Vaccine 33S (2015) B47–B51
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Review
Challenges in early clinical development of adjuvanted vaccines Giovanni Della Cioppa a,∗ , Ingileif Jonsdottir b,c , David Lewis d a
GlaxoSmithKline Via Fiorentina 1, I-53100 Siena, Italy Landspitali, The National University Hospital, Hringbraut, 101 Reykjavik, Iceland c University of Iceland, Hringbraut, 101 Reykjavik, Iceland d Clinical Research Centre, University of Surrey, Egerton Road, Guildford CU2 7XP, UK b
a r t i c l e
i n f o
Keywords: Vaccine Adjuvant Dose Schedule Exploratory End-points
a b s t r a c t A three-step approach to the early development of adjuvanted vaccine candidates is proposed, the goal of which is to allow ample space for exploratory and hypothesis-generating human experiments and to select dose(s) and dosing schedule(s) to bring into full development. Although the proposed approach is more extensive than the traditional early development program, the authors suggest that by addressing key questions upfront the overall time, size and cost of development will be reduced and the probability of public health advancement enhanced. The immunogenicity end-points chosen for early development should be critically selected: an established immunological parameter with a well characterized assay should be selected as primary end-point for dose and schedule finding; exploratory information-rich end-points should be limited in number and based on pre-defined hypothesis generating plans, including system biology and pathway analyses. Building a pharmacodynamic profile is an important aspect of early development: to this end, multiple early (within 24 h) and late (up to one year) sampling is necessary, which can be accomplished by sampling subgroups of subjects at different time points. In most cases the final target population, even if vulnerable, should be considered for inclusion in early development. In order to obtain the multiple formulations necessary for the dose and schedule finding, “bed-side mixing” of various components of the vaccine is often necessary: this is a complex and underestimated area that deserves serious research and logistical support. © 2015 Elsevier Ltd. All rights reserved.
1. Dose and schedule finding: a three step approach Clinical development of a vaccine candidate is a lengthy, expensive and risky process. Failures during or at the end of the large confirmatory (phase III) clinical trials are problematic from a financial, public health and ethical perspective. Hence, it is critical that efficacy and/or safety issues be identified early in order to redirect or stop the clinical program before the start of confirmatory trials. The likelihood of success can be maximized by front-loading the earlier stages of clinical development. To this end, at least seven interrelated questions should be addressed in order to advance a candidate adjuvanted vaccine to full development and eventually regulatory approval: i. What antigen dose(s)? ii. What adjuvant dose(s)? iii. What ratio(s) of antigen to adjuvant dose?
∗ Corresponding author. Tel.: +39 0577 245314. E-mail address: [email protected] (G. Della Cioppa). http://dx.doi.org/10.1016/j.vaccine.2015.02.031 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
iv. v. vi. vii.
What dosing schedule(s)? What end-points? What time points? What target population?
The complexity of early development is increased further if the candidate vaccine contains multiple antigens and if more than one candidate adjuvant is considered. We will focus on questions v, vi and vii in the following sections. In this section we propose a structured methodological approach to an early clinical program (phase I and II) aimed at selecting antigen dose, adjuvant dose, antigen to adjuvant dose ratio and dosing schedule to take into large confirmatory (pivotal) phase III studies required for registration (late stage development). Dose and schedule selection are often neglected in vaccine development plans, as there typically is an urge to push candidate vaccines into late stage development as quickly as possible, for scientific, public health and/or financial reasons. This is unfortunate as uncertainties about dose and dosing schedule often haunt vaccines development programs throughout their life cycle, and may
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Step 1 First in human • • •
Exclude frequent major safety issues Establish tolerated dose range Explore research & translational questions
Step 2 Dose finding •
•
Select dose(s) of antigen(s) and adjuvant for confirmatory (phase III) trials Explore research & translational questions Step 3 Schedule finding • •
Select dosing schedule(s) for confirmatory (phase III) trials Explore research & translational questions
Fig. 1. A three step approach to the early development of adjuvanted vaccines.
