pH-dependent Antibody Research Articles

An anti-CD98 antibody displaying pH-dependent Fc-mediated tumour-specific activity against multiple cancers in CD98-humanized mice.

The cell-surface glycoprotein CD98-a subunit of the LAT1/CD98 amino acid transporter-is an attractive target for cancer immunotherapies, but its widespread expression has hampered the development of CD98-targeting antibody therapeutics. Here we report that an anti-CD98 antibody, identified via the screening of phage-display libraries of CD98 single-chain variable fragments with mutated complementarity-determining regions, preserves the physiological function of CD98 and elicits broad-spectrum crystallizable-fragment (Fc)-mediated anti-tumour activity (requiring Fcγ receptors for immunoglobulins, macrophages, dendritic cells and CD8+ T cells, as well as other components of the innate and adaptive immune systems) in multiple xenograft and syngeneic tumour models established in CD98-humanized mice. We also show that a variant of the anti-CD98 antibody with pH-dependent binding, generated by solving the structure of the antibody-CD98 complex, displayed enhanced tumour-specific activity and pharmacokinetics. pH-dependent antibody variants targeting widely expressed antigens may lead to superior therapeutic outcomes.

A cross-reactive pH-dependent EGFR antibody with improved tumor selectivity and penetration obtained by structure-guided engineering

The clinical use of anti-EGFR antibody-based cancer therapy has been limited by antibody-EGFR binding in normal tissues, so developing pH-dependent anti-EGFR antibodies that selectively bind with EGFR in tumors-by taking advantage of the acidity of tumor microenvironment relative to normal tissues-may overcome these limitations. Here, we generated pH-dependent anti-EGFR antibodies with cross-species reactivity for human and mouse EGFR, and we demonstrate that pH-dependent antibodies exhibit tumor-selective binding by binding strongly to EGFR under acidic conditions (pH 6.5) but binding weakly under neutral (pH 7.4) conditions. Based on screening a non-immune human antibody library and antibody affinity maturation, we initially generated antibodies with cross-species reactivity for human and mouse EGFR. A structure model was subsequently constructed and interrogated for hotspots affecting pH-dependent binding, which supported development of a cross-reactive pH-dependent anti-EGFR antibody, G532. Compared with its non-pH-dependent antibody variant, G532 exhibits improved tumor selectivity, tumor penetration, and antitumor activity. Thus, beyond showing that pH-dependentanti-EGFR antibodies can overcome multiple limitationswith antibody-based cancer therapies targeting EGFR, our study illustrates a structure-guided antibody-antigenbinding pH-dependency engineering strategy to enhance antibody tumor selectivity and tumor penetration, which can inform the future development of antibody-based cancer therapies targeting other ubiquitously expressed molecules.

A stepwise mutagenesis approach using histidine and acidic amino acid to engineer highly pH-dependent protein switches.

Antibody-based drugs can be highly toxic, because they target normal tissue as well as tumor tissue. The pH value of the extracellular microenvironments around tumor tissues is lower than that of normal tissues. Therefore, antibodies that engage in pH-dependent binding at slightly acidic pH are crucial for improving the safety of antibody-based drugs. Thus, we implemented a stepwise mutagenesis approach to engineering pH-dependent antibodies capable of selective binding in the acidic microenvironment in this study. The first step involved single-residue histidine scanning mutagenesis of the antibody's complementarity-determining regions to prescreen for pH-dependent mutants and identify ionizable sensitive hot-spot residues that could be substituted by acidic amino acids to obtain pH-dependent antibodies. The second step involved single-acidic amino acid residue substitutions of the identified residues and the assessment of pH-dependent binding. We identified six ionizable sensitive hot-spot residues using single-histidine scanning mutagenesis. Nine pH-dependent antibodies were isolated using single-acidic amino acid residue mutagenesis at the six hot-spot residue positions. Relative to wild-type anti-CEA chimera antibody, the binding selectivity of the best performing mutant was improved by approximately 32-fold according to ELISA and by tenfold according to FACS assay. The mutant had a high affinity in the pH range of 5.5–6.0. This study supports the development of pH-dependent protein switches and increases our understanding of the role of ionizable residues in protein interfaces. The stepwise mutagenesis approach is rapid, general, and robust and is expected to produce pH-sensitive protein affinity reagents for various applications.

Programmable Bivalent Peptide-DNA Locks for pH-Based Control of Antibody Activity.

