Silencing of the humoral immune response against an enzyme therapeutic with a peptide biomaterial strategy

Introduction: : Enzyme drug products are an attractive therapeutic class because they demonstrate high specificity towards their substrate, low off-target effects, substrate turnover necessitating less of the product, and are metabolized into biologically available molecules. There are, however, common challenges that often hinder enzymes from realizing their therapeutic potential. A major challenge is immunogenicity, which results from the host developing anti-drug antibodies (ADAs). Emergence of ADAs may compromise the therapeutic by altering its pharmacokinetics, directly blocking its binding pocket, or more serious anaphylactic shock. Strategies for reducing enzyme immunogenicity often involve significant rounds of testing for each new therapeutic. We envision a strategy that would be agnostic to the protein or enzyme that is being delivered, instead leveraging the material as a means for evading the emergence of ADAs. We have previously developed a co-assembling peptide material (CATCH) which can retain an enzyme within it by recombinantly tagging the enzyme with a CATCH peptide at its N- or C- terminus. This approach has been demonstrated with a number of enzymes, however a recurrent theme is the emergence of ADAs. To combat this, we have developed a strategy that is intended to directly block processing and loading of the co-delivered enzyme within antigen-presenting cells. Leupeptin, a well-known pan serine/cysteine protease inhibitor, has been modified and appended to the CATCH sequence. We first show that the CATCH-leupeptin variants still retain their inhibitory properties, then test this in a mouse model of immunogenicity with a therapeutic enzyme.

Materials and

Methods: : CATCH(4K+), CATCH(6E-), CATCH(6K+), and CATCH(6D-) along with two CATCH-leupeptin variants of each peptide, were synthesized using standard solid-phase synthesis techniques. Adenosine synthase A (AdsA) with a CATCH(6E-) tag, will serve as the model enzyme. 2 μM recombinant CATCH(6E-)-AdsA was first mixed with the anionic CATCH peptide and then this mixture was combined with an equivalent volume of cationic CATCH peptide solution (1 mM or 12 mM of total peptide). The mixture was allowed to equilibrize over an hour before injections. The only pairings studied here were variations of CATCH(4K+/6E-) and CATCH(6K+/6D-). Fluorescence imaging was done by adding thioflavin T to glass slides spotted with the prepared material. Inhibitory constants of the eight CATCH-leupeptin variants were defined using the same assay, where BAPNA is a trypsin labile molecule that fluoresces when cleaved. Each peptide’s ability to inhibit this was measured over time, and the IC50s calculated. For ADA experiments, 100 μL of each CATCH formulation was injected subcutaneously into the scruff of C57BL/6J mice (n = 4-5) using a 25G needle on day 0, with a booster of 100 μL administered on day 28. Sera were collected weekly until the study conclusion criteria were met. Collected sera diluted to 1:100, 1:1000, and 1:10000 were then tested for antibodies against AdsA using an ELISA for total mouse IgG.

Results, Conclusions, and Discussions:: Imaging with thioflavin T confirmed that these new CATCH-leupeptin variants retain their ability to form beta-sheet materials, where at 1 mM the CATCH(4K+/6E-)-Leu_V1 formed a flocculate and the CATCH(6K+/6D-)-Leu_V1 formed spherulitic structures, consistent with prior reports (Figure 1a). All eight CATCH-leupeptin variants retained the inhibitory properties of the leupeptin as measured by defining a half-maximal inhibitory concentration (IC50), with some versions performing relatively better (Table 1, Figure 1b-c). We first probed how these new pairs performed at the 1 mM concentration, where previous studies have shown that ADAs against AdsA are elevated by Day 49 (Figure 1d). All four pairs were tested, and by Day 35 all except for CATCH(4K+/6E-)-Leu_V2 had measurable titers. By day 49 all groups had elevated ADAs against AdsA (Figure 2a-b), although the CATCH-leupeptin pairs generally showed lower ELISA readouts versus the Day 49, 0% leupeptin group. Additionally, there was high variability within each experimental group, exemplified by the large SEM bars. This coupled with the IC50 data suggests that there may be a minor effect at this concentration, but the dose of CATCH-leupeptin was too low to fully suppress ADA emergence.

Next, we tested the hydrogel state, where we found that injections of 12 mM CATCH(4K+/6E-)-Leu_V1 led to no measurable ADAs up to Day 49 (Figure 2c). If the CATCH-leupeptin content is halved, we find that measurable antibodies appear early on in the trial and remain elevated (Figure 2d). When compared to the base CATCH(4K+/6E-) gel with no leupeptin content, we find that ADAs at Day 42 have been drawn down by the highest dose of CATCH-leupeptin (Figure 2e). This set of data suggests that CATCH(4K+/6E-)-Leu_V1 in the hydrogel state prevents the emergence of ADAs directed against AdsA. Ongoing studies will explore the other three pairs at the gel state, histological analysis of recruited cells, and residence times. Overall, the data outlined illustrates that CATCH-leupeptin gels are an effective vehicle for an enzyme therapeutic and can actively suppress ADA emergence against the therapeutic.

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