result in inconclusive late stage results, longer time to approval or late stage discontinuation of development. We propose here a three step approach to the early development of adjuvanted vaccines (Fig. 1). This approach also allows ample space and time for information-rich exploratory and translational clinical research, aimed at hypothesis generation in humans. We believe that in many instances early testing in humans can replace animal experiments of low power and uncertain predictive value. Clearly, each vaccine development has its own complexities and nuances. Hence, the approach we are proposing should always be adapted to individual vaccine candidates. Step 1: “first in human” This is the study where the adjuvanted vaccine candidate is administered for the first time in humans. “First in human” may apply to one or more of the vaccine components, or to the combination itself. A dose escalation study design is recommended [1]: study subjects are split into 4–6 cohorts of 10–30 subjects each. In some dose-escalation designs, at each dose level subjects are randomized to active or placebo (1:2 or 1:3 ratio) and placebo subjects from all dose levels are taken together in the final analysis [1]. The starting dose should typically be lower than the dose expected to elicit adequate immunogenicity from pre-clinical experiments and/or clinical testing of individual components. A safety review board, ideally independent of the investigators and sponsor, should be established to assess the safety and tolerability of each dose level and decide whether dose escalation may take place. Whereas some pre-defined decision rules are useful, the safety review board must have the liberty to prevent dose escalation based on the overall emerging safety and tolerability profile. When the target population of the vaccine is different from healthy adults, as is often the case (children, elderly subjects, subjects with disease), the design of this first step is more complex. We believe on ethical and scientific grounds that it is advisable to consider switching to the target population as soon as practical (see Section 3 below). The tolerated dose range in this “first -in- human” study will be used for dose finding in Step 2. As mentioned above, this first study also serves another important function: it allows a number of research and translational
medicine questions to be tested outside the strict constraints of later stages of clinical development. If there is a clear and well defined driver for the development of a given vaccine candidate, e.g. reducing systemic tolerability compared to existing vaccines, then go–no go criteria can already be built in at this stage. Step 2: antigen and adjuvant dose finding Factorial design studies [2,3] are in our view an advantageous methodological approach to dose finding of combination vaccines. A factorial design allows us to test, in one study, the contribution to the outcome of two or more factors and several levels for each factor [3]. In our case, the antigen(s) and the adjuvant(s) are the factors, and the doses to be tested for each factor are the levels. For example, if we take the least complex scenario of only one antigen and one adjuvant, and decide to test four doses of both antigen and adjuvant, the study will have two factors, with four levels each, i.e. a total of 16 cells (a cell combines one level of the first factor and one of the second factor). This is a so called two by four (2 × 4) factorial design, as outlined in Table 1. In designing a dose finding factorial trial, the following aspects should be considered: • Whereas there is a legitimate pressure to minimize the number of participants, every effort should be made to test at least four doses of the antigen and four of the adjuvant. This will build a reasonable dose response curve and reduce the risk that all tested doses are on the flat parts of the curve (assuming a typical sigmoid dose-response curve).
Table 1 Example of dose finding 2 × 4 factorial design for an adjuvanted vaccine candidate. Antigen dose
Adjuvant dose
A B C D
1
2
3
4
Cell 1A Cell 1B Cell 1C Cell 1D
Cell 2A Cell 2B Cell 2C Cell 2D
Cell 3A Cell 3B Cell 3C Cell 3D
Cell 4A Cell 4B Cell 4C Cell 4D
G. Della Cioppa et al. / Vaccine 33S (2015) B47–B51
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Table 2 Schedule-finding design with one antigen (Ant)/adjuvant (Adj) dose level and two schedules. Schedule
Dose
10Ant/20Adj
I 2–4 moa
II 2–6 moa
III 2–4-6 moa
IV 2–3-4 moa
Cell IA
Cell IIA
Cell IIIA
Cell IVA
moa, months of age.
Table 3 Schedule-finding 2 × 4 factorial design with two antigen (Ant)/adjuvant (Adj) dose levels and four schedules. Schedule
Dose
A 5Ant/10Adj B 15Ant/5Adj
I 2–4 moa
II 2–6 moa
III 2–4–6 moa
IV 2–3–4 moa
Cell IA Cell IB
Cell IIA Cell IIB
Cell IIIA Cell IIIB
Cell IVA Cell IVB
moa, months of age.
• Practical reasons may render a full factorial design impossible, often because of inability to obtain all the necessary dose combinations either by pre-formulation or “bed-side mixing” (see Section 4 below). In this case, an incomplete factorial design, i.e. a factorial design where not all cells are tested, may still be valid. Clearly, the greater the number of empty cells, the less reliable the conclusions. • Sample size calculations and conclusions must not be based on multiple pairwise comparisons of cells, but on the overall shape and position of the dose response curve(s). • It is critical that the primary end-point used for the dose selection be pre-defined and sufficiently robust. In vaccine studies, typically the primary end-point is represented by a biological marker of immune response, e.g. serum concentration of a specific antibody. Although it is acceptable that the assay used to measure the selected antibody or other biomarker may not be fully validated, it should be in an advanced stage of development and sufficiently reliable. An effort should be made to improve the assays during the study to allow for a reliable evaluation of the end-points. However, it is not unusual that the key assay is at an earlier stage of development than the vaccine candidate. In this situation it may be wise to delay the start of clinical trials to wait for a sufficient maturity of the assay. • Safety and tolerability assessments will always complement the primary immunogenicity end-point in selecting the dose(s) of antigen and adjuvant. • It is not uncommon that a vaccine candidate contains more than one antigen, e.g. multiple serotypes of the target bacterium or virus. Although factorial designs with more than two factors are possible, the number of cells and the overall sample size rapidly increase to unmanageable levels. Hence we advise for a more pragmatic approach. For example, if we have a three antigen candidate vaccine (A, B and C) and wish to test a dose range of 5–100 mcg for each antigen with four dose levels, we include in the factorial design combinations of the same dose for each antigen, e.g. A5/B5/C5 mcg A15/B15/C15 mcg, A20/B20/C20 mcg, A40/B40/C40 mcg, A100/B100/C100 mcg. In the end, the dose selected may be different for each of the three antigens, e.g. 15 mcg for A and B and 40 mc for C. Formulation of a 15/15/40 mcg combination will then be necessary before clinical development can progress. It should be noted that the starting doses of each component of combinations do not necessarily have to be of the same dose if available preclinical or clinical data suggest unequal starting doses.
The final result of Step 2 is the selection of one or two antigenadjuvant dose combinations to test with different dosing schedules.