The ability to control antibody activity by pH has important applications in diagnostics, therapeutic antibody targeting, and antibody-guided imaging. Here, we report the rational design of bivalent peptide–DNA ligands that allow pH-dependent control of antibody activity. Our strategy uses a pH-responsive DNA triple helix to control switching from a tight-binding bivalent peptide–DNA lock into a weaker-binding monovalent ligand. Different designs are introduced that allow antibody activation at both basic and acidic pHs, either autonomously or in the presence of an additional oligonucleotide trigger. The pH of antibody activation could be precisely tuned by changing the DNA triple helix sequence. The peptide–DNA locks allowed pH-dependent antibody targeting of tumor cells both in bulk and for single cells confined in water-in-oil microdroplets. The latter approach enables high-throughput antibody-mediated detection of single tumor cells based on their distinctive metabolic activity.

Acidification of plasma for detection of pH-dependent antibodies

Abstract Most antibodies to blood group antigens react optimally at a pH range of 6.5–7.5, similar to that of normal plasma or serum. Some antibodies, however, including anti-M, react preferentially or exclusively in an acidic environment with a pH below 6.5. Antibodies with anti-M specificity often show dosage. They can be weakly reactive and even look like nonspecific reactivity at the antihuman globulin phase especially when immediate spin and/or room temperature testing is not part of routine screening. Acidification of serum or plasma may help to identify these antibodies as clinically insignificant versus as an unidentified antibody for which clinical significance is unknown.

SKY59: A Long-Lasting Engineered Anti-C5 Antibody for Subcutaneously Injectable Anti-Complement Therapy

Modulating the complement system is a promising approach in drug discovery for overcoming uncontrolled complement activation, which is known to cause various disorders. While these disorders can be effectively treated by inhibiting complement C5 with a therapeutic antibody, the high concentration of C5 in plasma requires a huge dosage and frequent intravenous administration of the antibody. We have generated SKY59, an engineered humanized anti-C5 monoclonal antibody that shows long-lasting neutralization of C5 in cynomolgus monkeys, which enables subcutaneous delivery or less frequent administration. We found that, although the effective duration of the antibody was prolonged by engineering a pH-dependent C5 binding, it was not sufficient to achieve subcutaneous delivery with less frequent administration. Here, we further improved the effectiveness of the pH-dependent anti-C5 antibody by applying a novel approach: surface charge engineering.Intravenous injection of 40 mg/kg CFA0322, a high affinity anti-C5 monoclonal antibody that is not pH-dependent, caused about 3-fold increase of total C5 concentration in the plasma of cynomolgus monkeys. This is because the C5-antibody immune complexes (ICs) have slower clearance than C5 alone. The increase in total C5 concentration then requires an even higher dosage of the antibody to neutralize the increased amount of C5.To reduce the required dosage of anti-C5 antibody, pH-dependent antibodies were identified from rabbit immunization. A humanized lead antibody, 305LO1, showed a pH-dependent binding property and reduced the plasma total C5 concentration compared to CFA0322. However, we still observed an increase in total C5 plasma concentration over the baseline. We assumed that this was due to a slow uptake rate of the C5-antibody ICs into the cell. We hypothesized that the surface charges of the ICs partially contributed to the slow uptake rate.Surface-charge engineered antibodies were generated by engineering both the variable and the constant regions. Both approaches successfully reduced the increase of plasma C5 in cynomolgus monkey and the combination of the two completely suppressed the accumulation. Interestingly, surface-charge engineering did not affect the pharmacokinetics of the antibody. The effect of surface-charge engineering on C5 reduction correlated clearly with the retention time of the C5-antibody ICs in cation exchange chromatography, indicating the contribution of positive surface charges of the ICs.After further engineering to optimize various properties such as immunogenicity and physicochemical properties, SKY59 was successfully generated. Our surface-charge engineered pH-dependent antibody suppressed the increase of total C5 concentration in vivo by accelerating the rate of IC uptake into cells, C5 release from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, SKY59 can be expected to provide significant benefits for patients with complement-mediated disorders. DisclosuresKitazawa:Chugai Pharmaceutical Co., Ltd: Employment, Equity Ownership, Patents & Royalties: Patents related to emicizumab.

