Journal of the Neurological Sciences 341 (2014) 22–27
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Natalizumab treatment shows no clinically meaningful effects on immunization responses in patients with relapsing-remitting multiple sclerosis☆ Michael Kaufman a,⁎, Gabriel Pardo b, Howard Rossman c, Marianne T. Sweetser d, Fiona Forrestal d, Petra Duda d a
Carolinas Health Care System, Charlotte, NC, USA OMRF Multiple Sclerosis Center of Excellence, 820 NE 15th Street, Oklahoma City, OK 73104, USA c Michigan Institute for Neurological Disorders, 28595 Orchard Lake Road, #200, Farmington Hills, MI 48334, USA d Biogen Idec Inc., 133 Boston Post Road, Weston, MA 02493, USA b
a r t i c l e
i n f o
Article history: Received 1 November 2013 Received in revised form 4 March 2014 Accepted 18 March 2014 Available online 26 March 2014 Keywords: Multiple sclerosis Natalizumab Immunization Vaccines Immunoglobulins Lymphocytes Immunity Humoral
a b s t r a c t Natalizumab is an immunomodulatory drug approved for the treatment of multiple sclerosis. This randomized, multicenter, open-label study evaluated natalizumab's effects on immunization responses to a recall antigen (tetanus toxoid [TT]) and a neoantigen (keyhole limpet hemocyanin [KLH]) in patients with relapsing forms of multiple sclerosis (MS). Natalizumab-naive relapsing MS patients were randomized (1:1; n = 30 per group) to receive TT and KLH immunizations either without natalizumab treatment (control) or after 6 months of natalizumab treatment (natalizumab group). An adequate response to immunization was defined as an increase to at least twofold in specific serum immunoglobulin G (IgG) 28 days after the first immunization. All evaluable patients achieved protective levels of anti-TT IgG antibodies, and the proportion of responders to this recall antigen, as well as to primary immunization with KLH, was similar in the presence and absence of natalizumab. This indicates that natalizumab treatment does not appear to affect responses to primary or secondary immunization in a clinically meaningful way. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Understanding immune responses with immunomodulatory therapy is important for the management of multiple sclerosis (MS) patients in clinical practice. Interferon beta-1a (Rebif®, EMD Serono Rockland, MA, USA) and teriflunomide (Aubagio®, Genzyme, Cambridge, MA, USA) do not appear to have an effect on immunization responses [1,2]. In contrast, fingolimod (Gilenya®, Novartis, East Hanover, NJ, USA) appeared to decrease the response to a pneumococcal vaccine, while responses to keyhole limpet hemocyanin (KLH) and influenza vaccines in patients treated with fingolimod were similar to or lower than responses in patients who received placebo [3–5]. In a small pilot study of patients treated with alemtuzumab (Lemtrada™, Genzyme, Cambridge, MA, USA) [6],
☆ Portions of this work were presented at the 63rd (2011) Annual Meeting of the American Academy of Neurology. ⁎ Corresponding author at: MS Center, Carolinas Medical Center, 1010 Edgehill Road North, Charlotte, NC 28207, USA. Tel.: +1 704 446 1900; fax: +1 704 446 6255. E-mail addresses: [email protected] (M. Kaufman), [email protected] (G. Pardo), [email protected] (H. Rossman), [email protected] (M.T. Sweetser), fi[email protected] (F. Forrestal), [email protected] (P. Duda).
http://dx.doi.org/10.1016/j.jns.2014.03.035 0022-510X/© 2014 Elsevier B.V. All rights reserved.
humoral responses to recall antigens (pneumococcal vaccine and diphtheria, tetanus, and poliomyelitis vaccine) and a novel antigen (Haemophilus influenza type b and meningococcal group C conjugate vaccine) were consistent with historical controls. However, compared with patients who were vaccinated at least 6 months after alemtuzumab treatment, response was achieved in a smaller proportion of patients who were vaccinated within 6 months of treatment [7]. To our knowledge, no immunization data are currently available for glatiramer acetate (Copaxone®, Teva Neuroscience, Kansas City, MO, USA), mitoxantrone (Novantrone®, EMD Serono, Inc, Darmstadt, Germany), or dimethyl fumarate (Tecfidera™, Biogen Idec, Cambridge, MA, USA). With respect to natalizumab (Tysabri®, Biogen Idec, Cambridge, MA, USA), one report showed no decrease in the response to influenza vaccination [8]. Natalizumab, an established therapy for MS and Crohn's disease, is a recombinant humanized monoclonal antibody (Ab) that inhibits binding of the α4 subunit of the α4β1 and α4β7 integrins on mononuclear leukocytes to their endothelial receptors and prevents trafficking of mononuclear leukocytes across vascular endothelium [9–11]. Treatment with natalizumab has been associated with herpes infections as well as with progressive multifocal leukoencephalopathy [9,12–14]. Both de novo Ab responses to neoantigens (primary responses) and recall responses to memory antigens (secondary responses) involve
M. Kaufman et al. / Journal of the Neurological Sciences 341 (2014) 22–27
activation of antigen-specific T and B cells in secondary lymphoid tissues [15]. While human and animal studies have shown that natalizumab increases the number of circulating lymphocytes, possibly due to trafficking effects [12,16–20], animal studies have not shown a significant effect of α4-integrin blockade on T and B cell trafficking into most lymphoid tissues [21]. Nonhuman primate studies have shown no significant effect of natalizumab on primary or secondary Ab responses [20]; however, it is important to evaluate its effects on primary and secondary immune responses in patients. Owing to the potential effects of α4-integrin blockade by natalizumab on lymphocyte trafficking through primary and secondary lymphoid organs, it is important to provide additional data on the impact of natalizumab therapy on vaccination response in MS patients [21]. The objective of this study was to evaluate the effect of natalizumab on T-cell-dependent Ab responses to a recall antigen (tetanus toxoid [TT]) and to a neoantigen (keyhole limpet hemocyanin [KLH]) in patients with relapsing forms of MS. TT was used in this United States (US) study to assess recall Ab responses, as the majority of the US population receive childhood TT immunizations [22]. KLH, a strongly immunogenic protein isolated from the giant keyhole limpet, was used to assess primary immunization responses [23]. 2. Materials and methods 2.1. Study design In this phase 4, randomized, multi center, open-label study, eligible MS patients, naive to natalizumab, were randomized (1:1) to receive TT and KLH immunizations either 2 months prior to natalizumab treatment or after 6 months of natalizumab treatment (Fig. 1). Patients enrolling in the study agreed to delay treatment with natalizumab for approximately 2 months if randomized to the control/immunization-only group. All nine US sites used an interactive voice response system to randomize patients at the baseline visit. Randomization was stratified by site. Planned enrollment was approximately 46 patients; 23 patients per group. The study protocol and amendments were reviewed and approved by each site's institutional review board. The study was performed in accordance with the Declaration of Helsinki and International Conference on Harmonisation Guideline on Good Clinical Practice and is registered with ClinicalTrials.gov, number NCT00536120. 2.2. Patients Patients 18–60 years old who were diagnosed with a relapsing form of MS, eligible to receive natalizumab per approved labeling, and
Natalizumab started
Natalizumab
M0
TT vaccine TT Ab assay
previously vaccinated against TT were eligible for the study. Written informed consent was obtained from each patient prior to evaluations performed for eligibility. Exclusion criteria included the following: [1] TT immunization b2 years prior to screening, [2] hypersensitivity to TT, KLH, or other immunizations or their components [3], significant infectious illness within 30 days prior to screening [4], prior treatment with natalizumab, rituximab, any murine protein, or any therapeutic monoclonal Ab [5], treatment with cyclophosphamide within 1 year prior to screening [6], treatment with intravenous (i.v.) or intramuscular (i.m.) immunoglobulin (Ig), or immunosuppressant medications within 6 months prior to screening [7], treatment with systemic corticosteroids within 4 weeks prior to screening, and [8] treatment with immunomodulatory medications (interferon-β and glatiramer acetate) within 2 weeks prior to screening.