Exploratory clinical research may also be included in Step 2, possibly in subgroups of subjects, considering the larger size of these studies compared to “first in man” (Step 1) studies. Step 3: schedule finding The immunogenicity, safety and tolerability, and practical use of adjuvanted vaccine candidates are determined not only by the dose level, but also by the number of doses needed (typically one to four) and the time interval between the doses, collectively referred here as dosing schedule. Although there may well be an interaction between dose levels and dosing schedule, it is generally impossible in practice to combine dose finding of antigen and adjuvant with schedule finding in one study. Therefore we recommend to conduct a separate schedule finding study (or studies, see below) using one or two antigen/adjuvant dose combinations selected in the previous step. Ideally one antigen/adjuvant dose should be selected from Step 2: Step 3 will be simpler and more schedules may be tested. Let’s assume we are developing a new adjuvanted vaccine in children and that we have selected in Step 2 a dose combining 10 mcg of the antigen and 20 mcg of the adjuvant given twice two months apart. The subsequent Step 3 schedule-finding trial could be designed as illustrated in Table 2. If instead it is deemed appropriate to select more than one antigen/adjuvant dose for schedule finding, then the design of the schedule finding study becomes more complicated and, as for Step 2, a factorial design may be the best approach. Going back to our putative vaccine development program in children, let’s assume we want to test two dose levels: 5 mcg antigen/10 mcg adjuvant (5Ant/10Adj) and 15 mcg antigen/5 mcg adjuvant (15Ant/5Adj). Then a two by four (2 × 4) factorial design could be considered as shown in Table 3 We recommend that no more than two antigen/adjuvant dose levels be brought from Step 2 to Step 3. As for Step 2, exploratory clinical research may also be included in Step 3. Outcome of the three step early development approach At the end of this three step approach to early development, an evidence based selection of dose and schedule will be obtained for confirmatory (phase III) trials. Occasionally there may be reasons to select more than one dose and/or schedule for confirmatory trials, for example when different countries impose different vaccination schedules. This choice should not be taken lightly as it renders pivotal trials longer, more expensive and risky. The proposed approach to early development also provides ample space for addressing exploratory, i.e. hypothesis-generating scientific questions directly in humans e.g. using a systems biology approach. We are aware that the approach we are suggesting is considerably more extensive than typically performed. We estimate that
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1500–2000 subjects and 2–3 years will be necessary to complete the three steps. Furthermore, considerable formulation work will be necessary (see Section 4 below). However, long standing experience in clinical development programs suggests to us that such an approach in the end is likely to increase the probability of success and significantly reduce the time and cost of bringing an adjuvanted vaccine candidate to regulatory approval and ultimately to people. 2. End-points In the selection of immunogenicity end-points for early development of adjuvanted vaccines, the researchers must address three sets of questions: How many? Which ones? When tested? 2.1. How many end-points? One immunogenicity end-point must be selected for the dose and schedule finding. The choice is more difficult when it comes to exploratory end-points. In exploratory setting researchers are tempted to include large numbers of measurements and end-points as a way to “explore” as much as possible. While this approach is understandable, it is fraught with complexities. It is true that exploratory and early clinical trials represent unique opportunities for researchers to address fundamental questions in the right species (humans). It is also true that in the exploratory setting (hypothesis generating) one can be less stringent compared to a confirmatory (hypothesis testing) setting, as there are fewer constrains when it comes to assumptions for sample size calculation, adjustments for multiple comparisons, validation of assays, etc. However, the inclusion of a very large number of end-points will inevitably create a “fishing expedition” effect, i.e. there will be a number of statistically significant results with no way to separate random variability from biological signals. In addition, the logistics of collecting, processing and testing of a large number of samples in a clinical trial setting is often underestimated by researchers and may lead to the “implosion” of a study, i.e. a situation where so many things go wrong that the validity of the overall study is jeopardized. Even for exploratory end-points it is essential that the researchers pre-define (with an appropriate written statistical analysis plan!) few hypothesis-generating questions and select end-points targeted at addressing those questions. Of great importance for the understanding of the biology of adjuvants are information-rich measurements for system biology and pathway analyses. Storage of samples for future measurements using new technologies may also be of great value. This should be adequately explained to study subjects and/or legal guardians in the informed consent form. 2.2. What end-points? The measurement technique must be reasonably well established and the assays used sufficiently robust to allow reliable conclusions. If the assay is too unstable, the variability due to the assay may well be greater than any biological response to the vaccine. The assays used for dose and schedule finding should be in an advanced stage of optimization and consultation with regulatory authorities may be important. 2.3. When should end-points be tested? Exploratory and early studies offer a great opportunity to study the kinetics of the immune response to the candidate vaccine. To this end, repeated measurements taken few hours or days after dosing are required. It is not necessary that samples for all time points be taken for all subjects included in the study: in addition to the main randomization to treatment groups, study subjects may
