Tyrosine Sulfation at Antibody Light Chain CDR-1 Increases Binding Affinity and Neutralization Potency to Interleukine-4.

Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.

Assembly of an activated rhodopsin-transducin complex in nanoscale lipid bilayers.

The formation and characterization of an activated complex of the visual pigment rhodopsin and its downstream signaling partner transducin have been the subject of intense focus by several research groups. While the subunit composition of the activated complex is still the subject of some controversy, our laboratory [Xie, G., D'Antona, A. M., Edwards, P. C., Fransen, M., Standfuss, J., Schertler, G. F. X., and Oprian, D. D. (2011) Biochemistry 50, 10399-10407] and that of Ernst et al. [Ernst, O. P., Gramse, V., Kolbe, M., Hofmann, K. P., and Heck, M. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 10859-10864] find that the two proteins are present in a 1/1 molar ratio. Unfortunately, these data could not distinguish a ratio of 1/1 from ratios of 2/2, 3/3, etc. For this reason, we reinvestigated the issue of stoichiometry of the activated complex, exploiting the ability of Nanodisc lipid bilayers to isolate single molecules of rhodopsin. We show here that the purified complex in Nanodiscs contains an activated rhodopsin with a covalently bound all-trans-retinal chromophore, that transducin has an empty nucleotide-binding pocket, that the isolated complex is active and dissociates upon addition of guanine nucleotide, and that the stoichiometry corresponds to exactly one molecule of rhodopsin and one molecule of transducin.

Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization.

Rapidly producing drug-like antibody therapeutics for lead molecule discovery and candidate optimization is typically accomplished by large-scale transient gene expression technologies (TGE) with cultivated mammalian cells. The TGE methodologies have been extensively developed over the past three decades, yet produce significantly lower yields than the stable cell line approach, facing the technical challenge of achieving universal high expression titers for a broad range of antibodies and therapeutics modalities. In this study, we explored various parameters for antibody production in the TGE cell host Expi293FTM and ExpiCHO-STM with the transfection reagents ExpiFectamineTM and polyethylenimine. We discovered that there are significant differences between Expi293FTM and ExpiCHO-STM cells with regards to DNA complex formation time and ratio, complex formation buffers, DNA complex uptake trafficking routes, responses to dimethyl sulfoxide and cell cycle inhibitors, as well as light-chain isotype expression preferences. This investigation mechanistically dissected the TGE processes and provided a new direction for future transient antibody production optimization.

A potential antibody repertoire diversification mechanism through tyrosine sulfation for biotherapeutics engineering and production.

The diversity of three hypervariable loops in antibody heavy chain and light chain, termed the complementarity-determining regions (CDRs), defines antibody's binding affinity and specificity owing to the direct contact between the CDRs and antigens. These CDR regions typically contain tyrosine (Tyr) residues that are known to engage in both nonpolar and pi stacking interaction with antigens through their complementary aromatic ring side chains. Nearly two decades ago, sulfotyrosine residue (sTyr), a negatively charged Tyr formed by Golgi-localized membrane-bound tyrosylprotein sulfotransferases during protein trafficking, were also found in the CDR regions and shown to play an important role in modulating antibody-antigen interaction. This breakthrough finding demonstrated that antibody repertoire could be further diversified through post-translational modifications, in addition to the conventional genetic recombination. This review article summarizes the current advances in the understanding of the Tyr-sulfation modification mechanism and its application in potentiating protein-protein interaction for antibody engineering and production. Challenges and opportunities are also discussed.

New Opportunities in Glycan Engineering for Therapeutic Proteins.

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins.

Large-Scale Transient Production in ExpiCHO-S™ with Enhanced N-Galactosylation-Sialylation and PEI-Based Transfection.

Large-scale transient expression in Chinese Hamster Ovary (CHO) cells provides a rapid protein production method with a potential start-to-end alignment advantage for biotherapeutics drug discovery. In this chapter, experimental protocols are illustrated for transient expression of therapeutic glycoproteins with improved galactosylation and sialylation in ExpiCHO-S™ system. To reduce the production cost, we also describe a novel procedure for PEI-mediated transfection in ExpiCHO-S™ cells that supports therapeutic protein expression comparable to the level with ExpiFectamine™-based transfection.

Impacts of fast production of afucosylated antibodies and Fc mutants in ExpiCHO-S™ for enhancing FcγRIIIa binding and NK cell activation.