2.3. Interventions Patients randomized to natalizumab treatment prior to immunization (natalizumab group) received natalizumab, 300 mg i.v. every 4 weeks for 6 months, starting at the baseline visit, and received immunizations after the seventh natalizumab infusion. Natalizumab treatment continued for 2 months after the first immunization during which their immune responses were measured. Patients randomized to the control group received immunizations beginning at the baseline visit and their immune responses were measured over the following 2 months. Both groups received a single intramuscular TT vaccine administered per approved label at the investigational site and both groups received three subcutaneous Immucothel® (KLH) (Biosyn Arzneimittel AG, Fellbach, Germany) vaccines, each separated by 2 weeks (Fig. 1). KLH was supplied by Biogen Idec. The first TT and KLH vaccines were given on the same day. For patients randomized to the natalizumab group, samples for Ab assessments were collected prior to natalizumab infusion, and all immunizations were administered after completion of the 1-hour natalizumab post-dosing observation period. All eligible patients from both groups could enroll in TYSABRI® Outreach: Unified Commitment to Health (TOUCH®) after the study ended.
2.4. Antibody measurement TT and KLH Ab levels were measured using an enzyme-linked immunosorbent assay by Focus Diagnostics (Cypress, CA, USA). The upper limit of detection for the anti-TT Ab assay was 7.0 IU/mL in this clinical assay, which is not validated for diluted samples.
M0
+14d
+28d
+56d
M6
M6+14d
M6+28d
M6+56d
X
X
X X
KLH vaccine KLH Ab assay
Control
23
X
X X
X
X
X
X
X
X
Fig. 1. Study design. Patients were randomized 1:1 to the control group or natalizumab group. Control group patients received immunizations shortly after randomization and agreed to delay initiation of natalizumab until after day +56; patients randomized to natalizumab were treated with natalizumab for 6 months prior to immunizations. In the natalizumab group, baseline levels were drawn at month 0 prior to the initiation of natalizumab therapy. Blood for pre-immunization anti-KLH and anti-TT Ab levels was drawn just prior to the first immunization in both groups; baseline and pre-immunization time points are equivalent in the control group. Patients received three KLH immunizations separated by 2 weeks, and one TT immunization. A pre-planned assessment of anti-KLH Ab levels occurred at 14 days (after one KLH immunization), 28 days (after two KLH immunizations), and 56 days (after three immunizations) after first immunization. A pre-planned assessment of anti-TT Ab levels occurred at 28 days and post hoc at 56 days after the first (only) immunization. M: month; d: days; KLH: keyhole limpet hemocyanin; TT: tetanus toxoid; Ab: antibody.
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2.5. Assessments Samples were collected on days 0, 14, 28, and 56 [2 months] after the first immunization. The proportion of responders to TT immunization at days 28 and 56 after the first (and only) TT immunization and the proportion of responders to KLH immunization at days 14, 28, and 56 after the first KLH immunization were assessed. Day 56 TT analyses were performed post hoc. A positive Ab response was defined as an increase to twofold or greater of the pre-immunization anti-TT or antiKLH immunoglobulin G (IgG) Ab level. This increase in Ab level is expected for patients with functional immune systems [24–26] (Fig. 1). Safety was assessed throughout the study. 2.6. Statistical analyses Outcomes after TT and KLH immunizations are reported as the difference in proportion of positive Ab responders (95% confidence interval [CI]) between the natalizumab group and the control group. Patients with anti-TT Ab levels greater than half the upper limit of quantitation (i.e., N 3.5 IU/mL) at pre-immunization could not be considered in the per-protocol analyses of responders because an increase to twofold of the pre-immunization Ab level was impossible to confirm. Patients with results N7.0 IU/mL (above the upper limit of the assay) were assigned a value of 7.0 IU/mL to allow for calculation of summary statistics. Ab responses were analyzed using the change from preimmunization levels obtained at month 0 for the control group, and at month 6, just before immunization, for the natalizumab group. Sensitivity analyses of anti-TT Ab and anti-KLH Ab responses included all available data regardless of protocol violations. The only patients excluded were those with missing Ab level results where response could not be assessed, e.g., due to the sample not being collected. For patients with pre-immunization anti-TT levels of N 3.5 IU/mL, a postimmunization level of N7 IU/mL was considered a positive response. Protocol deviations causing exclusion from primary per-protocol analyses but inclusion in these sensitivity analyses included delayed immunization or samples collected outside of pre-specified window, immunization by incorrect route, and sample collection after use of prohibited medications. It was possible to be eligible for some analyses while being excluded from others. For example, a patient who missed a KLH immunization could still be included in the analysis of anti-TT responses, or a patient who had a prohibited medication at the time of day + 56 sampling could still be included in day +14 and +28 analyses. 3. Results 3.1. Patients 3.1.1. Patient disposition Planned enrollment was approximately 46 patients — 23 patients per group. Per protocol, it was possible to replace patients who withdrew. This was done by increasing the total number of patients randomized rather than by recruiting patients to the required pre-specified groups. The number of eligible patients and the amount of eligible data were reduced further as a result of protocol deviations concerning the administration of KLH and TT. Therefore, in order to ensure sufficient patient numbers in each group to perform the analysis of the primary endpoint at day 28, a total of 60 patients were recruited — 30 to each group. Patient randomization was stratified by site, and the study was balanced across treatment arms both within each site and overall. Twenty-four (80%) of 30 natalizumab patients completed the study and 22 (73%) received all nine natalizumab infusions. Three natalizumab patients (10%) developed persistent anti-natalizumab Abs, a proportion similar to the results in the natalizumab phase 3 AFFIRM study (6%) [9]. Two of the three patients developed persistent anti-natalizumab Abs prior to the scheduled immunizations and were not immunized, as required by the protocol, as the anti-natalizumab