also be randomized to different time points. For example, assume we want to assess the immune response to a candidate vaccine at the following time-points post-dose: 2 h, 4 h, 8 h, 12 h, 18 h, 24 h, 30 h, 36 h, 4d, 5d, 6d, 7d. The number of samples for each subject would be untenable. However, study subjects could be randomized to one of three sampling schedules, e.g. • 2 h, 12 h, 30 h, 5d • 4 h, 18 h, 36 h, 6d • 8 h, 24 h, 4d, 7d Clearly, the number of subjects assigned to each time-point will be small and therefore the variability will increase. Hence, balance must be used in deciding how many time points should be chosen and how many subjects per time-point. However, in the context of the factorial designs illustrated above different cells may be merged to explore the early kinetics. This would increase the sample size for each time point. The long term persistence of the immune response is another question worth addressing in exploratory trials. In this case multiple sampling at late time-points, say 6 months, 9 months, 1 year and some times beyond, will be necessary. This is often a problem because of the implication of waiting that long to release the results of the study. However if one includes the late time points in a separate, stand-alone extension protocol, this issue can be circumvented. 3. Special populations Adjuvanted vaccines often target populations who are expected to have a reduced immune response compared to healthy adults. Such vulnerable populations include young children, the very old, immunocompromised subjects and patients with chronic diseases. Should the exploratory, dose and schedule finding trials described above be conducted in such vulnerable populations? This is a difficult question, which carries important ethical concerns. In our view, the answer may well be “yes”. The selection of the dose and dosing schedule, as well as the relevant exploratory hypothesis-generating questions, must be conducted in the target population, whenever the population is sufficiently large (small target populations, e.g. patients with rare diseases, are a different matter, which we will not discuss here). The “first in human” dosing may well be conducted in healthy, non-elderly volunteers, but then the study would have to be repeated in the target population. Healthy adults and the target population may also be combined in the same study: each level of the dose escalation is carried out one first in healthy adults and then, if no meaningful safety issues emerge, in the target population. Other design features that allow early switch to the target population include run in-phases in non-elderly adults volunteers and age de-escalation or escalation designs. Alternatively, bridging studies to show that the immune response in non-elderly healthy adults is similar to that of the target population may be included in the early stages of development: if successful it may be acceptable to conduct dose- and formulationfinding studies in non-elderly adults. This approach may be taken for example when the target population is represented by pregnant women, because there is plausible that pregnant women may will respond in a way similar to non-pregnant women. For most populations targeted by adjuvanted vaccine candidates, however, this is not the case and early shift to the target population is recommended on both scientific and ethical grounds. In most cases the key dose and schedule finding trials must be conducted in the target population. To this end, reproductive and carcinogenicity toxicology may have to be conducted earlier than usual.
G. Della Cioppa et al. / Vaccine 33S (2015) B47–B51
4. Formulations and bed-side mixing A critical condition for the proposed approach to dose and schedule finding studies is the availability of numerous formulations to allow for all antigen/adjuvant dose combinations included in the studies. Although ideally formulations will be pre-prepared for each antigen-adjuvant dose combination, this is often impossible in practice. Thus, so called “bed-side mixing” is necessary to reduce the number of formulations. “Bed-side mixing” is the extemporaneous creation of the required formulation shortly before dosing by serial dilutions and/or mixing of individual components. It may take place literally at the bed-side by clinical staff, or in an adjacent pharmacy by pharmacists. This is a difficult and underestimated field, which should be the object of more investment as well as serious regulatory and scientific research, both by the pharmaceutical companies and the academic sites involved in early clinical research. More formulation work is necessary in the Technical Development departments of pharmaceutical companies involved in vaccine development before the start of clinical trials. This requires a change in mindset and earlier allocation of significant financial and human resources. Academic sites involved in early development often lack the facilities, equipment, licenses, and know-how for bed-side mixing: this gap must be fixed and adequate quality controls put in place, possibly with the technical and financial support of the sponsors. “Mobile pharmacies” for bed-side mixing, i.e. a set up that can be moved from site to site, is a concept worth exploring to reduce costs. The regulatory status of bed-side mixing is also ambiguous: in some countries mixing multiple components may fall under the regulatory umbrella of “pharmaceutical manufacture”, which requires specific licenses and is something that may preferably be avoided, whereas in other countries this
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does not seem to be a problem. Uneven regulations in this field would clearly shift early clinical research to countries with clearer and simpler regulations. Finally, clinical investigators and site staff must be adequately trained on the bed-side mixing procedures, as any mistake or even doubt in this process would seriously jeopardize the reliability of dose and schedule finding results. Author contributions All the authors were involved in the production and revision of this manuscript, and the decision to submit for publication. Acknowledgements This paper is based on a workshop in the context of the Symposium “Enhancing Vaccine Immunity and Value”, Siena, Italy, 12–13 July 2014, funded by Novartis Vaccines and Diagnostics. The Authors thank the participants of the symposium for their insight and contributions. Conflicts of interest: G.D.C. is a full time employee of GlaxoSmithKline (GSK); D.L. has received research grants to his employer from Novartis Vaccines. References [1] Bacchieri A, Della Cioppa G. Dose-escalation and dose-titration. In: Fundamentals of Clinical Research: Bridging Medicine, Statistics and Operations, Chapter 10.4. Springer-Verlag; 2007. p. 244–6. [2] Bryar DP, Piantadosi SP. Factorial designs for randomized clinical trials. Cancer Treat Rep 1985;69:1055–62. [3] Bacchieri A, Della Cioppa G. Factorial designs. In: Fundamentals of Clinical Research: Bridging Medicine, Statistics and Operations, Chapter 10.8. SpringerVerlag; 2007. p. 263–70.
