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Fusion of engineered albumin with factor IX Padua extends half-life and improves coagulant activity.

ABSTRACT: The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, which resulted in improved HSA release. Second, insertion of the FIX R338L (Padua) substitution conferred hyperactive features (sevenfold higher specific activity) as for FIX Padua alone. Furthermore, we exploited an engineered HSA (QMP), which conferred enhanced human (h)FcRn binding [dissociation constant (K_D ) 0·5 nM] over wild-type FIX-HSA (K_D 164·4 nM). In hFcRn transgenic mice, Padua-QMP displayed a significantly prolonged half-life (2·7 days, P < 0·0001) versus FIX-HSA (1 day). Overall, we developed a novel FIX-HSA protein with improved activity and extended half-life. These combined properties may result in a prolonged functional profile above the therapeutic threshold, and thus in a potentially widened therapeutic window able to improve HB therapy. This rational engineering of both partners may pave the way for new fusion strategies for the design of engineered biotherapeutics.

SUBMITTER: Lombardi S

PROVIDER: S-EPMC8362221 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Fusion of engineered albumin with factor IX Padua extends half-life and improves coagulant activity.

Lombardi Silvia S Aaen Kristin H KH Nilsen Jeannette J Ferrarese Mattia M Gjølberg Torleif T TT Bernardi Francesco F Pinotti Mirko M Andersen Jan T JT Branchini Alessio A

British journal of haematology 20210609 2

The short half-life of coagulation factor IX (FIX) for haemophilia B (HB) therapy has been prolonged through fusion with human serum albumin (HSA), which drives the neonatal Fc receptor (FcRn)-mediated recycling of the chimera. However, patients would greatly benefit from further FIX-HSA half-life extension. In the present study, we designed a FIX-HSA variant through the engineering of both fusion partners. First, we developed a novel cleavable linker combining the two FIX activation sites, whic ...[more]

PMID: 34109608

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Project description:BackgroundCytokines have been promising cancer immunotherapeutics for decades, yet only two are licensed to date. Interleukin-12 (IL-12) is a potent regulator of cell-mediated immunity that activates NK cells and interferon-γ (IFNγ) production. It plays a central role in multiple pathways that can enhance cancer cell death and modify the tumor microenvironment (TME). Attempts to dose rIL-12 were initially successful but IFNγ toxicity in Phase 2 complicated further development in the late 1990s. Since then, better dosing strategies have been developed, but none have achieved the level of cancer control seen in preclinical models. We set out to develop a novel strategy to deliver fully functional IL-12 and other biologics to the TME by binding albumin, taking advantage of its ability to be concentrated and retained in the tumor.MethodsSingle-chain variable fragments (scFv) were identified from a human phage display library that bound human, mouse, and cynomolgus macaque serum albumin, both at physiologic and acidic conditions. These were taken through a series of steps to identify strongly binding molecules that don't interfere with the normal physiology of albumin to bind FcRn, giving it prolonged half-life in serum, along with SPARC/GP60, which allows albumin to target the TME. A final molecule was chosen and a single mutation was made that minimizes the potential for immunogenicity. This fully human albumin-binding (FHAB®) domain was characterized and manufacturing processes were developed to bring the first drug candidate into the clinic.ResultsOnce identified, the murine form of mIL12-FHAB was studied preclinically to understand its mechanism of action and biodistribution. It was found to be much more efficient at blocking tumor growth compared to murine IL-12, while stimulating significant IFNγ production with minimal toxicity. SON-1010, which uses the human IL-12 sequence, passed through all of the characterization and required toxicology and is currently being studied in the clinic.ConclusionsWe identified and developed a platform technology with prolonged half-life that can target IL-12 and other immune modulators to the TME. Safety and efficacy are being studied using SON-1010 as monotherapy and in combination with checkpoint blockade strategies.

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Fusion of engineered albumin with factor IX Padua extends half-life and improves coagulant activity.

Publications

Fusion of engineered albumin with factor IX Padua extends half-life and improves coagulant activity.

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