Abs may have led to increased natalizumab clearance and confounding effects on the analyses. 3.1.2. Patient demographics and baseline characteristics The majority of patients were white (88%) and female (85%). The median age was 40 years for control patients and 45 years for natalizumab patients. Both groups had wide ranges for baseline demographic variables but had comparable disease histories (Table 1). Prior therapy with glatiramer acetate, i.m. interferon (IFN) β-1a, and subcutaneous (s.c.) IFNβ-1a were each reported by 42% of patients overall. IFNβ-1b use was reported by 27% of patients. Some patients had more than one prior therapy. Previous use of i.m. IFNβ-1a was higher in patients randomized to the natalizumab group than to the control group (53% vs 30%), while prior use of glatiramer acetate was higher in the control group (50% vs 33%). Few patients had used other MS treatments, including chronic MS-related corticosteroid treatment (10%) and mitoxantrone (7%). 3.2. Ab response to TT (recall response) and KLH (neoantigen response) vaccines Patients who withdrew from the study prior to immunization and/ or sample collection or met pre-specified criteria were excluded from the primary Ab analyses. Concomitant medication use was similar in both groups during the immunization period. As a result of childhood immunization, up to 90% of adults in the United States have protective levels of anti-TT Abs (≥ 0.15 IU/mL), and repeat TT immunization in adulthood should cause a memory immune response with increased anti-TT Ab levels [15,27]. At days + 28 and + 56, the proportions of responders to TT immunization were similar between the two groups. Sensitivity analyses that included all available data from immunized patients, including those with pre-immunization anti-TT Ab levels N3.5 IU/L where a post-immunization level of N 7 IU/mL was considered a positive response, showed similar results (Table 2). The one non-responder to TT (per protocol) at day + 28 in the natalizumab group showed a clear response at day + 56. This patient had high protective anti-TT Ab levels of 3.16 IU/mL at baseline and 2.36 IU/mL at pre-immunization, which increased at day + 28 to 4.32 IU/mL and further to N7.0 IU/mL at day +56. Thus, although this patient was a non-responder per protocol, the patient might be considered a clinical responder. Four of the five patients randomized to natalizumab, who were excluded from the primary anti-TT analyses due to pre-immunization anti-TT Ab levels N3.5 IU/L, had anti-TT levels N7.0 IU/L at day +28. Median anti-TT Ab levels, both pre-immunization and postimmunization, were similar between the evaluable patients in both the control and natalizumab groups. One patient in the natalizumab group had a low anti-TT Ab level (0.03 U/L) pre-immunization, which may not have been protective. All patients had protective levels of anti-TT Abs post-immunization. The median (quartiles) anti-TT Ab
Table 1 Patient demographics and disease history. Characteristic
Control (n = 30)
Natalizumab (n = 30)
Age (years), median (range) Female, n (%) White, n (%) Weight (kg), median (range) Body mass index (kg/m2), median (range) Time since MS diagnosis (years), median (range) No. of relapses in prior 12 months, median (range) EDSS score, median (range)
40 (24–60) 28 (93) 26 (87) 75.3 (43.1–114.6) 26.7 (16.4–39.5) 5 (1–19)
45 (23–56) 23 (77) 27 (90) 80.4 (40.8–124.0) 29.2 (15.0–43.9) 6 (1–29)
1 (0–3)
1 (0–4)
3 (0–7)
2.75 (1–6)
MS: multiple sclerosis; EDSS: Expanded Disability Status Scale.
M. Kaufman et al. / Journal of the Neurological Sciences 341 (2014) 22–27
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Table 2 Analyses of patients responding to TT and KLH immunizations. Responders, n (%)
Day +14
Per-protocol analysis — anti-TT Control Natalizumab Difference in proportions (95% CI) Sensitivity analysis — anti-TTa Control Natalizumab Difference in proportions (95% CI) Per-protocol analysis — anti-KLH Control Natalizumab Difference in proportions (95% CI) Sensitivity analysis — anti-KLHa Control Natalizumab Difference in proportions (95% CI)
Day +28
Day +56
24/24 (100) 15/16 (94) −0.06 (−0.18, 0.06)
21/22 (95) 14/15 (93) −0.02 (−0.17, 0.13)
28/28 (100) 20/22 (91) −0.09 (−0.21, 0.03)
25/27 (93) 20/23 (87) −0.06 (−0.23, 0.11)
9/24 (38) 5/20 (25) −0.13 (−0.40, 0.15)
19/23 (83) 17/19 (89) 0.07 (−0.14, 0.28)
21/21 (100) 15/17 (88) −0.12 (−0.27, 0.04)
10/28 (36) 6/23 (26) −0.10 (−0.35, 0.16)
23/28 (82) 18/22 (82) −0.00 (−0.22, 0.21)
27/27 (100) 19/22 (86) −0.14 (−0.28, 0.01)
KLH: keyhole limpet hemocyanin; TT: tetanus toxoid; CI: confidence interval. a Sensitivity analysis used all available data, regardless of protocol violations. Only patients with missing pre-immunization values were excluded. For TT sensitivity analyses patients were considered responders if the anti-TT Ab level was twice the pre-immunization level or if the anti-TT Ab level was N7.0 IU/mL, regardless of the pre-immunization level.
levels (IU/mL) for evaluable control and natalizumab patients, respectively, were 1.41 (0.52, 1.80) and 1.54 (1.30, 2.36) at pre-immunization, 7.00 (7.00, 7.00) and 7.00 (4.21, 7.00) at day +28, and 7.00 (6.41, 7.00) and 6.31 (3.87, 7.00) at day +56. The proportions of responders to KLH were similar over time, and post hoc sensitivity analyses that included all available data showed comparable results (Table 2). The two non-responders to KLH immunization in the evaluable natalizumab patients at day + 28 did not develop Abs through day + 56. A review of these patients' records did not reveal any confounding factors to explain the lack of response and both patients mounted Ab responses to TT. The geometric mean (95% CI) anti-KLH Ab levels (μg/mL) at pre-immunization were similar
for control and natalizumab patients: 0.78 (0.55–1.13) and 0.72 (0.48–1.09), respectively. The geometric mean (95% CI) anti-KLH Ab levels (μg/mL) for evaluable control and natalizumab patients, respectively, were 1.92 (1.06–3.48) and 1.13 (0.61–2.12) at day + 14, 11.12 (5.52–22.42) and 7.04 (3.51–14.11) at day + 28, and 32.90 (17.39–62.25) and 27.12 (10.54–69.80) at day + 56. Ab concentrations plotted for evaluable patients from preimmunization through day + 56 in reverse cumulative distribution plots demonstrated no clinically relevant difference between the control and natalizumab groups in response to TT or KLH immunization (Figs. 2 and 3). On the vertical axis, reverse cumulative distribution plots display the percentage of patients having an Ab concentration
Natalizumab
Pre-immunization
100
Day +28 100 Cumulative percentage
Cumulative percentage
Control
80 60 40 20
80 60 40 20
0
0
0
1
2
3
4
5
6
7
0
1
Anti-TT concentration (IU/mL)
2
3
4
5
6
7
Anti-TT concentration (IU/mL)
Cumulative percentage
Day +56
100 80 60 40 20 0 0
1
2
3
4
5
6
7
Anti-TT concentration (IU/mL) Fig. 2. Reverse cumulative distribution functions* of observed anti-TT Ab concentration. Graphs display similar reverse cumulative distribution curves of anti-TT IgG Ab concentration for evaluable natalizumab-treated and control patients pre-immunization and at day +28 and day +56 after TT immunization. *On the vertical axis, reverse cumulative distribution plots display the percentage of patients having an Ab concentration greater than the level shown on the horizontal axis. Groups with essentially the same distribution of responses will produce curves with roughly the same shapes and positions [28]. TT: tetanus toxoid; Ab: antibody; IgG: immunoglobulin G.
