TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life.

TrYbe® is an Fc-free therapeutic antibody format, capable of engaging up to three targets simultaneously, with long in vivo half-life conferred by albumin binding. This format is shown by small-angle X-ray scattering to be conformationally flexible with favorable 'reach' properties. We demonstrate the format's broad functionality by co-targeting of soluble and cell surface antigens. The benefit of monovalent target binding is illustrated by the lack of formation of large immune complexes when co-targeting multivalent antigens. TrYbes® are manufactured using standard mammalian cell culture and protein A affinity capture processes. TrYbes® have been formulated at high concentrations and have favorable drug-like properties, including stability, solubility, and low viscosity. The unique functionality and inherent developability of the TrYbe® makes it a promising multi-specific antibody fragment format for antibody therapy.

The rational design of affinity-attenuated OmCI for the purification of complement C5.

Complement component C5 is the target of the mAb eculizumab and is the focus of a sustained drug discovery effort to prevent complement-induced inflammation in a range of autoimmune diseases. The immune evasion protein OmCI binds to and potently inactivates C5; this tight-binding interaction can be exploited to affinity-purify C5 protein from serum, offering a vastly simplified protocol compared with existing methods. However, breaking the high-affinity interaction requires conditions that risk denaturing or activating C5. We performed structure-guided in silico mutagenesis to identify prospective OmCI residues that contribute significantly to the binding affinity. We tested our predictions in vitro, using site-directed mutagenesis, and characterized mutants using a range of biophysical techniques, as well as functional assays. Our biophysical analyses suggest that the C5-OmCI interaction is complex with potential for multiple binding modes. We present single mutations that lower the affinity of OmCI for C5 and combinations of mutations that significantly decrease or entirely abrogate formation of the complex. The affinity-attenuated forms of OmCI are suitable for affinity purification and allow elution under mild conditions that are nondenaturing or activating to C5. We present the rational design, biophysical characterization, and experimental validation of affinity-reduced forms of OmCI as tool reagents to enable the affinity purification of C5.

IgG light chain-independent secretion of heavy chain dimers: consequence for therapeutic antibody production and design.

Rodent monoclonal antibodies with specificity towards important biological targets are developed for therapeutic use by a process of humanisation. This process involves the creation of molecules, which retain the specificity of the rodent antibody but contain predominantly human coding sequence. Here, we show that some humanised heavy chains (HCs) can fold, form dimers and be secreted even in the absence of a light chain (LC). Quality control of recombinant antibody assembly in vivo is thought to rely upon folding of the HC CH1 domain. This domain acts as a switch for secretion, only folding upon interaction with the LC CL domain. We show that the secreted heavy-chain dimers contain folded CH1 domains and contribute to the heterogeneity of antibody species secreted during the expression of therapeutic antibodies. This subversion of the normal quality control process is dependent on the HC variable domain, is prevalent with engineered antibodies and can occur when only the Fab fragments are expressed. This discovery will have an impact on the efficient production of both humanised antibodies and the design of novel antibody formats.

Cell surface dynamics and cellular distribution of endogenous FcRn.

A major role for FcRn is the salvage of pinocytosed IgG and albumin from a degradative fate in lysosomes. FcRn achieves this by binding IgG in a pH-dependent manner in acidic endosomes and recycling it to the plasma membrane to be released at neutral pH. This is important in maintaining high serum IgG and albumin levels and has the potential to be exploited to modulate the pharmacokinetics of antibody-based therapeutics. Although FcRn is responsible for the recycling of IgG, the dynamic behaviour of endogenous FcRn is not well understood. Our data shows that the majority of endogenous receptor is distributed throughout the endosomal system and is present only at a low percentage on the plasma membrane at steady state. A significant fraction of FcRn at the cell surface appears to be endocytosis resistant while the remainder can undergo rapid endocytosis. To maintain surface levels of the receptor, endocytosed FcRn is replaced with FcRn from the internal pool. This unexpected complexity in FcRn cell surface dynamics has led us to propose a model for FcRn trafficking that should be taken into account when targeting FcRn at the cell surface for therapeutic purposes.