Book Review: ADME and Translational Pharmacokinetics/Pharmacodynamics of Therapeutic Proteins

Edited by Honghui Zhou and Frank-Peter Theil John Wiley and Sons, Inc. 472 pp, hardcover ISBN: 978-1-118-89864-2 Reviewed by Balaji Agoram For more than a decade, clinical pharmacology (CP), absorption-distribution metabolism, and excretion (ADME), and pharmacokinetics-pharmacodynamics (PKPD) properties of biologics have received particular attention from experts within the field through increasing numbers of publications and the occasional book dealing with general aspects of CP or PK science.1, 2 While these publications have aided better understanding of this subject within the general PKPD population, a more thorough discussion of these aspects in the context of the underlying biochemical, biological, and technological processes that influence these properties for biologics has been lacking. This new book is the first to emerge with the ambitious objective of providing a “grand tour” of all things biologics ADME/PKPD/CP with an understandable focus on monoclonal antibodies (mAbs)—the dominant member of the biologics family. At the outset, one is impressed with the scope of the book; it provides an outline of very diverse topics, starting with the basics of protein engineering and how these are used to design and manipulate the ADME properties of recombinant and synthetic proteins (Chapters 2 and 3). Chapters such as the one on immunogenicity (Chapter 11) provide a good background for the reason behind the differences in development strategy for biologics. The authors have to be congratulated for their endeavor to stitch these topics together in a single book—although the organization of the chapters with these specialized non-PK topics spread out among the more PK-focused ones left this reader slightly confused. In spite of the breadth of the science covered in the book, most of the chapters that deal with subjects not many PK practitioners will be familiar with are relatively short and crisp, dealing only with the core principles. This helps provide an overview of these topics without delving into depth and thus turn off a casual reader. Also, a wide variety of biologic modalities are covered in different chapters—antibody-drug-conjugates (ADC), bispecific antibodies, pH-dependent antibodies, blood–brain barrier-penetrating biologics—which will help break up the myth of a monolithic biologic modality. With such a wide range of topics, even an experienced scientific practitioner in this area is likely to find something new to engage them and expand their knowledge. But the cores of the book are the CP and PKPD aspects, which deal with principles of ADME and PKPD modeling for biologics and how they impact preclinical development, first-in-human dose selection, and clinical development. These are dealt with in great detail and are comprehensive. Another welcome inclusion in this book are specific chapters on the development strategy for biologics (Chapters 22, 24, 25). These chapters outline typical differences in the clinical development strategy for biologics compared to typical small molecules. More information on preclinical development—especially focusing on PK and drug metabolism aspects—would also have been useful, especially since this is a stated aim in the foreword. The main criticism from this reader was that the PKPD information, while being comprehensive, felt repetitive in some aspects, e.g., the ADME of biologics repeatedly appears in various chapters with different depths (6, 7, 10, 15…); target-mediated drug disposition alone is dealt with in two different chapters and is summarized in others as well. Furthermore, examples such as fixed vs. weight-based dosing (Chapter 9) and extension of target engagement to biomarkers and clinical response (Chapter 13) are general pharmacometric issues better addressed in a general book on pharmacometrics rather than in a book specifically dealing with biologics (the dosing question is also dealt with twice and could have been edited out in Chapter 25). Perhaps in a future edition the editors would consider using the saved space from removing these redundancies to expand on some of the other emerging topics, such as the use of systems modeling in the design of mAb derivatives such as bispecifics and ADCs—an approach distinctly different from small molecules—or expand on some topics already presented such as tumor penetration of mAbs and preclinical development of biologics. More focus on non-mAb biologics—synthetic vs. natural peptides/proteins, etc.—would all be welcome additions to this tome. In summary, this is an excellent and comprehensive book for beginners to the CP/PK area to acquaint themselves with the area of biologics (specifically mAb) CP/PKPD principles and for experienced CP/PK scientists for reference.

Long lasting neutralization of C5 by SKY59, a novel recycling antibody, is a potential therapy for complement-mediated diseases

Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.

ID: 75: Unraveling the properties required for an effective therapeutic antibody targeting a chemokine