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Control
Natalizumab
Cumulative percentage
Cumulative percentage
Pre-immunization
100 80 60 40 20 0 0
Day +14
100 80 60 40 20 0
25 50 75 100 125 150 175 200 225 250 275
0
Anti-KLH concentration (µg/mL)
Anti-KLH concentration (µg/mL) Cumulative percentage
Cumulative percentage
Day +28
100 80 60 40 20 0 0
25 50 75 100 125 150 175 200 225 250 275
25 50 75 100 125 150 175 200 225 250 275
Day +56
100 80 60 40 20 0 0
Anti-KLH concentration (µg/mL)
25 50 75 100 125 150 175 200 225 250 275
Anti-KLH concentration (µg/mL)
Fig. 3. Reverse cumulative distribution functions* of observed anti-KLH Ab concentration. Graphs display similar reverse cumulative distribution curves of anti-KLH IgG Ab concentration for evaluable natalizumab-treated and control patients at pre-immunization and at day +14, day +28, and day +56 after the first KLH immunization. Pre-immunization, all control and natalizumab patients had Ab concentrations b15 μg/mL. *On the vertical axis, reverse cumulative distribution plots display the percentage of patients having an Ab concentration greater than the level shown on the horizontal axis. Groups with essentially the same distribution of responses will produce curves with roughly the same shapes and positions [28]. KLH: keyhole limpet hemocyanin; Ab: antibody; IgG: immunoglobulin G.
greater than the level shown on the horizontal axis. Groups with essentially the same distribution of responses will produce curves with roughly the same shapes and positions [28]. Safety analyses showed that immunizations and natalizumab were well tolerated, and no unexpected events were observed (data not shown). 4. Discussion Natalizumab has proven efficacy in reducing central nervous system inflammation in MS [9]; however, its effects on primary and secondary humoral immune responses have not been well studied. This study was not designed to detect small differences between treatment arms, but to investigate whether there is a clinically relevant alteration of the immune response with natalizumab treatment. The proportions of responders to recall antigen (TT) and neoantigen (KLH) immunization were similar in control and natalizumab-treated patients, indicating that natalizumab therapy does not have a major effect on the response to these primary and secondary vaccinations. This is supported by the fact that the anti-KLH and anti-TT Ab levels are similarly increased in response to these vaccinations in the natalizumab and control groups in the reverse cumulative distribution analyses. The sample size in our study and especially the imbalance between the two groups may raise questions about the reliability of the data. Treatment groups were well balanced overall and by site at randomization. However, enrollment beyond the originally planned study size was needed to compensate for [1] a higher than expected number of discontinuations in the natalizumab group, possibly due to the longer study participation in that group; [2] exclusion of data due to protocol deviations; and [3] the need to exclude patients with high preimmunization anti-TT Ab levels from the primary analysis. This led to an imbalance in evaluable patients between the two treatment groups. However, multiple sensitivity analyses confirmed the findings based on the primary analysis. Therefore, we consider our data to be adequate
and believe that our conclusion provides important information for patients receiving natalizumab therapy. In addition, our results confirm those of Vågberg et al., who found no significant difference between placebo and natalizumab-treated relapsing-remitting MS patients in the levels of serum IgG in response to influenza A or B vaccination [8]. Our data are also consistent with preclinical data showing that natalizumab does not affect responses to TT and KLH vaccination in monkeys [21]. In summary, our data indicate that natalizumab does not impair the humoral response to recall immunization with TT and de novo immunization with KLH in a clinically meaningful way [12]. Funding This study was funded by Biogen Idec Inc. Conflict of interest statement Dr. Kaufman has received honoraria and research support from Biogen Idec, has received financial support from Bayer, EMD Serono, Novartis, and Teva, and is a consultant for Department of Defense. Dr. Pardo has received consulting fees from Acorda, Biogen Idec, Novartis, and Teva; he has received compensation for speaking from Acorda, Bayer HealthCare, Biogen Idec, EMD Serono, Novartis, Pfizer, and Teva. Dr. Rossman has no financial relationships to disclose. Drs. Sweetser and Duda and Ms. Forrestal are employees of Biogen Idec and own stock. Acknowledgments Biogen Idec provided funding for editorial support in the development of this manuscript; Britt Anderson, PhD, from Infusion Communications wrote the first draft of the manuscript based on input from authors, and Jackie Cannon from Infusion Communications copyedited
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and styled the manuscript per journal requirements. Biogen Idec reviewed and provided feedback on the manuscript to the authors. The authors had full editorial control of the manuscript and provided their final approval of all content. References [1] Schwid SR, Decker MD, Lopez-Bresnahan M. Immune response to influenza vaccine is maintained in patients with multiple sclerosis receiving interferon beta-1a. Neurology Dec 27 2005;65(12):1964–6. [2] Bar-Or A, Freedman MS, Kremenchutzky M, Menguy-Vacheron F, Bauer D, Jodl S, et al. Teriflunomide effect on immune response to influenza vaccine in patients with multiple sclerosis. Neurology Aug 6 2013;81(6):552–8. [3] Gilenya (fingolimod). [prescribing information] East Hanover, NJ: Novartis Pharmaceuticals Corporation; 2012. [4] Boulton C, Meiser K, David OJ, Schmouder R. Pharmacodynamic effects of steadystate fingolimod on antibody response in healthy volunteers: a 4-week, randomized, placebo-controlled, parallel-group, multiple-dose study. J Clin Pharmacol Dec 2012;52(12):1879–90. [5] Mehling M, Hilbert P, Fritz S, Durovic B, Eichin D, Gasser O, et al. Antigen-specific adaptive immune responses in fingolimod-treated multiple sclerosis patients. Ann Neurol 2011;69(2):408–13. [6] Genzyme. European commission approves genzyme's multiple sclerosis treatment Lemtrada™ (alemtuzumab) . Accessed September 20, 2013. [7] McCarthy CL, Tuohy O, Compston DA, Kumararatne DS, Coles AJ, Jones JL. Immune competence after alemtuzumab treatment of multiple sclerosis. Neurology Sep 3 2013;81(10):872–6. [8] Vågberg M, Kumlin U, Svenningsson A. Humoral immune response to influenza vaccine in natalizumab-treated MS patients. Neurol Res Sep 2012;34(7):730–3. [9] Polman CH, O'Connor PW, Havrdova E, Hutchinson M, Kappos L, Miller DH, et al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med Mar 2 2006;354(9):899–910. [10] Sandborn WJ, Colombel JF, Enns R, Feagan BG, Hanauer SB, Lawrance IC, et al. Natalizumab induction and maintenance therapy for Crohn's disease. N Engl J Med Nov 3 2005;353(18):1912–25. [11] Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman L, Karin N. Prevention of experimental autoimmune encephalomyelitis by antibodies against alpha 4 beta 1 integrin. Nature Mar 5 1992;356(6364):63–6. [12] Tysabri (natalizumab). [prescribing information] Cambridge, MA: Biogen Idec, Inc.; 2013. [13] Clifford DB, De Luca A, Simpson DM, Arendt G, Giovannoni G, Nath A. Natalizumabassociated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: lessons from 28 cases. Lancet Neurol Apr 2010;9(4):438–46.