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Review
Challenges in early clinical development of adjuvanted vaccines Giovanni Della Cioppa a,∗ , Ingileif Jonsdottir b,c , David Lewis d a
GlaxoSmithKline Via Fiorentina 1, I-53100 Siena, Italy Landspitali, The National University Hospital, Hringbraut, 101 Reykjavik, Iceland c University of Iceland, Hringbraut, 101 Reykjavik, Iceland d Clinical Research Centre, University of Surrey, Egerton Road, Guildford CU2 7XP, UK b
a r t i c l e
i n f o
Keywords: Vaccine Adjuvant Dose Schedule Exploratory End-points
a b s t r a c t A three-step approach to the early development of adjuvanted vaccine candidates is proposed, the goal of which is to allow ample space for exploratory and hypothesis-generating human experiments and to select dose(s) and dosing schedule(s) to bring into full development. Although the proposed approach is more extensive than the traditional early development program, the authors suggest that by addressing key questions upfront the overall time, size and cost of development will be reduced and the probability of public health advancement enhanced. The immunogenicity end-points chosen for early development should be critically selected: an established immunological parameter with a well characterized assay should be selected as primary end-point for dose and schedule finding; exploratory information-rich end-points should be limited in number and based on pre-defined hypothesis generating plans, including system biology and pathway analyses. Building a pharmacodynamic profile is an important aspect of early development: to this end, multiple early (within 24 h) and late (up to one year) sampling is necessary, which can be accomplished by sampling subgroups of subjects at different time points. In most cases the final target population, even if vulnerable, should be considered for inclusion in early development. In order to obtain the multiple formulations necessary for the dose and schedule finding, “bed-side mixing” of various components of the vaccine is often necessary: this is a complex and underestimated area that deserves serious research and logistical support. © 2015 Elsevier Ltd. All rights reserved.
1. Dose and schedule finding: a three step approach Clinical development of a vaccine candidate is a lengthy, expensive and risky process. Failures during or at the end of the large confirmatory (phase III) clinical trials are problematic from a financial, public health and ethical perspective. Hence, it is critical that efficacy and/or safety issues be identified early in order to redirect or stop the clinical program before the start of confirmatory trials. The likelihood of success can be maximized by front-loading the earlier stages of clinical development. To this end, at least seven interrelated questions should be addressed in order to advance a candidate adjuvanted vaccine to full development and eventually regulatory approval: i. What antigen dose(s)? ii. What adjuvant dose(s)? iii. What ratio(s) of antigen to adjuvant dose?
∗ Corresponding author. Tel.: +39 0577 245314. E-mail address: [email protected] (G. Della Cioppa). http://dx.doi.org/10.1016/j.vaccine.2015.02.031 0264-410X/© 2015 Elsevier Ltd. All rights reserved.
iv. v. vi. vii.
What dosing schedule(s)? What end-points? What time points? What target population?
The complexity of early development is increased further if the candidate vaccine contains multiple antigens and if more than one candidate adjuvant is considered. We will focus on questions v, vi and vii in the following sections. In this section we propose a structured methodological approach to an early clinical program (phase I and II) aimed at selecting antigen dose, adjuvant dose, antigen to adjuvant dose ratio and dosing schedule to take into large confirmatory (pivotal) phase III studies required for registration (late stage development). Dose and schedule selection are often neglected in vaccine development plans, as there typically is an urge to push candidate vaccines into late stage development as quickly as possible, for scientific, public health and/or financial reasons. This is unfortunate as uncertainties about dose and dosing schedule often haunt vaccines development programs throughout their life cycle, and may
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G. Della Cioppa et al. / Vaccine 33S (2015) B47–B51
Step 1 First in human • • •
Exclude frequent major safety issues Establish tolerated dose range Explore research & translational questions
Step 2 Dose finding •
•
Select dose(s) of antigen(s) and adjuvant for confirmatory (phase III) trials Explore research & translational questions Step 3 Schedule finding • •
Select dosing schedule(s) for confirmatory (phase III) trials Explore research & translational questions
Fig. 1. A three step approach to the early development of adjuvanted vaccines.
