079 108.2 (94.93) 35.3 (35.72) 75.16 (7.383)
3.605 (0.9560)⁎ 1.42–5.15 3.132, 4.077 122.5 (90.20) 37.1 (25.85) 74.22 (6.373)
0.01 (0.197)+ −0.337, 0.363 14.4 (57.72) 1.8 (19.59) −0.94 (4.037)
22.27 (5.432)
22.01 (6.162)
−0.27 (2.911)
CI = confidence interval. a Difference is defined as week 5 CD4+:CD8+ ratio minus baseline CD4+:CD8+ ratio. ⁎ For the secondary endpoint, p b 0.0001 (1-sided, 1-sample t-test for H0: μ b1 vs H1: μ ≥ 1).
4. Discussion In this investigation, a single 450-mg vedolizumab dose did not alter the mean CSF CD4+:CD8+ ratio or affect mean CSF CD4+ and CD8+ cell counts in healthy volunteers. Serum vedolizumab concentrations at week 5 corresponding to postdose CSF assessments exceeded mean trough concentrations observed in a maintenance dosing regimen associated with efficacy in UC and CD and resulted in continuous target saturation (Feagan et al., 2013; Sandborn et al., 2013). Therefore, the absence of CSF changes cannot be attributed to insufficient exposure or target saturation. As prespecified, the subject who developed antivedolizumab antibodies was excluded to minimize confounding by altered PK resulting from drug sensitization. There were no drug-related AEs. These human CSF data are consistent with results in healthy monkeys following prolonged exposure to high-dose vedolizumab (Fedyk et al., 2012). Contrasting effects of vedolizumab and natalizumab on T-lymphocyte function have been further elucidated in monkeys with experimental autoimmune encephalitis (EAE), wherein natalizumab inhibited the formation of EAE while vedolizumab did not (Haanstra et al., 2013). Clinical studies in patients with MS treated with natalizumab have shown significant changes in the CSF CD4+:CD8+ ratio (as defined by ratio b1) (Stuve et al., 2006a, 2006b); no corresponding ratio change was seen with vedolizumab. Natalizumab has also been shown to cause peripheral lymphocytosis, stimulating B-cell release from the bone marrow, which represents a potential reservoir of neurotropic strains of JC virus (Major, 2011). In contrast, vedolizumab does not cause lymphocytosis (Parikh et al., 2010) or obvious changes in circulating B-cell populations (Data on file, Millennium Pharmaceuticals Inc., a Takeda company).
Fig. 1. CD4+:CD8+ ratio of individual subjects before and after vedolizumab exposure (n = 13). Each dot represents the CD4+:CD8+ ratio of an individual subject; blue lines connect the CD4+:CD8+ ratios of a subject before and after vedolizumab exposure; solid black line represents the mean ratios; dotted line corresponds to a ratio of 1.
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Fig. 2. Median and interquartile range (IQ) CSF lymphocyte counts at baseline and week 5 (n = 13). Center horizontal line in each box corresponds to median; upper and lower box margins correspond to 75th and 25th percentiles. The lower bar indicates the data point that is the lower quartile minus 1.5 times the IQ; the upper bar indicates the data point that is the upper quartile plus 1.5 times the IQ. The isolated data points (+) indicate values that are beyond these thresholds.
These data suggest a link between alterations in CNS lymphocyte homeostasis and impaired CNS immune surveillance, creating a permissive environment for PML. The absence of detectable effects on CNS T lymphocytes by selective targeting of α4β7 integrin provides a plausible mechanistic basis for a lower or absent PML risk with vedolizumab (Sathish et al., 2013). Consistent with this, no PML cases were reported during the vedolizumab development program, which comprised ~3000 subjects, many with prolonged exposure. If the premise of gutselective targeting as a means of achieving disease control while averting PML holds, it could translate to a meaningful safety advantage. Limitations of the current research include the choice of healthy volunteers. This differs from natalizumab studies in that the MS population displays an extensive T-lymphocyte infiltrate in CSF at baseline (Stuve et al., 2006a). Healthy volunteers were considered appropriate for this study because inflammatory bowel disease (IBD) patients do not exhibit CSF pleocytosis and to minimize confounding by concomitant immunosuppressants required to manage (IBD). Another is the use of a single dose and 5-week endpoint. Although pharmacologically relevant drug concentrations were present throughout the investigational period, it is possible that vedolizumab could require longer exposure to affect CSF lymphocytes. However, this is unlikely for several reasons. Hickey (1999) has shown mature lymphocytes readily cross into the CSF; without antigenic stimulation, these cells exit after 24 h. This constant cellular turnover results in low levels of CSF lymphocytes and, on average, a CD4+:CD8+ ratio of ~3 in healthy individuals. Moreover, the effect of natalizumab can occur within a day of administration of a single dose (Stuve et al., 2006a). Thus, a potential effect of vedolizumab on CSF lymphocyte populations should be rapid if trafficking into this compartment is dependent on the α4β7 integrin.
Table 2 Peripheral blood memory T-lymphocyte (RO+) percentages (n = 13).
Vedolizumab exposure did not affect the CSF CD4+:CD8+ ratio or cell counts in healthy volunteers. These results support the concept that the α4β7 integrin is unlikely to affect CNS immune surveillance, making PML an unlikely consequence of pharmacologic modulation of this pathway. Disclosures Catherine Milch, Tim Wyant, Jing Xu, Whitney Kent, and Irving Fox are employees of Millennium Pharmaceuticals Inc., a Takeda company. Asit Parikh is an employee of Takeda Pharmaceuticals International Inc. Dr. Berger serves on PML Adjudication Committees for Millennium, Johnson and Johnson, and Amgen; has served on scientific advisory boards or as a consultant for Amgen, Bayer, Biogen Idec, Eisai, Genzyme, GlaxoSmithKline, Genentech, Novartis, and Pfizer; has received speaking fees from CMSC, AAN, Bayer, and Biogen Idec; serves as an associate editor of the Journal of Neurovirology and serves on the editorial boards of ISRN Education, Multiple Sclerosis and Related Disorders, Neuroscience, World Journal of Rheumatology; and receives research support from the PML Consortium. This study was supported by Millennium Pharmaceuticals Inc., a Takeda company. Medical writing assistance was provided by Ms. Dorothy Scott, and editorial assistance was provided by Audrey Suh, PharmD, of Takeda Pharmaceuticals International Inc. Acknowledgments The authors wish to acknowledge critical review of this manuscript by Dr. Brian G. Feagan (Western University, London, Ont, Canada) and Dr. Eric Fedyk (Takeda Pharmaceuticals International Inc., Deerfield, IL). Medical writing assistance was provided by Ms. Dorothy Scott, Takeda Pharmaceuticals International, Inc., Deerfield, IL. References
CD4+CD45RO+ (%) Mean (SD) Median CD8+CD45RO+ (%) Mean (SD) Median
Baseline
Week 5
Mean (SD)
Mean (SD)
27.8 (4.98) 28.6
27.1 (5.02) 26.2
11.2 (3.40) 12.5
10.8 (2.98) 10.7
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