David Rabuka

Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag

The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Cotranslational modification of the proteins by FGE produced products bearing a unique aldehyde group. Proteins bearing this “aldehyde tag” were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. We applied the technique to site-specific modification of monoclonal antibodies, the fastest growing class of biopharmaceuticals, as well as membrane-associated and cytosolic proteins expressed in mammalian cells.

Site-specific chemical protein conjugation using genetically encoded aldehyde tags

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

Noncovalent Cell Surface Engineering: Incorporation of Bioactive Synthetic Glycopolymers into Cellular Membranes

The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. Here we demonstrate that cell surfaces can be engineered to display synthetic bioactive polymers at defined densities by exogenous membrane insertion. The polymers were designed to mimic native cell-surface mucin glycoproteins, which are defined by their dense glycosylation patterns and rod-like structures. End-functionalization with a hydrophobic anchor permitted incorporation into the membranes of live cultured cells. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Their diffusion properties mirrored those of many natural membrane-associated biomolecules. Furthermore, the membrane-bound glycopolymers were internalized into early endosomes similarly to endogenous membrane components and were capable of specific interactions with protein receptors. This system provides a platform to study cell-surface phenomena with a degree of chemical control that cannot be achieved using conventional biological tools.

Synthesis of Heterobifunctional Protein Fusions Using Copper-Free Click Chemistry and the Aldehyde Tag

Heterobifunctional protein fusions are gaining interest as next-generation biopharmaceuticals.1–5 Combining proteins with disparate functions can enable multidrug therapy with a single chemical entity,6, 7 add a targeting element to an otherwise nonspecific therapeutic,8, 9 or improve the pharmacokinetic profile of a rapidly cleared molecule.10, 11 Indeed, heterobifunctional proteins, such as immunoglobulin G (IgG) Fc domain fusions, are among the top-selling biotherapeutics on the market today.12 These biomolecules are primarily generated as genetic fusions. The DNA sequences that encode the individual protein components are fused in tandem to direct the expression of a single polypeptide that comprises the two proteins joined together at their N and C termini, respectively. However, this limited topology is not ideal for every protein combination, as some polypeptides require unmodified termini for optimal bioactivity13 or can suffer from expression difficulties as a result of folding and processing issues.3, 14, 15

Aldehyde Tag Coupled with HIPS Chemistry Enables the Production of ADCs Conjugated Site-Specifically to Different Antibody Regions with Distinct in Vivo Efficacy and PK Outcomes

It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody–drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C–C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

Synthesis and Microcontact Printing of Dual End-Functionalized Mucin-like Glycopolymers for Microarray Applications†

Click to view: Glycopolymers can be used to display glycans on microarrays in native-like architectures. The structurally uniform alkyne-terminated mucin mimetic glycopolymers (see picture; TR = fluorophore) were printed on azide-functionalized chips by microcontact printing in the presence of a copper catalyst. The surface-bound glycopolymers bind lectins in a ligand-specific manner.

Hydrazino-Pictet-Spengler ligation as a biocompatible method for the generation of stable protein conjugates.

Aldehyde- and ketone-functionalized biomolecules have found widespread use in biochemical and biotechnological fields. They are typically conjugated with hydrazide or aminooxy nucleophiles under acidic conditions to yield hydrazone or oxime products that are relatively stable, but susceptible to hydrolysis over time. We introduce a new reaction, the hydrazino-Pictet-Spengler (HIPS) ligation, which has two distinct advantages over hydrazone and oxime ligations. First, the HIPS ligation proceeds quickly near neutral pH, allowing for one-step labeling of aldehyde-functionalized proteins under mild conditions. Second, the HIPS ligation product is very stable (>5 days) in human plasma relative to an oxime-linked conjugate (∼1 day), as demonstrated by monitoring protein-fluorophore conjugates by ELISA. Thus, the HIPS ligation exhibits a combination of product stability and speed near neutral pH that is unparalleled by current carbonyl bioconjugation chemistries.

Saccharide Display on Microtiter Plates

New insight into the importance of carbohydrates in biological systems underscores the need for rapid synthetic and screening procedures for them. Development of an organic synthesis-compatible linker that would attach saccharides to microtiter plates was therefore undertaken to facilitate research in glycobiology. Galactosyllipids containing small, hydrophobic groups at the anomeric position were screened for noncovalent binding to microtiter plates. When the lipid component was a saturated hydrocarbon between 13 and 15 carbons in length, the monosaccharide showed complete retention after aqueous washing and could be utilized in biological assays. This alkyl chain was also successfully employed with more complex oligosaccharides in biological assays. In light of these findings, this method of attachment of oligosaccharides to microtiter plates should be highly efficacious to high-throughput synthesis and analyses of carbohydrates in biological assays.

Synthetic Trehalose Glycolipids Confer Desiccation Resistance to Supported Lipid Monolayers

Lipid-derived desiccation resistance in membranes is a rare, unique ability previously observed only with trehalose dimycolate (TDM), an abundant mycobacterial glycolipid. Here we present the first synthetic trehalose glycolipids capable of providing desiccation protection to membranes of which they are constituents. The synthetic glycolipids consist of a simple trehalose disaccharide headgroup, similar to TDM, with hydrophobic tail groups of two 15- or 18-carbon chains. The synthetic trehalose glycolipids protected supported monolayers of phospholipids against dehydration even as minority components of the overall membrane, down to as little as 20 mol % trehalose glycolipid as assessed by assays of membrane fluidity. The dependence of the desiccation protection on the synthetic trehalose glycolipid fraction is nearly identical to that of TDM. The striking similarity of the desiccation resistance observed with TDM and the synthetic trehalose glycolipids, despite the variety of hydrophobic tail structures employed, suggests that interactions between the trehalose headgroup and surrounding molecules are the determining factor in dehydration protection.

Control of the Molecular Orientation of Membrane-Anchored Biomimetic Glycopolymers

Quantifying and controlling the orientation of surface-bound macromolecules is crucial to a wide range of processes in areas as diverse as biology, materials science, and nanotechnology. Methods capable of directing orientation, as well as an understanding of the underlying physical mechanisms are, however, lacking. In this paper, we describe experiments in which the conformations of structurally well-defined polymers anchored to fluid lipid membranes were probed using Fluorescence Interference Contrast Microscopy (FLIC), an optical technique that provides topographic information with few-nanometer precision. The novel rodlike polymers mimic the architecture of mucin glycoproteins and feature a phospholipid tail for membrane incorporation and a fluorescent optical probe for FLIC imaging situated at the opposite termini of the densely glycosylated polymeric backbones. We find that the orientation of the rigid, approximately 30 nm long glycopolymers depends profoundly on the properties of the optical reporter. Molecules terminated with Alexa Fluor 488 projected away from the lipid bilayer by 11 ± 1 nm, consistent with entropy-dominated sampling of the membrane-proximal space. Molecules terminated with Texas Red lie flat at the membrane (height, 0 ± 2 nm), implying that interactions between Texas Red and the bilayer dominate the polymers’ free energy. These results demonstrate the design of macromolecules with specific orientational preferences, as well as nanometer-scale measurement of their orientation. Importantly, they reveal that seemingly minute changes in molecular structure, in this case fluorophores that comprise only 2% of the total molecular weight, can significantly alter the molecule’s presentation to the surrounding environment.