This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.

Preparation of an activated rhodopsin/transducin complex using a constitutively active mutant of rhodopsin.

The interaction of rhodopsin and transducin has been the focus of study for more than 30 years, but only recently have efforts to purify an activated complex in detergent solution materialized. These efforts have used native rhodopsin isolated from bovine retina and employed either sucrose density gradient centrifugation or size exclusion chromatography to purify the complex. While there is general agreement on most properties of the activated complex, subunit stoichiometry is not yet settled, with rhodopsin/transducin molar ratios of both 2/1 and 1/1 reported. In this report, we introduce methods for preparation of the complex that include use of recombinant rhodopsin, so as to take advantage of mutations that confer constitutive activity and enhanced thermal stability on the protein, and immunoaffinity chromatography for purification of the complex. We show that chromatography on ConA-Sepharose can substitute for the immunoaffinity column and that bicelles can be used instead of detergent solution. We demonstrate the following: that rhodopsin has a covalently bound all-trans-retinal chromophore and therefore corresponds to the active metarhodopin II state; that transducin has an empty nucleotide-binding pocket; that the isolated complex is active and dissociates upon addition of guanine nucleotide; and finally that the stoichiometry corresponds reproducibly to a 1/1 molar ratio of rhodopsin to transducin.

Recent Advances in the Molecular Design and Applications of Multispecific Biotherapeutics.

Recombinant protein-based biotherapeutics drugs have transformed clinical pipelines of the biopharmaceutical industry since the launch of recombinant insulin nearly four decades ago. These biologic drugs are structurally more complex than small molecules, and yet share a similar principle for rational drug discovery and development: That is to start with a pre-defined target and follow with the functional modulation with a therapeutic agent. Despite these tremendous successes, this "one target one drug" paradigm has been challenged by complex disease mechanisms that involve multiple pathways and demand new therapeutic routes. A rapidly evolving wave of multispecific biotherapeutics is coming into focus. These new therapeutic drugs are able to engage two or more protein targets via distinct binding interfaces with or without the chemical conjugation to large or small molecules. They possess the potential to not only address disease intricacy but also exploit new therapeutic mechanisms and assess undruggable targets for conventional monospecific biologics. This review focuses on the recent advances in molecular design and applications of major classes of multispecific biotherapeutics drugs, which include immune cells engagers, antibody-drug conjugates, multispecific tetherbodies, biologic matchmakers, and small-scaffold multispecific modalities. Challenges posed by the multispecific biotherapeutics drugs and their future outlooks are also discussed.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Automated pH and conductivity conditioning using feedback control to support a two-step continuous purification process.

As discovery research organizations push more molecules and new modalities through their company pipelines, there comes a need to widen purification development and production bandwidth by increasing automation and throughput. Continuous processing technologies have the unique property of reducing manufacturing floor space and reducing costs. We can speed development and production by implementing automation and continuous process technologies early in discovery research. Here we describe an automated continuous instrument made up of an ÄKTA™ pcc for initial capture by protein A, an ÄKTA pure 150 retrofitted to automatically condition protein A eluate, and a second ÄKTA pure 150 built for flow-through anion exchange chromatography. The continuous instrument we have designed and built recirculates protein A eluate from the ÄKTA pcc in a closed loop while signals from the pH and conductivity meters direct addition of titrant for accurate and precise adjustments to the pH and salt concentration. The instrument is run without user intervention and can be used continuously for production or for development as a tool for screening running conditions on the anion exchange step.

Modeling and mitigation of high-concentration antibody viscosity through structure-based computer-aided protein design.

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins.

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019.

Parallel loading and complete automation of a 3-step mAb purification process for multiple samples using a customized preparative chromatography instrument with networked pumps.

Advancement in high-throughput screening methods of novel therapeutic proteins for early stage research and development, specifically mAbs, have given mid-scale (milligram to gram scale) purification groups access to more of these molecules. The available purification technologies built to support mid-scale production was not efficient or versatile enough to keep up with this surge. To remedy this problem, we have designed and built a custom instrument using an ÄKTA Pure. This system enables parallel processing up to 5 samples and has the versatility to perform 1- to 3-step purification processes in a single queue. Furthermore, a unique purification scheme can be selected for each of the five samples in the queue. Overall processing time has reduced by 83% compared to manual, non-parallel load methods. Here, we describe our novel approach and demonstrate the flexibility, speed and efficiency of the instrument.

