Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation.

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.

Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is an established, powerful tool for investigating protein-ligand interactions, protein folding, and protein dynamics. However, HDX-MS is still an emergent tool for quality control of biopharmaceuticals and for establishing dynamic similarity between a biosimilar and an innovator therapeutic. Because industry will conduct quality control and similarity measurements over a product lifetime and in multiple locations, an understanding of HDX-MS reproducibility is critical. To determine the reproducibility of continuous-labeling, bottom-up HDX-MS measurements, the present interlaboratory comparison project evaluated deuterium uptake data from the Fab fragment of NISTmAb reference material (PDB: 5K8A ) from 15 laboratories. Laboratories reported ∼89 800 centroid measurements for 430 proteolytic peptide sequences of the Fab fragment (∼78 900 centroids), giving ∼100% coverage, and ∼10 900 centroid measurements for 77 peptide sequences of the Fc fragment. Nearly half of peptide sequences are unique to the reporting laboratory, and only two sequences are reported by all laboratories. The majority of the laboratories (87%) exhibited centroid mass laboratory repeatability precisions of ⟨ sLab⟩ ≤ (0.15 ± 0.01) Da (1σx̅). All laboratories achieved ⟨sLab⟩ ≤ 0.4 Da. For immersions of protein at THDX = (3.6 to 25) °C and for D2O exchange times of tHDX = (30 s to 4 h) the reproducibility of back-exchange corrected, deuterium uptake measurements for the 15 laboratories is σreproducibility15 Laboratories( tHDX) = (9.0 ± 0.9) % (1σ). A nine laboratory cohort that immersed samples at THDX = 25 °C exhibited reproducibility of σreproducibility25C cohort( tHDX) = (6.5 ± 0.6) % for back-exchange corrected, deuterium uptake measurements.

Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins.

Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy.

Both in vivo data in preclinical cancer models and in vitro data with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional properties in vivo.

Optimization of an Enzymatic Antibody-Drug Conjugation Approach Based on Coenzyme A Analogs.

Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.

Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Phosphopantetheinyl Transferases.

Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.

Estimation of Hydrogen-Exchange Protection Factors from MD Simulation Based on Amide Hydrogen Bonding Analysis.

Hydrogen exchange (HX) studies have provided critical insight into our understanding of protein folding, structure, and dynamics. More recently, hydrogen exchange mass spectrometry (HX-MS) has become a widely applicable tool for HX studies. The interpretation of the wealth of data generated by HX-MS experiments as well as other HX methods would greatly benefit from the availability of exchange predictions derived from structures or models for comparison with experiment. Most reported computational HX modeling studies have employed solvent-accessible-surface-area based metrics in attempts to interpret HX data on the basis of structures or models. In this study, a computational HX-MS prediction method based on classification of the amide hydrogen bonding modes mimicking the local unfolding model is demonstrated. Analysis of the NH bonding configurations from molecular dynamics (MD) simulation snapshots is used to determine partitioning over bonded and nonbonded NH states and is directly mapped into a protection factor (PF) using a logistics growth function. Predicted PFs are then used for calculating deuteration values of peptides and compared with experimental data. Hydrogen exchange MS data for fatty acid synthase thioesterase (FAS-TE) collected for a range of pHs and temperatures was used for detailed evaluation of the approach. High correlation between prediction and experiment for observable fragment peptides is observed in the FAS-TE and additional benchmarking systems that included various apo/holo proteins for which literature data were available. In addition, it is shown that HX modeling can improve experimental resolution through decomposition of in-exchange curves into rate classes, which correlate with prediction from MD. Successful rate class decompositions provide further evidence that the presented approach captures the underlying physical processes correctly at the single residue level. This assessment is further strengthened in a comparison of residue resolved protection factor predictions for staphylococcal nuclease with NMR data, which was also used to compare prediction performance with other algorithms described in the literature. The demonstrated transferable and scalable MD based HX prediction approach adds significantly to the available tools for HX-MS data interpretation based on available structures and models.

Isotope-Coded Labeling for Accelerated Protein Interaction Profiling Using MS.

Protein interaction surface mapping using MS is widely applied but comparatively resource-intensive. Here, a workflow adaptation for use of isotope-coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible.

Site-specific dual labeling of proteins by using small orthogonal tags at neutral pH.

To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FcεRI, which is a key event in allergic disease.

Feature based retention time alignment for improved HDX MS analysis.

An algorithm for retention time alignment of mass shifted hydrogen-deuterium exchange (HDX) data based on an iterative distance minimization procedure is described. The algorithm performs pairwise comparisons in an iterative fashion between a list of features from a reference file and a file to be time aligned to calculate a retention time mapping function. Features are characterized by their charge, retention time and mass of the monoisotopic peak. The algorithm is able to align datasets with mass shifted features, which is a prerequisite for aligning hydrogen-deuterium exchange mass spectrometry datasets. Confidence assignments from the fully automated processing of a commercial HDX software package are shown to benefit significantly from retention time alignment prior to extraction of deuterium incorporation values.

Subzero temperature chromatography for reduced back-exchange and improved dynamic range in amide hydrogen/deuterium exchange mass spectrometry.

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.

Fragmentation hydrogen exchange mass spectrometry: a review of methodology and applications.

The ability of proteins to function is intricately connected to proper folding and other environmental parameters. Methods and tools that are able to report back on structure and dynamics in native or otherwise desired environment are of utmost importance as they can be used to connect structure and function and provide us with deeper mechanistic understanding of the underlying principles involved. Besides, they might be useful in the verification of material quality and provide assurance of efficacy and soundness of experimental observations made with such materials. Hydrogen exchange mass spectrometry is one of those tools. It combines the universality of a "native" chemical labeling approach with an almost equally universally applicable detection scheme based on mass spectrometry that has already revolutionized proteomic research as a whole in the past. This review focuses on the concepts of the exchange method, advances in methodology that have made specifically the fragmentation hydrogen exchange mass spectrometry approach so successful, broadly surveys some of the application space, and highlights the most recent use in the area of biopharmaceutical development.

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197.

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.

Simultaneous purification and site-specific modification of pyrroline-carboxy-lysine proteins.

Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.

Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy poly(ethylene glycol) (mPEG) polymers.

Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs.

Site-specific protein modifications through pyrroline-carboxy-lysine residues.

Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.

D-Ornithine coopts pyrrolysine biosynthesis to make and insert pyrroline-carboxy-lysine.

D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.

Expanding the genetic repertoire of the methylotrophic yeast Pichia pastoris.

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.