result in inconclusive late stage results, longer time to approval or late stage discontinuation of development. We propose here a three step approach to the early development of adjuvanted vaccines (Fig. 1). This approach also allows ample space and time for information-rich exploratory and translational clinical research, aimed at hypothesis generation in humans. We believe that in many instances early testing in humans can replace animal experiments of low power and uncertain predictive value. Clearly, each vaccine development has its own complexities and nuances. Hence, the approach we are proposing should always be adapted to individual vaccine candidates. Step 1: “first in human” This is the study where the adjuvanted vaccine candidate is administered for the first time in humans. “First in human” may apply to one or more of the vaccine components, or to the combination itself. A dose escalation study design is recommended [1]: study subjects are split into 4–6 cohorts of 10–30 subjects each. In some dose-escalation designs, at each dose level subjects are randomized to active or placebo (1:2 or 1:3 ratio) and placebo subjects from all dose levels are taken together in the final analysis [1]. The starting dose should typically be lower than the dose expected to elicit adequate immunogenicity from pre-clinical experiments and/or clinical testing of individual components. A safety review board, ideally independent of the investigators and sponsor, should be established to assess the safety and tolerability of each dose level and decide whether dose escalation may take place. Whereas some pre-defined decision rules are useful, the safety review board must have the liberty to prevent dose escalation based on the overall emerging safety and tolerability profile. When the target population of the vaccine is different from healthy adults, as is often the case (children, elderly subjects, subjects with disease), the design of this first step is more complex. We believe on ethical and scientific grounds that it is advisable to consider switching to the target population as soon as practical (see Section 3 below). The tolerated dose range in this “first -in- human” study will be used for dose finding in Step 2. As mentioned above, this first study also serves another important function: it allows a number of research and translational
medicine questions to be tested outside the strict constraints of later stages of clinical development. If there is a clear and well defined driver for the development of a given vaccine candidate, e.g. reducing systemic tolerability compared to existing vaccines, then go–no go criteria can already be built in at this stage. Step 2: antigen and adjuvant dose finding Factorial design studies [2,3] are in our view an advantageous methodological approach to dose finding of combination vaccines. A factorial design allows us to test, in one study, the contribution to the outcome of two or more factors and several levels for each factor [3]. In our case, the antigen(s) and the adjuvant(s) are the factors, and the doses to be tested for each factor are the levels. For example, if we take the least complex scenario of only one antigen and one adjuvant, and decide to test four doses of both antigen and adjuvant, the study will have two factors, with four levels each, i.e. a total of 16 cells (a cell combines one level of the first factor and one of the second factor). This is a so called two by four (2 × 4) factorial design, as outlined in Table 1. In designing a dose finding factorial trial, the following aspects should be considered: • Whereas there is a legitimate pressure to minimize the number of participants, every effort should be made to test at least four doses of the antigen and four of the adjuvant. This will build a reasonable dose response curve and reduce the risk that all tested doses are on the flat parts of the curve (assuming a typical sigmoid dose-response curve).
Table 1 Example of dose finding 2 × 4 factorial design for an adjuvanted vaccine candidate. Antigen dose
Adjuvant dose
A B C D
1
2
3
4
Cell 1A Cell 1B Cell 1C Cell 1D
Cell 2A Cell 2B Cell 2C Cell 2D
Cell 3A Cell 3B Cell 3C Cell 3D
Cell 4A Cell 4B Cell 4C Cell 4D
G. Della Cioppa et al. / Vaccine 33S (2015) B47–B51
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Table 2 Schedule-finding design with one antigen (Ant)/adjuvant (Adj) dose level and two schedules. Schedule
Dose
10Ant/20Adj
I 2–4 moa
II 2–6 moa
III 2–4-6 moa
IV 2–3-4 moa
Cell IA
Cell IIA
Cell IIIA
Cell IVA
moa, months of age.
Table 3 Schedule-finding 2 × 4 factorial design with two antigen (Ant)/adjuvant (Adj) dose levels and four schedules. Schedule
Dose
A 5Ant/10Adj B 15Ant/5Adj
I 2–4 moa
II 2–6 moa
III 2–4–6 moa
IV 2–3–4 moa
Cell IA Cell IB
Cell IIA Cell IIB
Cell IIIA Cell IIIB
Cell IVA Cell IVB
moa, months of age.
• Practical reasons may render a full factorial design impossible, often because of inability to obtain all the necessary dose combinations either by pre-formulation or “bed-side mixing” (see Section 4 below). In this case, an incomplete factorial design, i.e. a factorial design where not all cells are tested, may still be valid. Clearly, the greater the number of empty cells, the less reliable the conclusions. • Sample size calculations and conclusions must not be based on multiple pairwise comparisons of cells, but on the overall shape and position of the dose response curve(s). • It is critical that the primary end-point used for the dose selection be pre-defined and sufficiently robust. In vaccine studies, typically the primary end-point is represented by a biological marker of immune response, e.g. serum concentration of a specific antibody. Although it is acceptable that the assay used to measure the selected antibody or other biomarker may not be fully validated, it should be in an advanced stage of development and sufficiently reliable. An effort should be made to improve the assays during the study to allow for a reliable evaluation of the end-points. However, it is not unusual that the key assay is at an earlier stage of development than the vaccine candidate. In this situation it may be wise to delay the start of clinical trials to wait for a sufficient maturity of the assay. • Safety and tolerability assessments will always complement the primary immunogenicity end-point in selecting the dose(s) of antigen and adjuvant. • It is not uncommon that a vaccine candidate contains more than one antigen, e.g. multiple serotypes of the target bacterium or virus. Although factorial designs with more than two factors are possible, the number of cells and the overall sample size rapidly increase to unmanageable levels. Hence we advise for a more pragmatic approach. For example, if we have a three antigen candidate vaccine (A, B and C) and wish to test a dose range of 5–100 mcg for each antigen with four dose levels, we include in the factorial design combinations of the same dose for each antigen, e.g. A5/B5/C5 mcg A15/B15/C15 mcg, A20/B20/C20 mcg, A40/B40/C40 mcg, A100/B100/C100 mcg. In the end, the dose selected may be different for each of the three antigens, e.g. 15 mcg for A and B and 40 mc for C. Formulation of a 15/15/40 mcg combination will then be necessary before clinical development can progress. It should be noted that the starting doses of each component of combinations do not necessarily have to be of the same dose if available preclinical or clinical data suggest unequal starting doses.