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[14] Rudick RA, Stuart WH, Calabresi PA, Confavreux C, Galetta SL, Radue EW, et al. Natalizumab plus interferon beta-1a for relapsing multiple sclerosis. N Engl J Med Mar 2 2006;354(9):911–23. [15] Janeway C, Murphy KM, Travers P, Walport M. Janeway's immunobiology (immunobiology: the immune system). 7th ed. London, UK: Garland Science; 2007. [16] Frisullo G, Iorio R, Plantone D, Marti A, Nociti V, Patanella AK, et al. CD4 + T-bet+, CD4 + pSTAT3+ and CD8 + T-bet + T cells accumulate in peripheral blood during NZB treatment. Mult Scler May 2011;17(5):556–66. [17] Miller DH, Khan OA, Sheremata WA, Blumhardt LD, Rice GP, Libonati MA, et al. A controlled trial of natalizumab for relapsing multiple sclerosis. N Engl J Med Jan 2 2003;348(1):15–23. [18] Planas R, Jelcic I, Schippling S, Martin R, Sospedra M. Natalizumab treatment perturbs memory- and marginal zone-like B-cell homing in secondary lymphoid organs in multiple sclerosis. Eur J Immunol Mar 2012;42(3):790–8. [19] Putzki N, Baranwal MK, Tettenborn B, Limmroth V, Kreuzfelder E. Effects of natalizumab on circulating B cells, T regulatory cells and natural killer cells. Eur Neurol 2010;63(5):311–7. [20] Wehner NG, Gasper C, Shopp G, Nelson J, Draper K, Parker S, et al. Immunotoxicity profile of natalizumab. J Immunotoxicol Jun 2009;6(2):115–29. [21] von Andrian UH, Mempel TR. Homing and cellular traffic in lymph nodes. Nat Rev Immunol Nov 2003;3(11):867–78. [22] Gergen PJ, McQuillan GM, Kiely M, Ezzati-Rice TM, Sutter RW, Virella G. A population-based serologic survey of immunity to tetanus in the United States. N Engl J Med Mar 23 1995;332(12):761–6. [23] Harris JR, Markl J. Keyhole limpet hemocyanin (KLH): a biomedical review. Micron Dec 1999;30(6):597–623. [24] Amlot PL, Hayes AE, Gray D, Gordon-Smith EC, Humphrey JH. Human immune responses in vivo to protein (KLH) and polysaccharide (DNP-Ficoll) neoantigens: normal subjects compared with bone marrow transplant patients on cyclosporine. Clin Exp Immunol Apr 1986;64(1):125–35. [25] Birdsall HH, Rossen RD. Characterization of anti-Fab' antibodies in human sera: identification of soluble immune complexes that contain hidden anti-KLH and blocking anti-immunoglobulins following immunization with keyhole limpet haemocyanin. Clin Exp Immunol Aug 1983;53(2):497–504. [26] Welch MJ, Fong S, Vaughan J, Carson D. Increased frequency of rheumatoid factor precursor B lymphocytes after immunization of normal adults with tetanus toxoid. Clin Exp Immunol Feb 1983;51(2):299–304. [27] Talan DA, Abrahamian FM, Moran GJ, Mower WR, Alagappan K, Tiffany BR, et al. Tetanus immunity and physician compliance with tetanus prophylaxis practices among emergency department patients presenting with wounds. Ann Emerg Med Mar 2004;43(3):305–14. [28] Reed GF, Meade BD, Steinhoff MC. The reverse cumulative distribution plot: a graphic method for exploratory analysis of antibody data. Pediatrics Sep 1 1995;96(3 Pt 2): 600–3.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns
Natalizumab treatment shows no clinically meaningful effects on immunization responses in patients with relapsing-remitting multiple sclerosis☆ Michael Kaufman a,⁎, Gabriel Pardo b, Howard Rossman c, Marianne T. Sweetser d, Fiona Forrestal d, Petra Duda d a
Carolinas Health Care System, Charlotte, NC, USA OMRF Multiple Sclerosis Center of Excellence, 820 NE 15th Street, Oklahoma City, OK 73104, USA c Michigan Institute for Neurological Disorders, 28595 Orchard Lake Road, #200, Farmington Hills, MI 48334, USA d Biogen Idec Inc., 133 Boston Post Road, Weston, MA 02493, USA b
a r t i c l e
i n f o
Article history: Received 1 November 2013 Received in revised form 4 March 2014 Accepted 18 March 2014 Available online 26 March 2014 Keywords: Multiple sclerosis Natalizumab Immunization Vaccines Immunoglobulins Lymphocytes Immunity Humoral
a b s t r a c t Natalizumab is an immunomodulatory drug approved for the treatment of multiple sclerosis. This randomized, multicenter, open-label study evaluated natalizumab's effects on immunization responses to a recall antigen (tetanus toxoid [TT]) and a neoantigen (keyhole limpet hemocyanin [KLH]) in patients with relapsing forms of multiple sclerosis (MS). Natalizumab-naive relapsing MS patients were randomized (1:1; n = 30 per group) to receive TT and KLH immunizations either without natalizumab treatment (control) or after 6 months of natalizumab treatment (natalizumab group). An adequate response to immunization was defined as an increase to at least twofold in specific serum immunoglobulin G (IgG) 28 days after the first immunization. All evaluable patients achieved protective levels of anti-TT IgG antibodies, and the proportion of responders to this recall antigen, as well as to primary immunization with KLH, was similar in the presence and absence of natalizumab. This indicates that natalizumab treatment does not appear to affect responses to primary or secondary immunization in a clinically meaningful way. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Understanding immune responses with immunomodulatory therapy is important for the management of multiple sclerosis (MS) patients in clinical practice. Interferon beta-1a (Rebif®, EMD Serono Rockland, MA, USA) and teriflunomide (Aubagio®, Genzyme, Cambridge, MA, USA) do not appear to have an effect on immunization responses [1,2]. In contrast, fingolimod (Gilenya®, Novartis, East Hanover, NJ, USA) appeared to decrease the response to a pneumococcal vaccine, while responses to keyhole limpet hemocyanin (KLH) and influenza vaccines in patients treated with fingolimod were similar to or lower than responses in patients who received placebo [3–5]. In a small pilot study of patients treated with alemtuzumab (Lemtrada™, Genzyme, Cambridge, MA, USA) [6],
☆ Portions of this work were presented at the 63rd (2011) Annual Meeting of the American Academy of Neurology. ⁎ Corresponding author at: MS Center, Carolinas Medical Center, 1010 Edgehill Road North, Charlotte, NC 28207, USA. Tel.: +1 704 446 1900; fax: +1 704 446 6255. E-mail addresses: [email protected] (M. Kaufman), [email protected] (G. Pardo), [email protected] (H. Rossman), [email protected] (M.T. Sweetser), fi[email protected] (F. Forrestal), [email protected] (P. Duda).
http://dx.doi.org/10.1016/j.jns.2014.03.035 0022-510X/© 2014 Elsevier B.V. All rights reserved.
