Elsevier

Journal of Controlled Release

Volume 229, 10 May 2016, Pages 37-47
Journal of Controlled Release

Single chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo

https://doi.org/10.1016/j.jconrel.2016.03.017Get rights and content

Abstract

The neonatal Fc receptor for IgG (FcRn) is considered critical for the regulation of endogenous IgG and serum albumin (SA) and their circulation half-life in vivo. Both IgG and SA can bind to FcRn tightly at acidic pH but not so much at neutral pH. Here we reported a few novel single chain antibody fragments (scFv) obtained based on screening of a phage library. FnAb-8 and FnAb-12 can bind to human FcRn with higher affinities than IgG at acidic pH but similar or lower affinities than IgG at pH 7.4. Fusion proteins consisted of the therapeutic peptide, GLP-1 (Glucagon-like peptide-1) connected to the N-terminus of FnAb-8 and FnAb-12, named as G8 and G12, were shown to retain the pH-dependent binding capabilities to FcRn while also bound to the GLP-1 receptor. In vivo efficacy studies in diet induced diabetes mice confirmed the GLP-1 receptor (GLP-1R) agonist activities and sustained blood sugar lowering effect. In vivo pharmacokinetics (PK) studies were performed in nonhuman primates and FnAb-8 was found to have circulation half-life several folds longer than what have been reported for scFvs. G8 may be developed into long acting GLP-1R agonists with great potentials in clinical applications.

Introduction

The neonatal Fc receptor (FcRn) is well known for having the functions of transporting IgGs across fetal and neonatal tissue barriers and regulating the rate of IgG and SA degradation throughout life [1], [2], [3]. It was firstly identified from the neonatal rodent gut which led to its designation as the neonatal Fc receptor (FcRn) [4], [5]. The expression of FcRn was originally believed to be restricted to those sites involved in the transport of maternal IgG from mother to fetus or neonatal [6], [7], [8]. However, the presence of FcRn have now been extensively reported in many tissues and cell types including the endothelial cells, epithelial cells, the majority of hematopoietic cells, and some specialized cells such as podocytes, keratinocytes, the blood brain barrier endothelia, and the ocular cells [9], [10], [11], [12], [13], [14].

Human FcRn was characterized as a heterodimer of one β2-microglobulin (14 kDa) light chain and one α heavy chain (46 kDa), structurally homologous to the MHC class I molecule [15], [16]. The binding of both IgG and SA to FcRn were found to be highly pH dependent, with high affinity at acidic pH and low affinity at neutral pH [17], [18], [19], [20]. They were protected from degradation based on a similar mechanism although through different binding sites [21], [22], [23]. Specifically, serum proteins including IgG and SA in circulation are taken up by myeloid cells or endothelial cells without FcRn binding near the cell surface when the pH is close to neutral. Entry of IgG and SA into cells is followed by accumulation in early endosomes where the acidic pH is permissive for FcRn binding. Then the early endosomes containing complex of FcRn and IgG/SA are sorted to recycle back to cell surface and IgG/SA released by exocytic processes close to the physiological pH [24], [25], [26], [27], [28].

Many studies had used the FcRn binding domains from SA or IgG and made fusion proteins containing these domains to achieve recycling and circulation extension [29], [30], [31], [32], there had been also some efforts made to improve drugs' circulation properties by engineering or developing new peptide or protein binding domains of FcRn [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]. But most of these attempts including Fc-fusion proteins could not match the endogenous IgGs' long circulation half-life. Higher binding affinity at endosomal pH may not always translate to half-life extension [44], [45]. Other factors including the binding sites, pH dependency, and possibly affinity thresholds at different pHs may exist that governed the FcRn mediated recycling process [46].

In this study, we seek to explore whether it is possible to generate small affinity antibody fragments, i.e., single chain variable fragment (scFv Abs), that can interact directly with human FcRn in a pH-dependent way. The scFv fragments should have higher binding affinity at pH 6.0 towards FcRn than those of hIgG or SA, but almost no binding at pH 7.4. A specific scFv screening scheme was devised and successfully implemented. The detailed characterizations of the screen results indicated they may have excellent recycling properties and could be used as a long circulation carrier for therapeutical proteins and peptides.

Section snippets

Materials

Human serum albumin (SA) was purchased from Abcam. Human IgG and HLA-A2 proteins, Lipofectamine2000, 293fectine and AF647 (AlexaFluor647) antibody labeling kit were from Invitrogen. Mouse anti-M13 phage antibody, anti-Poly-histidine tag antibody and GLP-1 were purchased from Genscript. GLP-1R-Fc, β2-microglobulin (β2M) protein, β2M expression vector, FcRn and HLA-A2 cDNAs were obtained from Sinobiological. Anti-hFcRn and anti-HLA-A2 antibodies were from Santa-Cruz. Strepavidin-AF647, anti-mouse

Screening and identification of pH-dependent hFcRn binders from a naïve human scFv phage library

The scFv phage library screening was carried out using soluble extracellular domain of FcRn. Since we are interested only in pH dependent binders, therefore we designed a binding at pH 6.0 and eluting at pH 7.4 panning strategy (Fig. 1A). In addition, because hFcRn naturally forms heterodimer with β2M, we had to use the hFcRn&β2M dimer protein as the panning target and β2M as the control. In order to avoid interferences from β2M binders, we adopted an improved subtractive panning strategy, as

Discussion

A major challenge for the therapeutic use of many peptides and proteins is their short blood half-life. Their fast clearance from circulation is mainly based on two mechanisms: renal filtration and lysosome degradation. There are in general two strategies to address these problems for prolongation of the drug's half-life: (i) to increase molecular size and its hydrodynamic volume by coupling polymers, carbohydrates or other proteins [52], [53], [54] and (ii) to take advantage of the recycling

Conclusion

In this study, we identified two candidate scFv structures that can bind to human FcRn with great specificity, high affinity at pH 6.0, and low to almost no binding at pH 7.4. They were shown to have extended circulation half-lives in cynomolgus monkeys. They can be used as a carrier protein to be fuse with the therapeutic peptide GLP-1 to improve its stability and activity duration in vivo.

Acknowledgments

We thank the financial supports from NSF China grant No. 81273465 and the Chinese Ministry of Sci. & Tech grant No. 2014AA020707.

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