The final result of Step 2 is the selection of one or two antigenadjuvant dose combinations to test with different dosing schedules.
Exploratory clinical research may also be included in Step 2, possibly in subgroups of subjects, considering the larger size of these studies compared to “first in man” (Step 1) studies. Step 3: schedule finding The immunogenicity, safety and tolerability, and practical use of adjuvanted vaccine candidates are determined not only by the dose level, but also by the number of doses needed (typically one to four) and the time interval between the doses, collectively referred here as dosing schedule. Although there may well be an interaction between dose levels and dosing schedule, it is generally impossible in practice to combine dose finding of antigen and adjuvant with schedule finding in one study. Therefore we recommend to conduct a separate schedule finding study (or studies, see below) using one or two antigen/adjuvant dose combinations selected in the previous step. Ideally one antigen/adjuvant dose should be selected from Step 2: Step 3 will be simpler and more schedules may be tested. Let’s assume we are developing a new adjuvanted vaccine in children and that we have selected in Step 2 a dose combining 10 mcg of the antigen and 20 mcg of the adjuvant given twice two months apart. The subsequent Step 3 schedule-finding trial could be designed as illustrated in Table 2. If instead it is deemed appropriate to select more than one antigen/adjuvant dose for schedule finding, then the design of the schedule finding study becomes more complicated and, as for Step 2, a factorial design may be the best approach. Going back to our putative vaccine development program in children, let’s assume we want to test two dose levels: 5 mcg antigen/10 mcg adjuvant (5Ant/10Adj) and 15 mcg antigen/5 mcg adjuvant (15Ant/5Adj). Then a two by four (2 × 4) factorial design could be considered as shown in Table 3 We recommend that no more than two antigen/adjuvant dose levels be brought from Step 2 to Step 3. As for Step 2, exploratory clinical research may also be included in Step 3. Outcome of the three step early development approach At the end of this three step approach to early development, an evidence based selection of dose and schedule will be obtained for confirmatory (phase III) trials. Occasionally there may be reasons to select more than one dose and/or schedule for confirmatory trials, for example when different countries impose different vaccination schedules. This choice should not be taken lightly as it renders pivotal trials longer, more expensive and risky. The proposed approach to early development also provides ample space for addressing exploratory, i.e. hypothesis-generating scientific questions directly in humans e.g. using a systems biology approach. We are aware that the approach we are suggesting is considerably more extensive than typically performed. We estimate that
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1500–2000 subjects and 2–3 years will be necessary to complete the three steps. Furthermore, considerable formulation work will be necessary (see Section 4 below). However, long standing experience in clinical development programs suggests to us that such an approach in the end is likely to increase the probability of success and significantly reduce the time and cost of bringing an adjuvanted vaccine candidate to regulatory approval and ultimately to people. 2. End-points In the selection of immunogenicity end-points for early development of adjuvanted vaccines, the researchers must address three sets of questions: How many? Which ones? When tested? 2.1. How many end-points? One immunogenicity end-point must be selected for the dose and schedule finding. The choice is more difficult when it comes to exploratory end-points. In exploratory setting researchers are tempted to include large numbers of measurements and end-points as a way to “explore” as much as possible. While this approach is understandable, it is fraught with complexities. It is true that exploratory and early clinical trials represent unique opportunities for researchers to address fundamental questions in the right species (humans). It is also true that in the exploratory setting (hypothesis generating) one can be less stringent compared to a confirmatory (hypothesis testing) setting, as there are fewer constrains when it comes to assumptions for sample size calculation, adjustments for multiple comparisons, validation of assays, etc. However, the inclusion of a very large number of end-points will inevitably create a “fishing expedition” effect, i.e. there will be a number of statistically significant results with no way to separate random variability from biological signals. In addition, the logistics of collecting, processing and testing of a large number of samples in a clinical trial setting is often underestimated by researchers and may lead to the “implosion” of a study, i.e. a situation where so many things go wrong that the validity of the overall study is jeopardized. Even for exploratory end-points it is essential that the researchers pre-define (with an appropriate written statistical analysis plan!) few hypothesis-generating questions and select end-points targeted at addressing those questions. Of great importance for the understanding of the biology of adjuvants are information-rich measurements for system biology and pathway analyses. Storage of samples for future measurements using new technologies may also be of great value. This should be adequately explained to study subjects and/or legal guardians in the informed consent form. 2.2. What end-points? The measurement technique must be reasonably well established and the assays used sufficiently robust to allow reliable conclusions. If the assay is too unstable, the variability due to the assay may well be greater than any biological response to the vaccine. The assays used for dose and schedule finding should be in an advanced stage of optimization and consultation with regulatory authorities may be important. 2.3. When should end-points be tested? Exploratory and early studies offer a great opportunity to study the kinetics of the immune response to the candidate vaccine. To this end, repeated measurements taken few hours or days after dosing are required. It is not necessary that samples for all time points be taken for all subjects included in the study: in addition to the main randomization to treatment groups, study subjects may
