Thermoresponsive hydrogel for long-acting delivery of structurally intact and biologically active Fab fragment and monoclonal antibody

Therapeutic antibodies are a cornerstone of modern medicine, providing targeted and effective treatments for a wide range of diseases. Antibody delivery, however, has long been recognized as a major bottleneck for safety and efficacy. We developed a hyaluronic acid-poloxamer 407-calcium (HAP-Ca²⁺) thermoresponsive hydrogel for the controlled, long-term delivery of a full-length human monoclonal antibody (mAb) and an antibody fragment (Fab). The hydrogel enabled sustained, biphasic release of the mAb without initial burst (∼60% released over 25 days, followed by 20% over 35 days), while the Fab showed a modest burst (∼15% on day 1) and gradual release over two months. Both antibodies retained thermal stability and structural integrity, confirmed by nanoscale differential scanning fluorometry (nanoDSF) and circular dichroism (CD). Microscale thermophoresis (MST) showed maintained Fab binding over one month (K_D ≈ 40 nM), while mAb EC50 increased from 0.108 nM to 0.338 nM. In vitro assays demonstrated biological activity for up to three weeks following hydrogel release. In vivo intravitreal administration in rabbits demonstrated that the hydrogel prolonged the vitreal half-life of mAb compared to PBS (7.0 ± 1.5 vs 5.1 ± 0.9 days), extending the time above estimated reference concentrations by approximately 5-11 days. Our results show that the HAP-Ca²⁺ thermoresponsive hydrogel can deliver Fab and mAb in a controlled, long-lasting manner while preserving their structure and activity, making it a strong candidate for sustained antibody delivery. More broadly, thermoresponsive hydrogels like this are emerging as powerful tools to enhance the safety and efficacy of antibody therapies. STATEMENT OF SIGNIFICANCE: Antibodies are among the most powerful medicines but delivering them safely and steadily remains a challenge. We designed a smart hydrogel that gradually releases both a full-length antibody and a smaller antibody fragment, avoiding sudden bursts that can cause side effects. Importantly, the antibodies retain their structure and activity for weeks after release. This represents an important step toward achieving controlled, long-term delivery for these complex molecules. More broadly, our study suggests that temperature-responsive hydrogels could serve as promising antibody delivery systems, an approach with the potential to make future therapies safer, longer-lasting, and more practical for real-world patient care.