The control of cell migration by chemokines is mediated by interactions with seven transmembrane G protein-coupled receptors and cell surface proteoglycans. Monoclonal antibodies (mAbs) are a successful class of drugs especially when targeting cytokines. However, there has been limited success when targeting a chemokine. In order to further define what is required to achieve therapeutic efficacy when targeting a chemokine with a mAb, we have dissected the characteristics of two anti-murine CXCL10 mAbs. Interestingly, despite having similar affinities and selectivity for CXCL10, they exhibit different key properties. Both mAbs are able to inhibit receptor mediated events such as chemotaxis in vitro, albeit with different potencies yet only one shows efficacy in several animal models of human disease. In addition, the PK of the two mAbs in mice was significantly different: the active mAb had a PK profile characteristic of IgGs with a T1/2 of 18 days whereas the other was rapidly cleared. In contrast to the belief that the active form of chemokine is that bound to cell surface GAGs, the mAb that showed activity in vivo did not recognize cell bound chemokine, whilst the mAb that was rapidly cleared did, leading to a proposal of a new paradigm in chemokine biology. Furthermore, in view of the unexpected abundance of chemokine protein, we have identified a pH dependent antibody which is could inhibit multiple target antigens, through release of the target in the endosomal compartment. Their head to head comparison in a CXCL10 dependent disease model will be presented.

De novo isolation of antibodies with pH-dependent binding properties

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.

Dynamic adsorption of monoclonal antibody layers on hydrophilic silica surface: A combined study by spectroscopic ellipsometry and AFM

Spectroscopic ellipsometry (SE) and atomic force microscopy (AFM) have been used to investigate the adsorption of a mouse monoclonal antibody (type IgG1, anti- β-hCG) on hydrophilic silica (bearing weak negative charges above pH 3), followed by the assessment of binding of human chorionic gonadotrophin (hCG). The antibody is a relatively large molecule with a molecular weight of 150 kDa and the isoelectric point (IP) around pH 6. The antibody adsorption was conducted at pH 4.0, 6.0 and 8.0 to examine the role of charge interaction. Ellipsometric results show that away from the IP, both initial adsorption rate and surface excess decreased, with the reduction at pH 8.0 being more pronounced than that at pH 4.0 due to the electrostatic repulsion not only between the charged antibody molecules within the adsorbed layer but also between antibody and the silica surface. Whilst parallel AFM measurements confirmed the main trend of pH dependent antibody adsorption, they also revealed the tendency of surface aggregation with increasing surface coverage. AFM height profiling at low surface coverage confirmed the “flat-on” orientation of adsorbed antibody molecules, consistent with the previous study by neutron reflection. Interestingly, the antibody height at pH 4.0 was found to be lower than that at pH 8.0, showing the influence from different electrostatic interactions under the two pH conditions. Subsequent hCG binding to the adsorbed antibodies was found to decrease with increasing surface coverage due to the steric hindrance. Under similar antibody surface coverage, the hCG binding ratio at pH 8 was higher than that at pH 4.0, a difference that could only be accounted for by the tighter surface confinement at pH 4.0.

Structural basis of pH-dependent antibody binding by the neonatal Fc receptor.

The neonatal Fc receptor (FcRn) mediates the transcytosis of maternal immunoglobulin G (IgG) across fetal and/or neonatal tissues for the acquisition of passive immunity. In adults, FcRn is involved in the maintenance of high serum IgG levels. Both processes are mediated by pH-dependent IgG binding to FcRn-FcRn binds to IgG with nanomolar affinity at pH 6, but shows no detectable binding at pH 7.5. At pH 6, FcRn is more thermally stable and the dissociation rate of its light chain is an order of magnitude slower than at pH 8.0. Comparison of the structures of FcRn at pH 6.5 and pH 8 allows an analysis of the structural basis for the receptor's pH-dependent ligand binding and stability. We have determined the structure of FcRn at pH 8 and compared it to a further refined version of the structure at pH 6.5. An extensive ordered carbohydrate structure is observed at both pH values. The two structures are very similar; thus the pH dependence of FcRn stability and affinity for IgG can be attributed to chemical properties of the structures themselves, rather than mechanisms that rely on conformational changes. The pH-dependent properties are mediated by electrostatic interactions involving histidine residues, which are more favorable for the protonated form of histidine that predominates at acidic pH values. No major conformational change is observed between the pH 6.5 and pH 8 structures of FcRn that could account for the differences in affinity for IgG. The pH dependence of IgG binding to FcRn can therefore primarily be attributed to titration of histidine residues on Fc that interact with anionic pockets on the receptor. The FcRn dimer, which is required for high affinity binding of IgG, is itself stabilized at acidic pH by histidine-mediated salt bridges and a sidechain rearrangement that creates a more favorable interaction with an anionic pocket at pH 6.5 relative to pH 8. FcRn dimerization is facilitated by reciprocal interactions in which carbohydrate from one receptor molecule binds to protein residues from the dimer-related receptor molecule to form a 'carbohydrate handshake'.