George Thom

Probing a protein-protein interaction by in vitro evolution

In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

BackgroundModelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies.MethodsBrain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting.ResultsUsing a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter.ConclusionsOur techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

In vitro potency, pharmacokinetic profiles, and pharmacological activity of optimized anti-IL-21R antibodies in a mouse model of lupus.

Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in VL CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Faslpr mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC0-∞) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Faslpr mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.

A variant human IgG1-Fc mediates improved ADCC

Ribosome display was applied to the Fc region of human immunoglobulin G (IgG1) to select for improved binding to human FcγRIIIa, the receptor expressed on human natural killer cells that mediates antibody-dependent cellular cytotoxicity (ADCC). A library of human Fcγ1 variants was generated using error-prone polymerase chain reaction, and subjected to multiple rounds of ribosome display selection against progressively decreasing concentrations of soluble human FcγRIIIa, to enrich for improved binders. Radioimmunoassay and alphascreen analyses of the aglycosylated IgG-Fc output revealed variants with improved binding to FcγRIIIa relative to wild-type IgG-Fc. Subsequent expression in human (HEK-EBNA) cells generated glycosylated IgGs with modified activity in ADCC assays. One particular variant, 125_B01 triggered enhanced ADCC (EC(50) up to four-fold reduced with increased maximal lysis) relative to wild-type antibody, having more equal levels of ADCC for each allotype (V158/F158) of FcγRIIIa. Deconvolution of individual replacements within the variant showed that improved function arose from the Phe243Leu replacement within the CH2 domain, rather than the CH3 domain replacements Thr393Ala or His433Pro. Surprisingly, the oligosaccharide profiles of 125_B01 indicated more oligosaccharide chains lacking fucose, or with bisecting N-acetylglucosamine relative to wild-type IgG1, which correlates with improved function and the replacement Phe243Leu that is a carbohydrate contact residue within the C(H)2 domain.

Brain penetration, target engagement, and disposition of the blood–brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood‐brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB‐crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high‐affinity, selective antibody antagonist of the metabotropic glutamate receptor‐1 (BBB‐mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB‐NiP). Both BBB‐NiP and BBB‐mGluR1 demonstrated a similar 20‐fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo. Comparative assessment of BBB‐NiP and BBB‐mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target‐mediated removal of the mGluR1‐engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose‐dependent inhibition of mGluR‐1‐mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved inmGluR1‐mediated pain processing. We demonstrate the feasibility of targeting central G‐protein‐coupled receptors using a BBB‐crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.—Webster, C. I., Caram‐Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood‐brain barrier‐crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1 FASEB J. 30, 1927–1940 (2016). www.fasebj.org

Enhanced delivery of IL-1 receptor antagonist to the central nervous system as a novel anti–transferrin receptor-IL-1RA fusion reverses neuropathic mechanical hypersensitivity

Abstract Neuropathic pain is a major unmet medical need, with only 30% to 35% of patients responding to the current standard of care. The discovery and development of novel therapeutics to address this unmet need have been hampered by poor target engagement, the selectivity of novel molecules, and limited access to the relevant compartments. Biological therapeutics, either monoclonal antibodies (mAbs) or peptides, offer a solution to the challenge of specificity as the intrinsic selectivity of these kinds of molecules is significantly higher than traditional medicinal chemistry–derived approaches. The interleukin-1 receptor system within the spinal cord has been implicated in the amplification of pain signals, and its central antagonism provides relief of neuropathic pain. Targeting the IL-1 system in the spinal cord with biological drugs, however, raises the even greater challenge of delivery to the central compartment. Targeting the transferrin receptor with monoclonal antibodies has proved successful in traversing the endothelial cell–derived blood–brain barrier and delivering proteins to the central nervous system. In this study, we describe a novel construct exemplifying an engineered solution to overcome these challenges. We have generated a novel anti–transferrin receptor-interleukin-1 receptor antagonist fusion that transports to the central nervous system and delivers efficacy in a model of nerve ligation–induced hypersensitivity. Approaches such as these provide promise for novel and selective analgesics that target the central compartment.

Optimization of CAT-354, a Therapeutic Antibody Directed Against Interleukin-13, Using Ribosome Display

In this case study, we describe the use of in vitro protein evolution with ribosome display to improve the potency of a human interleukin-13-neutralising antibody by a factor of over 200-fold and derive a therapeutic candidate, CAT-354, for the treatment of asthma. A combination of directed and random mutagenesis enabled the identification of highly potent neutralising antibodies and highlighted the advantage of the ribosome display protein evolution approach in identifying beneficial mutations across the entire sequence space. This chapter describes in detail the process followed to achieve a successful in vitro affinity maturation outcome using ribosome display technology.

Isolation of blood-brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system

ABSTRACT The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity

The blood–brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re‐directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter‐study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR‐binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics‐selected reaction monitoring and fluorescent imaging with markers of early‐ and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co‐localized with late‐endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co‐localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.

A peptide derived from melanotransferrin delivers a protein-based interleukin 1 receptor antagonist across the BBB and ameliorates neuropathic pain in a preclinical model:

Delivery of biologic drugs across the blood–brain barrier is becoming a reality. However, the solutions often involve the assembly of complex multi-specific antibody molecules. Here we utilize a simple 12 amino-acid peptide originating from the melanotransferrin (MTf) protein that has shown improved brain delivery properties. 3D confocal fluorescence microscopic analysis demonstrated brain parenchymal localisation of a fluorescently labelled antibody (NIP228) when chemically conjugated to either the MTf peptide or full-length MTf protein. Measurement of plasma kinetics demonstrated the MTf peptide fusions had very similar kinetics to an unmodified NIP228 control antibody, whereas the fusion to MTf protein had significantly reduced plasma exposure most likely due to a higher tissue distribution in the periphery. Brain exposure for the MTf peptide fusions was significantly increased for the duration of the study, exceeding that of the fusions to full length MTf protein. Using a neuropathic pain model, we have demonstrated that fusions to interleukin-1 receptor antagonist (IL-1RA) are able to induce significant and durable analgesia following peripheral administration. These data demonstrate that recombinant and chemically conjugated MTf-based brain delivery vectors can deliver therapeutic levels of drug to the central nervous system.