humoral responses to recall antigens (pneumococcal vaccine and diphtheria, tetanus, and poliomyelitis vaccine) and a novel antigen (Haemophilus influenza type b and meningococcal group C conjugate vaccine) were consistent with historical controls. However, compared with patients who were vaccinated at least 6 months after alemtuzumab treatment, response was achieved in a smaller proportion of patients who were vaccinated within 6 months of treatment [7]. To our knowledge, no immunization data are currently available for glatiramer acetate (Copaxone®, Teva Neuroscience, Kansas City, MO, USA), mitoxantrone (Novantrone®, EMD Serono, Inc, Darmstadt, Germany), or dimethyl fumarate (Tecfidera™, Biogen Idec, Cambridge, MA, USA). With respect to natalizumab (Tysabri®, Biogen Idec, Cambridge, MA, USA), one report showed no decrease in the response to influenza vaccination [8]. Natalizumab, an established therapy for MS and Crohn's disease, is a recombinant humanized monoclonal antibody (Ab) that inhibits binding of the α4 subunit of the α4β1 and α4β7 integrins on mononuclear leukocytes to their endothelial receptors and prevents trafficking of mononuclear leukocytes across vascular endothelium [9–11]. Treatment with natalizumab has been associated with herpes infections as well as with progressive multifocal leukoencephalopathy [9,12–14]. Both de novo Ab responses to neoantigens (primary responses) and recall responses to memory antigens (secondary responses) involve
M. Kaufman et al. / Journal of the Neurological Sciences 341 (2014) 22–27
activation of antigen-specific T and B cells in secondary lymphoid tissues [15]. While human and animal studies have shown that natalizumab increases the number of circulating lymphocytes, possibly due to trafficking effects [12,16–20], animal studies have not shown a significant effect of α4-integrin blockade on T and B cell trafficking into most lymphoid tissues [21]. Nonhuman primate studies have shown no significant effect of natalizumab on primary or secondary Ab responses [20]; however, it is important to evaluate its effects on primary and secondary immune responses in patients. Owing to the potential effects of α4-integrin blockade by natalizumab on lymphocyte trafficking through primary and secondary lymphoid organs, it is important to provide additional data on the impact of natalizumab therapy on vaccination response in MS patients [21]. The objective of this study was to evaluate the effect of natalizumab on T-cell-dependent Ab responses to a recall antigen (tetanus toxoid [TT]) and to a neoantigen (keyhole limpet hemocyanin [KLH]) in patients with relapsing forms of MS. TT was used in this United States (US) study to assess recall Ab responses, as the majority of the US population receive childhood TT immunizations [22]. KLH, a strongly immunogenic protein isolated from the giant keyhole limpet, was used to assess primary immunization responses [23]. 2. Materials and methods 2.1. Study design In this phase 4, randomized, multi center, open-label study, eligible MS patients, naive to natalizumab, were randomized (1:1) to receive TT and KLH immunizations either 2 months prior to natalizumab treatment or after 6 months of natalizumab treatment (Fig. 1). Patients enrolling in the study agreed to delay treatment with natalizumab for approximately 2 months if randomized to the control/immunization-only group. All nine US sites used an interactive voice response system to randomize patients at the baseline visit. Randomization was stratified by site. Planned enrollment was approximately 46 patients; 23 patients per group. The study protocol and amendments were reviewed and approved by each site's institutional review board. The study was performed in accordance with the Declaration of Helsinki and International Conference on Harmonisation Guideline on Good Clinical Practice and is registered with ClinicalTrials.gov, number NCT00536120. 2.2. Patients Patients 18–60 years old who were diagnosed with a relapsing form of MS, eligible to receive natalizumab per approved labeling, and
Natalizumab started
Natalizumab
M0
TT vaccine TT Ab assay
previously vaccinated against TT were eligible for the study. Written informed consent was obtained from each patient prior to evaluations performed for eligibility. Exclusion criteria included the following: [1] TT immunization b2 years prior to screening, [2] hypersensitivity to TT, KLH, or other immunizations or their components [3], significant infectious illness within 30 days prior to screening [4], prior treatment with natalizumab, rituximab, any murine protein, or any therapeutic monoclonal Ab [5], treatment with cyclophosphamide within 1 year prior to screening [6], treatment with intravenous (i.v.) or intramuscular (i.m.) immunoglobulin (Ig), or immunosuppressant medications within 6 months prior to screening [7], treatment with systemic corticosteroids within 4 weeks prior to screening, and [8] treatment with immunomodulatory medications (interferon-β and glatiramer acetate) within 2 weeks prior to screening.
2.3. Interventions Patients randomized to natalizumab treatment prior to immunization (natalizumab group) received natalizumab, 300 mg i.v. every 4 weeks for 6 months, starting at the baseline visit, and received immunizations after the seventh natalizumab infusion. Natalizumab treatment continued for 2 months after the first immunization during which their immune responses were measured. Patients randomized to the control group received immunizations beginning at the baseline visit and their immune responses were measured over the following 2 months. Both groups received a single intramuscular TT vaccine administered per approved label at the investigational site and both groups received three subcutaneous Immucothel® (KLH) (Biosyn Arzneimittel AG, Fellbach, Germany) vaccines, each separated by 2 weeks (Fig. 1). KLH was supplied by Biogen Idec. The first TT and KLH vaccines were given on the same day. For patients randomized to the natalizumab group, samples for Ab assessments were collected prior to natalizumab infusion, and all immunizations were administered after completion of the 1-hour natalizumab post-dosing observation period. All eligible patients from both groups could enroll in TYSABRI® Outreach: Unified Commitment to Health (TOUCH®) after the study ended.
2.4. Antibody measurement TT and KLH Ab levels were measured using an enzyme-linked immunosorbent assay by Focus Diagnostics (Cypress, CA, USA). The upper limit of detection for the anti-TT Ab assay was 7.0 IU/mL in this clinical assay, which is not validated for diluted samples.
M0
+14d
+28d
+56d
M6
M6+14d
M6+28d
M6+56d
X
X
X X
KLH vaccine KLH Ab assay
Control
23
X
X X
X
X
X
X
X
X
Fig. 1. Study design. Patients were randomized 1:1 to the control group or natalizumab group. Control group patients received immunizations shortly after randomization and agreed to delay initiation of natalizumab until after day +56; patients randomized to natalizumab were treated with natalizumab for 6 months prior to immunizations. In the natalizumab group, baseline levels were drawn at month 0 prior to the initiation of natalizumab therapy. Blood for pre-immunization anti-KLH and anti-TT Ab levels was drawn just prior to the first immunization in both groups; baseline and pre-immunization time points are equivalent in the control group. Patients received three KLH immunizations separated by 2 weeks, and one TT immunization. A pre-planned assessment of anti-KLH Ab levels occurred at 14 days (after one KLH immunization), 28 days (after two KLH immunizations), and 56 days (after three immunizations) after first immunization. A pre-planned assessment of anti-TT Ab levels occurred at 28 days and post hoc at 56 days after the first (only) immunization. M: month; d: days; KLH: keyhole limpet hemocyanin; TT: tetanus toxoid; Ab: antibody.
