Therapeutic Intervention in Multiple Sclerosis with Alpha B-Crystallin: A Randomized Controlled Phase IIa Trial

Abstract

As a molecular chaperone and activator of Toll-like receptor 2-mediated protective responses by microglia and macrophages, the small heat shock protein alpha B-crystallin (HspB5) exerts therapeutic effects in different animal models for neuroinflammation, including the model for multiple sclerosis (MS). Yet, HspB5 can also stimulate human antigen-specific memory T cells to release IFN-γ, a cytokine with well-documented detrimental effects during MS. In this study, we explored in a Phase IIa randomized clinical trial the therapeutic application of HspB5 in relapsing-remitting MS (RR-MS), using intravenous doses sufficient to support its protective effects, but too low to trigger pathogenic memory T-cell responses. These sub-immunogenic doses were selected based on in vitro analysis of the dose-response profile of human T cells and macrophages to HspB5, and on the immunological effects of HspB5 in healthy humans as established in a preparatory Phase I study. In a 48-week randomized, placebo-controlled, double-blind Phase IIa trial, three bimonthly intravenous injections of 7.5, 12.5 or 17.5 mg HspB5 were found to be safe and well tolerated in RR-MS patients. While predefined clinical endpoints did not differ significantly between the relatively small groups of MS patients treated with either HspB5 or placebo, repeated administration especially of the lower doses of HspB5 led to a progressive decline in MS lesion activity as monitored by magnetic resonance imaging (MRI), which was not seen in the placebo group. Exploratory linear regression analysis revealed this decline to be significant in the combined group receiving either of the two lower doses, and to result in a 76% reduction in both number and total volumes of active MRI lesions at 9 months into the study. These data provide the first indication for clinical benefit resulting from intervention in RR-MS with HspB5.

Trial registration: ClinicalTrials.gov Phase I: NCT02442557; Phase IIa: NCT02442570.

Conflict of interest statement

Competing Interests: JMvN, MB, PJN and RV are paid employees of Delta Crystallon BV; EHGV is a paid consultant of Delta Crystallon BV. JMvN holds equity in Delta Crystallon BV. Intervention in multiple sclerosis with HspB5 is the subject of international patent application PCT/NL2014/050383.

Figures

Fig 1. Phase IIa study profile.

Fig 2. Selection of therapeutic intravenous doses of HspB5 in humans.

Therapeutic application of HspB5 in humans relies on activation of M2-like protective microglia and macrophage responses, while avoiding pathogenic IFN-γresponses by memory T cells. In Fig 2A, the existence of a substantial HspB5-reactive memory T-cell repertoire in humans is illustrated by a representative fresh PBMC sample from a healthy subject. Proliferative responses to HspB5 of CD45RO+ memory T cells in this sample are reflected by proliferation-induced dilution of the fluorescent tracer CFSE over 9 days in culture. With CFSEdim CD45RO+ T cells representing proliferated memory T cells, Fig 1A illustrates that a substantial part of all human memory T cells, an estimated one in about every 4,000 cells, do indeed respond to HspB5. In Fig 2B, it is illustrated, again with a representative example, that human T cells are triggered by HspB5 to release IFN-γ, but only when the HspB5 concentration exceeds a threshold of about 20 μg/mL. The protective macrophage response, on the other hand, is already activated by markedly lower μg/mL-concentrations of HspB5, as exemplified by IL-10 secretion as marker for protective responses. At these low sub-immunogenic concentrations, HspB5 can therefore be therapeutically exploited in humans. In Fig 3C, it is shown that single intravenous doses of up to 37.5 mg HspB5 leads to such sub-immunogenic peak serum concentrations in humans, remaining well below the 20-μg/mL threshold. Fig 3C shows the mean ± standard deviation peak serum concentrations of HspB5 found in groups of 8 healthy subjects 10–20 min after receiving varying amounts of intravenous HspB5 during the Phase I study.

Fig 3. Effects of intravenous HspB5 on peripheral T-cell reactivity to HspB5 itself, and tetanus toxoid.

Fig 3 illustrates the proliferative response in vitro of CD4+ (A) as well as CD45RO+ memory T-cells (B) to either 50 μg/mL HspB5 or 0.2 μg/mL tetanus toxoid as a control antigen, at various time points after a single intravenous dose of 12.5 mg HspB5 in 8 healthy subjects, as evaluated using a CFSE assay during Phase I. Percentages of proliferated CFSEdim cells are expressed relative to all other lymphocytes detected by flowcytometry in the sample after 9 days in culture, including CD4/CD45RO-negative cells. The results not only confirm that following an intravenous dose of 12.5 mg HspB5, its levels remain at sub-immunogenic levels, but even illustrate significant suppressive effect on in vitro T-cell reactivity that is markedly more lasting for HspB5-reactive T cells than for tetanus toxoid-reactive T cells. Background proliferation found in cultures without any antigen was well below 1% in all cases. *: p< 0.05; ***: p<0.005.

Fig 4. The impact of intravenous HspB5 on active MRI lesions in RR-MS patients.

RR-MS patients received three bimonthly intravenous administrations of either PBS as placebo, or HspB5 at the indicated doses, and mean numbers of Gd+ T1 lesions for each group were evaluated by MRI over 36 weeks. Fig 4 shows mean values ± standard deviation, as calculated for the full analysis set of patients, including readings for 7 to 8 subjects at different time points for each group except for the 7.5-mg group, which included readings for 4 to 8 subjects. Linear regression analysis was used to evaluate the trends of change in each group over 36 weeks; p values reflect the probability that the deviation of such trends from a horizontal line is a chance event. Arrows indicate times of administration of placebo or HspB5.

Fig 5. Decline in MRI lesions RR-MS patients treated with the two lower doses of HspB5.

RR-MS patients received three bimonthly intravenous administrations of either 7.5 or 12.5 mg HspB5, and mean numbers as well as total volumes of Gd+ T1 lesions for the combined groups were evaluated by MRI over the course of 36 weeks. Data show mean values ± standard deviation, as calculated for the full analysis set of patients, including readings for 11 to 14 subjects at different time points. Linear regression analysis was used to evaluate the trends of change over 36 weeks; p values reflect the probability that the deviation of such trends from a horizontal line is a chance event. Arrows indicate times of administration of HspB5.