also be randomized to different time points. For example, assume we want to assess the immune response to a candidate vaccine at the following time-points post-dose: 2 h, 4 h, 8 h, 12 h, 18 h, 24 h, 30 h, 36 h, 4d, 5d, 6d, 7d. The number of samples for each subject would be untenable. However, study subjects could be randomized to one of three sampling schedules, e.g. • 2 h, 12 h, 30 h, 5d • 4 h, 18 h, 36 h, 6d • 8 h, 24 h, 4d, 7d Clearly, the number of subjects assigned to each time-point will be small and therefore the variability will increase. Hence, balance must be used in deciding how many time points should be chosen and how many subjects per time-point. However, in the context of the factorial designs illustrated above different cells may be merged to explore the early kinetics. This would increase the sample size for each time point. The long term persistence of the immune response is another question worth addressing in exploratory trials. In this case multiple sampling at late time-points, say 6 months, 9 months, 1 year and some times beyond, will be necessary. This is often a problem because of the implication of waiting that long to release the results of the study. However if one includes the late time points in a separate, stand-alone extension protocol, this issue can be circumvented. 3. Special populations Adjuvanted vaccines often target populations who are expected to have a reduced immune response compared to healthy adults. Such vulnerable populations include young children, the very old, immunocompromised subjects and patients with chronic diseases. Should the exploratory, dose and schedule finding trials described above be conducted in such vulnerable populations? This is a difficult question, which carries important ethical concerns. In our view, the answer may well be “yes”. The selection of the dose and dosing schedule, as well as the relevant exploratory hypothesis-generating questions, must be conducted in the target population, whenever the population is sufficiently large (small target populations, e.g. patients with rare diseases, are a different matter, which we will not discuss here). The “first in human” dosing may well be conducted in healthy, non-elderly volunteers, but then the study would have to be repeated in the target population. Healthy adults and the target population may also be combined in the same study: each level of the dose escalation is carried out one first in healthy adults and then, if no meaningful safety issues emerge, in the target population. Other design features that allow early switch to the target population include run in-phases in non-elderly adults volunteers and age de-escalation or escalation designs. Alternatively, bridging studies to show that the immune response in non-elderly healthy adults is similar to that of the target population may be included in the early stages of development: if successful it may be acceptable to conduct dose- and formulationfinding studies in non-elderly adults. This approach may be taken for example when the target population is represented by pregnant women, because there is plausible that pregnant women may will respond in a way similar to non-pregnant women. For most populations targeted by adjuvanted vaccine candidates, however, this is not the case and early shift to the target population is recommended on both scientific and ethical grounds. In most cases the key dose and schedule finding trials must be conducted in the target population. To this end, reproductive and carcinogenicity toxicology may have to be conducted earlier than usual.
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4. Formulations and bed-side mixing A critical condition for the proposed approach to dose and schedule finding studies is the availability of numerous formulations to allow for all antigen/adjuvant dose combinations included in the studies. Although ideally formulations will be pre-prepared for each antigen-adjuvant dose combination, this is often impossible in practice. Thus, so called “bed-side mixing” is necessary to reduce the number of formulations. “Bed-side mixing” is the extemporaneous creation of the required formulation shortly before dosing by serial dilutions and/or mixing of individual components. It may take place literally at the bed-side by clinical staff, or in an adjacent pharmacy by pharmacists. This is a difficult and underestimated field, which should be the object of more investment as well as serious regulatory and scientific research, both by the pharmaceutical companies and the academic sites involved in early clinical research. More formulation work is necessary in the Technical Development departments of pharmaceutical companies involved in vaccine development before the start of clinical trials. This requires a change in mindset and earlier allocation of significant financial and human resources. Academic sites involved in early development often lack the facilities, equipment, licenses, and know-how for bed-side mixing: this gap must be fixed and adequate quality controls put in place, possibly with the technical and financial support of the sponsors. “Mobile pharmacies” for bed-side mixing, i.e. a set up that can be moved from site to site, is a concept worth exploring to reduce costs. The regulatory status of bed-side mixing is also ambiguous: in some countries mixing multiple components may fall under the regulatory umbrella of “pharmaceutical manufacture”, which requires specific licenses and is something that may preferably be avoided, whereas in other countries this
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does not seem to be a problem. Uneven regulations in this field would clearly shift early clinical research to countries with clearer and simpler regulations. Finally, clinical investigators and site staff must be adequately trained on the bed-side mixing procedures, as any mistake or even doubt in this process would seriously jeopardize the reliability of dose and schedule finding results. Author contributions All the authors were involved in the production and revision of this manuscript, and the decision to submit for publication. Acknowledgements This paper is based on a workshop in the context of the Symposium “Enhancing Vaccine Immunity and Value”, Siena, Italy, 12–13 July 2014, funded by Novartis Vaccines and Diagnostics. The Authors thank the participants of the symposium for their insight and contributions. Conflicts of interest: G.D.C. is a full time employee of GlaxoSmithKline (GSK); D.L. has received research grants to his employer from Novartis Vaccines. References [1] Bacchieri A, Della Cioppa G. Dose-escalation and dose-titration. In: Fundamentals of Clinical Research: Bridging Medicine, Statistics and Operations, Chapter 10.4. Springer-Verlag; 2007. p. 244–6. [2] Bryar DP, Piantadosi SP. Factorial designs for randomized clinical trials. Cancer Treat Rep 1985;69:1055–62. [3] Bacchieri A, Della Cioppa G. Factorial designs. In: Fundamentals of Clinical Research: Bridging Medicine, Statistics and Operations, Chapter 10.8. SpringerVerlag; 2007. p. 263–70.