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2.5. Assessments Samples were collected on days 0, 14, 28, and 56 [2 months] after the first immunization. The proportion of responders to TT immunization at days 28 and 56 after the first (and only) TT immunization and the proportion of responders to KLH immunization at days 14, 28, and 56 after the first KLH immunization were assessed. Day 56 TT analyses were performed post hoc. A positive Ab response was defined as an increase to twofold or greater of the pre-immunization anti-TT or antiKLH immunoglobulin G (IgG) Ab level. This increase in Ab level is expected for patients with functional immune systems [24–26] (Fig. 1). Safety was assessed throughout the study. 2.6. Statistical analyses Outcomes after TT and KLH immunizations are reported as the difference in proportion of positive Ab responders (95% confidence interval [CI]) between the natalizumab group and the control group. Patients with anti-TT Ab levels greater than half the upper limit of quantitation (i.e., N 3.5 IU/mL) at pre-immunization could not be considered in the per-protocol analyses of responders because an increase to twofold of the pre-immunization Ab level was impossible to confirm. Patients with results N7.0 IU/mL (above the upper limit of the assay) were assigned a value of 7.0 IU/mL to allow for calculation of summary statistics. Ab responses were analyzed using the change from preimmunization levels obtained at month 0 for the control group, and at month 6, just before immunization, for the natalizumab group. Sensitivity analyses of anti-TT Ab and anti-KLH Ab responses included all available data regardless of protocol violations. The only patients excluded were those with missing Ab level results where response could not be assessed, e.g., due to the sample not being collected. For patients with pre-immunization anti-TT levels of N 3.5 IU/mL, a postimmunization level of N7 IU/mL was considered a positive response. Protocol deviations causing exclusion from primary per-protocol analyses but inclusion in these sensitivity analyses included delayed immunization or samples collected outside of pre-specified window, immunization by incorrect route, and sample collection after use of prohibited medications. It was possible to be eligible for some analyses while being excluded from others. For example, a patient who missed a KLH immunization could still be included in the analysis of anti-TT responses, or a patient who had a prohibited medication at the time of day + 56 sampling could still be included in day +14 and +28 analyses. 3. Results 3.1. Patients 3.1.1. Patient disposition Planned enrollment was approximately 46 patients — 23 patients per group. Per protocol, it was possible to replace patients who withdrew. This was done by increasing the total number of patients randomized rather than by recruiting patients to the required pre-specified groups. The number of eligible patients and the amount of eligible data were reduced further as a result of protocol deviations concerning the administration of KLH and TT. Therefore, in order to ensure sufficient patient numbers in each group to perform the analysis of the primary endpoint at day 28, a total of 60 patients were recruited — 30 to each group. Patient randomization was stratified by site, and the study was balanced across treatment arms both within each site and overall. Twenty-four (80%) of 30 natalizumab patients completed the study and 22 (73%) received all nine natalizumab infusions. Three natalizumab patients (10%) developed persistent anti-natalizumab Abs, a proportion similar to the results in the natalizumab phase 3 AFFIRM study (6%) [9]. Two of the three patients developed persistent anti-natalizumab Abs prior to the scheduled immunizations and were not immunized, as required by the protocol, as the anti-natalizumab
Abs may have led to increased natalizumab clearance and confounding effects on the analyses. 3.1.2. Patient demographics and baseline characteristics The majority of patients were white (88%) and female (85%). The median age was 40 years for control patients and 45 years for natalizumab patients. Both groups had wide ranges for baseline demographic variables but had comparable disease histories (Table 1). Prior therapy with glatiramer acetate, i.m. interferon (IFN) β-1a, and subcutaneous (s.c.) IFNβ-1a were each reported by 42% of patients overall. IFNβ-1b use was reported by 27% of patients. Some patients had more than one prior therapy. Previous use of i.m. IFNβ-1a was higher in patients randomized to the natalizumab group than to the control group (53% vs 30%), while prior use of glatiramer acetate was higher in the control group (50% vs 33%). Few patients had used other MS treatments, including chronic MS-related corticosteroid treatment (10%) and mitoxantrone (7%). 3.2. Ab response to TT (recall response) and KLH (neoantigen response) vaccines Patients who withdrew from the study prior to immunization and/ or sample collection or met pre-specified criteria were excluded from the primary Ab analyses. Concomitant medication use was similar in both groups during the immunization period. As a result of childhood immunization, up to 90% of adults in the United States have protective levels of anti-TT Abs (≥ 0.15 IU/mL), and repeat TT immunization in adulthood should cause a memory immune response with increased anti-TT Ab levels [15,27]. At days + 28 and + 56, the proportions of responders to TT immunization were similar between the two groups. Sensitivity analyses that included all available data from immunized patients, including those with pre-immunization anti-TT Ab levels N3.5 IU/L where a post-immunization level of N 7 IU/mL was considered a positive response, showed similar results (Table 2). The one non-responder to TT (per protocol) at day + 28 in the natalizumab group showed a clear response at day + 56. This patient had high protective anti-TT Ab levels of 3.16 IU/mL at baseline and 2.36 IU/mL at pre-immunization, which increased at day + 28 to 4.32 IU/mL and further to N7.0 IU/mL at day +56. Thus, although this patient was a non-responder per protocol, the patient might be considered a clinical responder. Four of the five patients randomized to natalizumab, who were excluded from the primary anti-TT analyses due to pre-immunization anti-TT Ab levels N3.5 IU/L, had anti-TT levels N7.0 IU/L at day +28. Median anti-TT Ab levels, both pre-immunization and postimmunization, were similar between the evaluable patients in both the control and natalizumab groups. One patient in the natalizumab group had a low anti-TT Ab level (0.03 U/L) pre-immunization, which may not have been protective. All patients had protective levels of anti-TT Abs post-immunization. The median (quartiles) anti-TT Ab
Table 1 Patient demographics and disease history. Characteristic
Control (n = 30)
Natalizumab (n = 30)
Age (years), median (range) Female, n (%) White, n (%) Weight (kg), median (range) Body mass index (kg/m2), median (range) Time since MS diagnosis (years), median (range) No. of relapses in prior 12 months, median (range) EDSS score, median (range)
40 (24–60) 28 (93) 26 (87) 75.3 (43.1–114.6) 26.7 (16.4–39.5) 5 (1–19)
45 (23–56) 23 (77) 27 (90) 80.4 (40.8–124.0) 29.2 (15.0–43.9) 6 (1–29)
1 (0–3)
1 (0–4)
3 (0–7)
2.75 (1–6)
MS: multiple sclerosis; EDSS: Expanded Disability Status Scale.
M. Kaufman et al. / Journal of the Neurological Sciences 341 (2014) 22–27
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Table 2 Analyses of patients responding to TT and KLH immunizations. Responders, n (%)
Day +14
Per-protocol analysis — anti-TT Control Natalizumab Difference in proportions (95% CI) Sensitivity analysis — anti-TTa Control Natalizumab Difference in proportions (95% CI) Per-protocol analysis — anti-KLH Control Natalizumab Difference in proportions (95% CI) Sensitivity analysis — anti-KLHa Control Natalizumab Difference in proportions (95% CI)
Day +28
Day +56
24/24 (100) 15/16 (94) −0.06 (−0.18, 0.06)
21/22 (95) 14/15 (93) −0.02 (−0.17, 0.13)
28/28 (100) 20/22 (91) −0.09 (−0.21, 0.03)
25/27 (93) 20/23 (87) −0.06 (−0.23, 0.11)
9/24 (38) 5/20 (25) −0.13 (−0.40, 0.15)
19/23 (83) 17/19 (89) 0.07 (−0.14, 0.28)
21/21 (100) 15/17 (88) −0.12 (−0.27, 0.04)
10/28 (36) 6/23 (26) −0.10 (−0.35, 0.16)
23/28 (82) 18/22 (82) −0.00 (−0.22, 0.21)
27/27 (100) 19/22 (86) −0.14 (−0.28, 0.01)
KLH: keyhole limpet hemocyanin; TT: tetanus toxoid; CI: confidence interval. a Sensitivity analysis used all available data, regardless of protocol violations. Only patients with missing pre-immunization values were excluded. For TT sensitivity analyses patients were considered responders if the anti-TT Ab level was twice the pre-immunization level or if the anti-TT Ab level was N7.0 IU/mL, regardless of the pre-immunization level.
