Intravascular hemolysis releases hemoglobin into the bloodstream, which can damage vascular and renal tissues due to its oxidative nature. Circulating haptoglobin acts as a primary defense by binding to free hemoglobin, forming a haptoglobin-hemoglobin (HpHb) complex that is then recognized and cleared by the CD163 scavenger receptor on macrophages. While the function and structure of HpHb complex are mostly well-defined, the molecular mechanism underlying its interaction with CD163 remains unclear. Here we report the cryo-electron microscopy structures of human CD163 in its unliganded state and in its complex with HpHb. These structures reveal that CD163 functions as a trimer, forming a composite binding site at its center for one protomer of the dimeric HpHb, resulting in a 3:1 binding stoichiometry. In the unliganded state, CD163 can also form a trimer, but in an autoinhibitory configuration that occludes the ligand binding site. Widespread electrostatic interactions mediated by calcium ions are pivotal in both pre-ligand and ligand-bound receptor assemblies. This calcium-dependent mechanism enables CD163/HpHb complexes to assemble and, once internalized, disassemble into individual components upon reaching the endosome, where low calcium and lower pH conditions prevail. Collectively, this study elucidates the molecular mechanism by which CD163-mediated endocytosis efficiently clears different isoforms of HpHb.

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.

Next-generation site-specific cysteine-based antibody–drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.

Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid profile improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using a combination of N-glycan engineering for enhanced protease resistance and improved solubility, Fc fusion for further half-life extension, and a single point mutation for improving manufacturability in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability profile that is compatible with subcutaneous (SC) administration. In particular, it showed a low systemic clearance (0.243 mL/hr/kg) and long terminal half-life (~200 hours for intact protein) in cynomolgus monkeys that approaches those of monoclonal antibodies. Furthermore, the superior PK properties translated into robust improvement in glucose tolerance and the effects lasted 14 days post single SC dose in ob/ob mice. PF-06645849 also caused greater body weight loss in DIO mice at lower and less frequent SC doses, compared to previous FGF21 analogue PF-05231023. In summary, the overall PK/PD and pharmaceutical profile of PF-06645849 offers great potential for development as weekly to twice-monthly SC administered therapeutic for chronic treatment of metabolic diseases.

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.

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.

Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme's poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation.

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.

Background:Antibody v-domains in scFv format often suffer from aggregation and stability issues that restrict formulation.Results:Structural and empirical analyses of an optimized scFv revealed that three VL-CDR3 mutations were sufficient to mediate significant stability and affinity improvements.Conclusion:scFv issues were resolved via removal of side-chain clashes at the VL/VH interface.Significance:CDR-restricted mutagenesis delivers stability-optimized molecules for high concentration dosing.Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.

This investigation used in vivo and in vitro tools to study pharmacokinetics and glycosylation of two monomeric antibodies produced either transiently by HEK293 cells or stably by Chinese hamster ovary cells, and demonstrated that higher in vivo clearance of human embryonic kidney antibody was due to higher glycosylation, thus higher mannose receptor mediated uptake.

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and >18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with <2% high molecular weight species present after 7 weeks at 4 °C and viscosity <15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

estrogen or neutralizing IL-6 monoclonal antibodies (Jilka William Somers1, Mark Stahl and et al., 1992). Mutant mice lacking IL-6 have normal Jasbir S.Seehra amounts of trabecular bone and ovariectomy does not Small Molecule Drug Discovery, Genetics Institute, Inc., cause bone loss or a change in the rate of remodeling 87 Cambridge Park Drive, Cambridge, MA 02140, USA (Poli et al., 1994). These studies strongly suggest that 1Corresponding author IL-6 plays an important role in post-menopausal bone loss. IL-6 belongs to a family which share remarkably similar Interleukin 6 (IL-6) has many biological activities structural features both for the cytokines and the receptors in vivo, and deregulation has been implicated in many to which they bind. These similarities also extend to the disease processes. IL-6, a 185 amino acid polypeptide sequential clustering events leading to signal transduction. was refolded, purified and crystallized. The crystals The closest members of the family include leukemia diffracted to beyond 1.9 Å and the structure was solved inhibitory factor (LIF), cillary neurotrophic factor (CNTF), using single isomorphous replacement. The X-ray oncostatin M and IL-11 (Yamasaki et al., 1988; Davis structure of IL-6 is composed of a four helix bundle et al., 1991; Gearing et al., 1991; Kishimoto et al., 1992, linked by loops and an additional mini-helix. 157 out 1994; Miyajima et al., 1992). IL-6 receptor consists of 185 residues are well defined in the final structure, of two polypeptides: the α chain (IL-6r), an 80 kDa with 18 N-terminal and 8 A–B loop amino acids transmembrane glycoprotein that binds IL-6 with low displaying no interpretable electron density. The threeaffinity, and the β chain (gp130), a 130 kDa transmembrane dimensional structure has been used to construct a glycoprotein that binds to the IL-6–IL-6r heterodimer to model of IL-6 interacting with the IL-6 receptor form the high affinity signal transducing complex (Taga (α-chain) and gp130 (β-chain) that gives new insight et al., 1989). The IL-6r is a transmembrane protein into the process of molecular recognition and signaling. composed of a cytokine binding type I domain (necessary Based on this model, we predict a fourth binding site and sufficient for binding IL-6; Yawata et al., 1993), an on IL-6, a low affinity IL-6–IL-6 interaction, which Ig-like domain and a short cytosolic domain (Yamasaki may be necessary for the sequential assembly of a et al., 1988) that is not required for signaling (Taga et al., functional hexameric IL-6 receptor complex. 1989). gp130 is also a transmembrane protein composed

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C(H)3-C(H)3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.

Glycosylation is one of the most complex post-translation modifications, commonly found in many cell surface and secreted eukaryotic proteins. 1-2% of the human transcriptome encodes proteins that link to glycosylation. Many protein-based biotherapeutics approved or in clinical trials are glycoproteins. The oligosaccharides covalently attached to therapeutic glycoproteins pose biological benefits as well as manufacturing challenges. The present chapter reviews the structure and function of glycosylation, glycoform patterns observed for the biotherapeutic proteins produced by various host systems, and analytic methods for the characterization of glycoforms. Recent advances in utilizing glycosylation as a strategy to improve biotherapeutics properties are also discussed.