Understanding the Adjuvanticity of Co-Assembled Peptide Hydrogels for Biotherapeutic Delivery

Introduction:: Sustained 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 clearance.¹ Understanding how the carrier used for sustained delivery contributes to the emergence of anti-drug antibodies holds promise for mitigating these responses. We are developing protein biotherapeutic carriers based on synthetic charge-complementary peptides that co-assemble into nanofibrillar hydrogels. 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 Protein biotherapeutics can be physically immobilized within CATCH hydrogels by fusing either CATCH(+) or CATCH(-) onto the protein terminus.² CATCH hydrogels are viscoelastic solids that undergo shear-thinning and recovery, which allows for minimally invasive delivery of protein biotherapeutic payloads that are retained at the site of injection for a longer duration than soluble payloads.³ However, fibrillar biomaterials like CATCH have been shown to induce a heightened level of adaptive immunity against biologics leading to anti-drug antibody production – i.e., the carrier acts as an adjuvant⁴. Using a library of CATCH(+/-) variants with different net charges, amino acid content, fibril network architectures, and hydrogel mechanics, we report on the features that govern carrier adjuvanticity for a candidate anti-inflammatory enzyme, adenosine synthase A (AdsA).


Materials and Methods:: CATCH peptides were synthesized using standard solid-phase synthesis techniques. The five CATCH peptide pairs studied were: CATCH(4K+/4E-), CATCH(4K+/6E-), CATCH(6K+/4E-), CATCH(6K+/6E-), and CATCH(6K+/6D-), where the number indicates the content of lysine (K+), glutamic acid (E-), or aspartic acid (D-) residues. AdsA with a CATCH(6E-) tag at 2 μM was mixed with the anionic CATCH peptide prior to hydrogel formation. Cationic and anionic CATCH solutions were combined at a 1:1 (v:v) ratio, upon which point they begin forming a viscoelastic hydrogel (total [CATCH] = 12 mM), soluble nanofibers (total [CATCH] = 1 mM), or soluble nanoparticles (total [CATCH] = 0.1 mM). Following a curing step, 100 μL of CATCH hydrogel or nanofibers was then injected subcutaneously into the scruff of C57BL/6J mice (n = 5) using a 25G needle at day 0, with a booster administered on day 28. Sera were collected pre-injection and then weekly thereafter through day 42. Sera of the five mice were then tested for development of antibodies against AdsA using an ELISA for total IgG. Sera were diluted to 1:100, 1:1000, and 1:10000 to measure antibody levels.


Results, Conclusions, and Discussions:: Measurable anti-AdsA total IgG levels were found for all CATCH peptide pairs up to a 1:10000 dilution (Figure 1). CATCH(6K+/6D-) showed the highest levels, indicating a greater concentration of IgG in the serum when compared to the other pairs. Notably, the net neutral CATCH(6K+/6E-) (Figure 1d) induced comparable total IgG levels as the anionic (Figure 1b) and cationic (Figure 1c) pairs, which suggests that charge is not a primary driver of adjuvanticity in this system in contrast to other systems⁵. Furthermore, CATCH(6K+/6E-) induced significantly lower total IgG levels than CATCH(6K+/6D-), despite only differing in one amino acid. This may be explained by viscoelastic properties of each hydrogel, where the stiffness of CATCH(6K+/6D-) is an order of magnitude greater than that of CATCH(6K+/6E-) (Table 1). Consistent with this, CATCH(4K+/4E-), which has comparable stiffness to CATCH(6K+/6D-) induced higher total IgG levels than all other pairs except for CATCH(6K+/6D-). These data suggested that stiffness may be a determinant of adjuvanticity.

Prior work has shown that decreasing CATCH peptide concentration correlates with decreasing stiffness³. This property was utilized to study how CATCH material stiffness affects adjuvanticity. CATCH(4K+/6E-) carriers in the nanofiber state ([peptide] = 1 mM) and granular nanoparticle state ([peptide] = 0.1 mM) induced lower anti-AdsA antibody levels than CATCH(4K+/6E-) in the hydrogel state, even when the total AdsA load was held constant (data not shown). This supported the suggestion that carrier mechanics govern adjuvanticity. However, hydrogel mechanics alone do not fully explain this relationship, given that CATCH(4K+/4E-) and CATCH(6K+/6D-) had similar stiffnesses but different total IgG levels (Figures 1a and 1e). Cryo-SEM images revealed that the CATCH(+/-) pairs form gel networks with different architectural features (Figure 2). In particular, the CATCH(6K+/6E-) pair and the charged pairs form entangled networks of thin fibrils, whereas CATCH(4K+/4E-) formed networks with smaller pore diameters and regions of dense mats³. Notably, the CATCH(6K+/6D-) pair formed a dramatically different architecture of plate-like structures that were not seen with any of the other CATCH peptide pairs⁶. Taken altogether, these data suggest that CATCH hydrogel adjuvanticity is governed by a complex relationship of hydrogel viscoelasticity and fibril network architecture.


Acknowledgements (Optional): :

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(6.) Liu, R. et al. unpublished