A cannabinoid receptor 1 mutation proximal to the DRY motif results in constitutive activity and reveals intramolecular interactions involved in receptor activation.

Activation of a G-protein-coupled receptor involves changes in specific microdomain interactions within the transmembrane region of the receptor. Here, we have focused on the role of L207, proximal to the DRY motif of the human cannabinoid receptor 1 in the interconversion of the receptor resting and active states. Ligand binding analysis of the mutant receptor L207A revealed an enhanced affinity for agonists (three- to six-fold) and a diminished affinity for inverse agonists (19- to 35-fold) compared to the wild-type receptor, properties characteristic of constitutive activity. To further examine whether this mutant adopts a ligand-independent, active form, treatment with GTPgammaS was used to inhibit G protein coupling. Under these conditions, the L207A receptor exhibited a 10-fold increase in affinity for the inverse agonist SR141716A, consistent with a shift away from an enhanced precoupled state. Analysis of the cellular activity of the L207A receptor showed elevated basal cyclic AMP accumulation relative to the wild type that is inhibited by SR141716A, consistent with receptor-mediated Gs precoupling. Using toxins to selectively abrogate Gs or Gi coupling, we found that CP55940 nonetheless induced only a Gi response suggesting a strong preference of this ligand-bound form for Gi in this system. Molecular dynamics simulations reveal that the single residue change of L207A impacts the association of TM3 and TM6 in the receptor by altering hydrophobic interactions involving L207, the salt bridge involving the Arg of the DRY motif, and the helical structure of TM6, consistent with events leading to activation. The structural alterations parallel those observed in models of a mutant CB(1) receptor T210I, with established constitutive activity (D'Antona, A.M., Ahn, K.H. and Kendall, D.A., 2006. Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry, 45, 5606-5617).

Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization.

Human cannabinoid receptor 1 (CB(1)) has attracted substantial interest as a potential therapeutic target for treating obesity and other obsessive disorders. An understanding of the mechanism governing the transition of the CB(1) receptor between its inactive and active states is critical for understanding how therapeutics can selectively regulate receptor activity. We have examined the importance of the Thr at position 210 in CB(1) in this transition, a residue predicted to be on the same face of the helix as the Arg of the DRY motif highly conserved in the G protein-coupled receptor superfamily. This Thr was substituted with Ile and Ala via mutagenesis, and the receptors, T210I and T210A, were expressed in HEK 293 cells. The T210I receptor exhibited enhanced agonist and diminished inverse agonist affinity relative to the wild type, consistent with a shift toward the active form. However, treatment with GTPgammaS to inhibit G protein coupling diminished the affinity change for the inverse agonist SR141716A. The decreased thermal stability of the T210I receptor and increased level of internalization of a T210I receptor-GFP chimera were also observed, consistent with constitutive activity. In contrast, the T210A receptor exhibited the opposite profile: diminished agonist and enhanced inverse agonist affinity. The T210A receptor was found to be more thermally stable than the wild type, and high levels of a T210A receptor-GFP chimera were localized to the cell surface as predicted for an inactive receptor form. These results suggest that T210 plays a key role in governing the transition between inactive and active CB(1) receptor states.

Membrane assembly of the cannabinoid receptor 1: impact of a long N-terminal tail.

The human cannabinoid receptor 1 (CB1) belongs to the G protein-coupled receptor (GPCR) family. Among the members of GPCR family, it has an exceptionally long extracellular N-terminal domain (N-tail) of 116 amino acids but has no typical signal sequence. This poses questions of how the long N-tail affects the biosynthesis of the receptor and of how it is inserted into the endoplasmic reticulum (ER) membrane. Here we have examined the process of membrane assembly of CB1 in the ER membrane and the maturation of the receptor from the ER to the plasma membrane. We find that the long N-tail cannot be efficiently translocated across the ER membrane, causing the rapid degradation of CB1 by proteasomes; this leads to a low level of expression of the receptor at the plasma membrane. The addition of a signal peptide at the N terminus of CB1 or shortening of the long N-tail greatly enhances the stability and cell surface expression of the receptor without affecting receptor binding to a cannabinoid ligand, CP-55,940. We propose that the N-tail translocation is a crucial early step in biosynthesis of the receptor and may play a role in regulating the stability and surface expression of CB1.