levels (IU/mL) for evaluable control and natalizumab patients, respectively, were 1.41 (0.52, 1.80) and 1.54 (1.30, 2.36) at pre-immunization, 7.00 (7.00, 7.00) and 7.00 (4.21, 7.00) at day +28, and 7.00 (6.41, 7.00) and 6.31 (3.87, 7.00) at day +56. The proportions of responders to KLH were similar over time, and post hoc sensitivity analyses that included all available data showed comparable results (Table 2). The two non-responders to KLH immunization in the evaluable natalizumab patients at day + 28 did not develop Abs through day + 56. A review of these patients' records did not reveal any confounding factors to explain the lack of response and both patients mounted Ab responses to TT. The geometric mean (95% CI) anti-KLH Ab levels (μg/mL) at pre-immunization were similar
for control and natalizumab patients: 0.78 (0.55–1.13) and 0.72 (0.48–1.09), respectively. The geometric mean (95% CI) anti-KLH Ab levels (μg/mL) for evaluable control and natalizumab patients, respectively, were 1.92 (1.06–3.48) and 1.13 (0.61–2.12) at day + 14, 11.12 (5.52–22.42) and 7.04 (3.51–14.11) at day + 28, and 32.90 (17.39–62.25) and 27.12 (10.54–69.80) at day + 56. Ab concentrations plotted for evaluable patients from preimmunization through day + 56 in reverse cumulative distribution plots demonstrated no clinically relevant difference between the control and natalizumab groups in response to TT or KLH immunization (Figs. 2 and 3). On the vertical axis, reverse cumulative distribution plots display the percentage of patients having an Ab concentration
Natalizumab
Pre-immunization
100
Day +28 100 Cumulative percentage
Cumulative percentage
Control
80 60 40 20
80 60 40 20
0
0
0
1
2
3
4
5
6
7
0
1
Anti-TT concentration (IU/mL)
2
3
4
5
6
7
Anti-TT concentration (IU/mL)
Cumulative percentage
Day +56
100 80 60 40 20 0 0
1
2
3
4
5
6
7
Anti-TT concentration (IU/mL) Fig. 2. Reverse cumulative distribution functions* of observed anti-TT Ab concentration. Graphs display similar reverse cumulative distribution curves of anti-TT IgG Ab concentration for evaluable natalizumab-treated and control patients pre-immunization and at day +28 and day +56 after TT immunization. *On the vertical axis, reverse cumulative distribution plots display the percentage of patients having an Ab concentration greater than the level shown on the horizontal axis. Groups with essentially the same distribution of responses will produce curves with roughly the same shapes and positions [28]. TT: tetanus toxoid; Ab: antibody; IgG: immunoglobulin G.
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M. Kaufman et al. / Journal of the Neurological Sciences 341 (2014) 22–27
Control
Natalizumab
Cumulative percentage
Cumulative percentage
Pre-immunization
100 80 60 40 20 0 0
Day +14
100 80 60 40 20 0
25 50 75 100 125 150 175 200 225 250 275
0
Anti-KLH concentration (µg/mL)
Anti-KLH concentration (µg/mL) Cumulative percentage
Cumulative percentage
Day +28
100 80 60 40 20 0 0
25 50 75 100 125 150 175 200 225 250 275
25 50 75 100 125 150 175 200 225 250 275
Day +56
100 80 60 40 20 0 0
Anti-KLH concentration (µg/mL)
25 50 75 100 125 150 175 200 225 250 275
Anti-KLH concentration (µg/mL)
Fig. 3. Reverse cumulative distribution functions* of observed anti-KLH Ab concentration. Graphs display similar reverse cumulative distribution curves of anti-KLH IgG Ab concentration for evaluable natalizumab-treated and control patients at pre-immunization and at day +14, day +28, and day +56 after the first KLH immunization. Pre-immunization, all control and natalizumab patients had Ab concentrations b15 μg/mL. *On the vertical axis, reverse cumulative distribution plots display the percentage of patients having an Ab concentration greater than the level shown on the horizontal axis. Groups with essentially the same distribution of responses will produce curves with roughly the same shapes and positions [28]. KLH: keyhole limpet hemocyanin; Ab: antibody; IgG: immunoglobulin G.
greater than the level shown on the horizontal axis. Groups with essentially the same distribution of responses will produce curves with roughly the same shapes and positions [28]. Safety analyses showed that immunizations and natalizumab were well tolerated, and no unexpected events were observed (data not shown). 4. Discussion Natalizumab has proven efficacy in reducing central nervous system inflammation in MS [9]; however, its effects on primary and secondary humoral immune responses have not been well studied. This study was not designed to detect small differences between treatment arms, but to investigate whether there is a clinically relevant alteration of the immune response with natalizumab treatment. The proportions of responders to recall antigen (TT) and neoantigen (KLH) immunization were similar in control and natalizumab-treated patients, indicating that natalizumab therapy does not have a major effect on the response to these primary and secondary vaccinations. This is supported by the fact that the anti-KLH and anti-TT Ab levels are similarly increased in response to these vaccinations in the natalizumab and control groups in the reverse cumulative distribution analyses. The sample size in our study and especially the imbalance between the two groups may raise questions about the reliability of the data. Treatment groups were well balanced overall and by site at randomization. However, enrollment beyond the originally planned study size was needed to compensate for [1] a higher than expected number of discontinuations in the natalizumab group, possibly due to the longer study participation in that group; [2] exclusion of data due to protocol deviations; and [3] the need to exclude patients with high preimmunization anti-TT Ab levels from the primary analysis. This led to an imbalance in evaluable patients between the two treatment groups. However, multiple sensitivity analyses confirmed the findings based on the primary analysis. Therefore, we consider our data to be adequate
and believe that our conclusion provides important information for patients receiving natalizumab therapy. In addition, our results confirm those of Vågberg et al., who found no significant difference between placebo and natalizumab-treated relapsing-remitting MS patients in the levels of serum IgG in response to influenza A or B vaccination [8]. Our data are also consistent with preclinical data showing that natalizumab does not affect responses to TT and KLH vaccination in monkeys [21]. In summary, our data indicate that natalizumab does not impair the humoral response to recall immunization with TT and de novo immunization with KLH in a clinically meaningful way [12]. Funding This study was funded by Biogen Idec Inc. Conflict of interest statement Dr. Kaufman has received honoraria and research support from Biogen Idec, has received financial support from Bayer, EMD Serono, Novartis, and Teva, and is a consultant for Department of Defense. Dr. Pardo has received consulting fees from Acorda, Biogen Idec, Novartis, and Teva; he has received compensation for speaking from Acorda, Bayer HealthCare, Biogen Idec, EMD Serono, Novartis, Pfizer, and Teva. Dr. Rossman has no financial relationships to disclose. Drs. Sweetser and Duda and Ms. Forrestal are employees of Biogen Idec and own stock. Acknowledgments Biogen Idec provided funding for editorial support in the development of this manuscript; Britt Anderson, PhD, from Infusion Communications wrote the first draft of the manuscript based on input from authors, and Jackie Cannon from Infusion Communications copyedited
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