PEGylation prolongs the pulmonary retention of an anti-IL-17A Fab' antibody fragment after pulmonary delivery in three different species.

The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Production, purification and biological characterization of mono-PEGylated anti-IL-17A antibody fragments.

The aim of this study was to maximize the yield of the production of mono-PEGylated anti-interleukin-17A (anti-IL-17A) antibody fragments using large (≥ 20 kDa) polyethylene glycol (PEG) chains. Particular attention was paid to selectively yield mono-PEGylated species to maintain the maximum possible functionality and to simplify the purification. Neutralization of IL-17A by antibody constructs might find application for the treatment of bronchial hyperreactivity. Amino-directed and sulfhydryl-directed PEGylation of the native antibody fragments were compared. The former was selected as it produced the most interesting construct in terms of yield and preservation of biological activity. In particular, the F(ab')2-PEG conjugate with one 40 kDa branched PEG prepared in this study was produced at a 42% yield. The conjugate presented only a slight decrease in its binding activity and in its in vitro inhibitory potency offering interesting perspectives for in vivo studies.

Towards a universal disulphide stabilised single chain Fv format: importance of interchain disulphide bond location and vL-vH orientation.

Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. Five of these disulphide bond locations were introduced into one scFv in order to compare their relative effects on expression, thermal stability, percent monomer formation and retention of antigen binding. The disulphide bond position vH44-vL100 was observed to enable the most favourable balance of biophysical properties. The vH44-vL100 disulphide bond was introduced into five additional scFv in both vL-vH and vH-vL orientations in order to investigate its general applicability. Data are presented to show the relative influence of scFv sequence, v-region organisation and interchain disulphide bond on expression yield, thermal stability and percent monomer. Introduction of the vH44-vL100 disulphide bond typically resulted in no or little increase in thermal stability and no change in percent monomer but did confer the benefit of permanently fixing monomer:dimer ratios during purification and analysis.

Alternative antibody Fab' fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering.

Antigen-binding fragments (Fab') of antibodies can be site specifically PEGylated at thiols using cysteine reactive PEG-maleimide conjugates. For therapeutic Fab'-PEG, conjugation with 40 kDa of PEG at a single hinge cysteine has been found to confer appropriate pharmacokinetic properties to enable infrequent dosing. Previous methods have activated the hinge cysteine using mildly reducing conditions in order to retain an intact interchain disulphide. We demonstrate that the final Fab-PEG product does not need to retain the interchain disulphide and also therefore that strongly reducing conditions can be used. This alternative approach results in PEGylation efficiencies of 88 and 94% for human and murine Fab, respectively. It also enables accurate and efficient site-specific multi-PEGylation. The use of the non-thiol reductant tris(2-carboxyethyl) phosphine combined with protein engineering enables us to demonstrate the mono-, di- and tri-PEGylation of Fab fragments with a range of PEG size. We present evidence that PEGylated and unPEGylated Fab' molecules that lack an interchain disulphide bond retain very high levels of chemical and thermal stability and normal performance in PK and efficacy models.

Engineering of Escherichia coli to improve the purification of periplasmic Fab' fragments: changing the pI of the chromosomally encoded PhoS/PstS protein.

Escherichia coli is a widely used host for the heterologous expression of proteins of therapeutic and commercial interest. The scale and speed at which it can be cultured can result in the rapid generation of large quantities of product. However, to achieve low costs of production a simple and robust purification process is also required. The general factors that impact on the cost of a purification process are the scale at which a process can be performed, the cost of the purification matrix, and the number and complexity of the chromatographic steps employed. Purification of Fab' fragments of antibodies from the periplasm of E. coli using ion exchange chromatography can result in the co-purification of E. coli host proteins having similar functional pI: such as the periplasmic phosphate binding protein, PhoS/PstS. In such circumstances, an additional chromatographic step is required to separate Fab' from PhoS. Here, we change the functional pI of the chromosomally encoded PhoS/PstS to effect its non-purification with Fab' fragments, enabling the removal of an entire chromatographic step. This exemplifies the strategy of the modification of host proteins with the aim of simplifying the production of heterologous proteins.