Poster W16 - Effect of dosing schedule on the immune response toward a supramolecular enzyme-peptide carrier

Introduction: Carrier-mediated delivery of protein biotherapeutics is attractive for increasing drug efficacy by way of extending drug half-life, but this prolonged exposure can lead to unwanted production of anti-drug antibodies that counteract protein action through neutralization or clearance1. Re-administration is often required for chronic conditions. Understanding how carrier composition and dosing schedule are interrelated will enable carrier re-design to mitigate anti-drug responses. We are developing an enzyme carrier based on synthetic charge-complementary peptides that co-assemble into nanofibrillar supramolecular structures, known as “CATCH(+/-)”, or co-assembling tags based on charge complementarity. The CATCH(+) and CATCH(-) peptides remain unassembled due to electrostatic repulsion when alone, but rapidly co-assemble into β-sheet structures when combined.2 Enzymes can be physically immobilized within CATCH biomaterials by fusing either CATCH(+) or CATCH(-) onto the protein terminus2. However, the material characteristics of CATCH products are variable. For example, different pairs at high concentrations result in hydrogels of varying stiffness. Conversely, pairs like CATCH(6K+/6D-) form unique micro-spherulitic structures at lower concentrations, where other CATCH (+/-) pairs form amorphous flocculates3. Although fibrillar biomaterials like CATCH have been shown to induce a heightened level of adaptive immunity against biologics leading to anti-drug antibody production, a clear understanding of the material factors influencing underlying these responses are lacking4. Using CATCH peptide pairs with distinct structural architectures , here we will report on relationships between dose and treatment schedule that inform future studies on the immune response toward re-administered biologics immobilized in a supramolecular carrier.

Materials and

Methods: CATCH peptides, CATCH(4K+), CATCH(6E-), CATCH(6K+) and CATCH(6D-) were synthesized using standard solid-phase synthesis techniques. 2 μM recombinant CATCH(6E-)-AdsA was first mixed with 1 mM anionic CATCH peptide and then this mixture was combined with an equivalent volume of 1 mM cationic CATCH peptide solution (1 mM total peptide). Under these conditions, the CATCH(4K+/6E-) pair forms a structure referred to as “amorphous flocculate”, whereas the CATCH(6K+/6D-) pair forms a structure referred to as “spherulites”. 100 μL of each CATCH formulation was then injected subcutaneously into the scruff of C57BL/6J mice (n = 5) using a 25G needle on day 0, with a booster (100 L or 50 L “half strength”) administered on day 28 and day 56 for the monthly injection schedule, or booster administered on days 14, 28, and 42 for the biweekly schedule (Schematic). 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: Biweekly injections of 1 mM CATCH(6K+/6D-) spherulites resulted in measurable antibodies that were first detected a week following the second dose (Day 22 in Figure 1). Levels moderately rose until the end of the study, where saturation of the assay was almost reached. When instead administered on a monthly injection schedule, IgG levels were undetectable up to Day 28 (Figure 2a) for both 1 mM CATCH(6K+/6D-) spherulites and 1 mM CATCH(4K+/6E-) amorphous flocculate. At Day 49, 3 weeks after the first booster, antibodies were raised in animals that received either 1 mM CATCH(6K+/6D-) spherulites or 1 mM CATCH(4K+/6E-) amorphous flocculate (Figure 2b), showing that repeated administration led to a heightened immune response. However, the antibody levels produced by this monthly injection schedule were lower than the levels produced by Day 27 with a bi-weekly dosing schedule of 1 mM CATCH(6K+/6D-) spherulites (Figure 2b). When the AdsA load was decreased to half-strength antibody levels remained undetectable through Day 42 with a monthly dosing schedule (Figure 3).
The two CATCH peptide pairs investigated in this study demonstrate that immunogenicity of a protein therapeutic is dependent on the dose, treatment schedule, and structural properties of the carrier. The CATCH(6K+/6D-) results in total show that a more frequent treatment schedule can induce a much stronger antibody response, but that this response can be mitigated by decreasing the enzyme dose in subsequent exposures. However, on the same dosing schedule, the choice of CATCH peptide pair also influences IgG production. By Day 49 on a monthly full dose schedule, the CATCH(4K+/6E-) amorphous flocculate outperforms the CATCH(6K+/6D-) spherulites. In vivo imaging data (not shown) demonstrate that the CATCH(4K+/6E-) amorphous flocculate is cleared from a subcutaneous injection site faster than the CATCH(6K+/6D-) spherulites, further supporting the observation that the frequency of exposure to the enzyme correlates with the magnitude of the immune response against the enzyme. Collectively, these data show that the CATCH(+/-) peptide system allows for systematic evaluations of carrier-mediated immunogenicity that will inform future efforts to minimize the generation of harmful anti-drug antibodies against re-administered biotherapeutics.

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