RESUMEN
Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Antineoplásicos , Benzodiazepinas , Neoplasias de la Mama , Inmunoconjugados , Humanos , Animales , Femenino , Neoplasias de la Mama/genética , Macaca fascicularis/genética , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , ADNRESUMEN
Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.
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Proteómica , Espectrometría de Masas en Tándem , Humanos , Estreptavidina , Reproducibilidad de los Resultados , Péptidos/metabolismo , Anticuerpos , Epítopos de Linfocito T , Desarrollo de MedicamentosRESUMEN
Polatuzumab vedotin (or POLIVY®), an antibody-drug conjugate (ADC) composed of a polatuzumab monoclonal antibody conjugated to monomethyl auristatin E (MMAE) via a cleavable dipeptide linker, has been approved by the United States Food and Drug Administration (FDA) for the treatment of diffuse large B-cell lymphoma (DLBCL). To support the clinical development of polatuzumab vedotin, we characterized the distribution, catabolism/metabolism, and elimination properties of polatuzumab vedotin and its unconjugated MMAE payload in Sprague Dawley rats. Several radiolabeled probes were developed to track the fate of different components of the ADC, with 125I and 111In used to label the antibody component and 3H to label the MMAE payload of the ADC. Following a single intravenous administration of the radiolabeled probes into normal or bile-duct cannulated rats, blood, various tissues, and excreta samples were collected over 7-14 days post-dose and analyzed for radioactivity and to characterize the metabolites/catabolites. The plasma radioactivity of polatuzumab vedotin showed a biphasic elimination profile similar to that of unconjugated polatuzumab but different from unconjugated radiolabeled MMAE, which had a fast clearance. The vast majority of the radiolabeled MMAE in plasma remained associated with antibodies, with a minor fraction as free MMAE and MMAE-containing catabolites. Similar to unconjugated mAb, polatuzumab vedotin showed a nonspecific distribution to multiple highly perfused organs, including the lungs, heart, liver, spleen, and kidneys, where the ADC underwent catabolism to release MMAE and other MMAE-containing catabolites. Both polatuzumab vedotin and unconjugated MMAE were mainly eliminated through the biliary fecal route (>90%) and a small fraction (<10%) was eliminated through renal excretion in the form of catabolites/metabolites, among which, MMAE was identified as the major species, along with several other minor species. These studies provided significant insight into ADC's absorption, distribution, metabolism, and elimination (ADME) properties, which supports the clinical development of POLIVY.
RESUMEN
Invasive Staphylococcus aureus infection is a leading cause of infectious disease-related deaths because S. aureus survives within host phagocytic cells, from which the bacteria are not adequately eliminated using current antibiotic treatments. Anti-S. aureus THIOMAB antibody-antibiotic conjugate (TAC), an anti-S. aureus antibody conjugated with antibiotic payload dmDNA31, was designed to deliver antibiotics into phagocytes, thereby killing intracellular S. aureus Herein, we present the distribution, metabolism/catabolism, and elimination properties for this modality. The tissue distribution of TAC and the release and elimination of its payload dmDNA31 were characterized in rats using multiple approaches. Intravenous injection of unconjugated [14C]dmDNA31 to rats resulted in a rapid clearance in both systemic circulation and tissues, with biliary secretion as the major route of elimination. Six major metabolites were identified. When [14C]dmDNA31 was conjugated to an antibody as TAC and administered to rat intravenously, a sustained exposure was observed in both systemic circulation and tissues. The dmDNA31 in blood and tissues mainly remained in conjugated form after administering TAC, although minimal deconjugation of dmDNA31 from TAC was also observed. Several TAC catabolites were identified, which were mainly eliminated through the biliary-fecal route, with dmDNA31 and deacetylated dmDNA31 as the most abundant catabolites. In summary, these studies provide a comprehensive characterization of the distribution, metabolism/catabolism, and elimination properties of TAC. These data fully support further clinical development of TAC for the invasive and difficult-to-treat S. aureus infection. SIGNIFICANCE STATEMENT: The present studies provide a comprehensive investigation of the absorption, distribution, metabolism/catabolism, and elimination of the first antibody-antibiotic conjugate developed for the treatment of an infectious disease. Although many antibody-drug conjugates are in development for various disease indications, only a limited amount of absorption, distribution, metabolism/catabolism, and elimination information is available in the literature. This study demonstrates the use of radiolabeling technology to delineate the absorption, distribution, metabolism/catabolism, and elimination properties of a complex modality and help address the key questions related to clinical pharmacological studies.
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Antibacterianos/farmacocinética , Anticuerpos Antibacterianos/farmacología , Inmunoconjugados/farmacocinética , Animales , Antibacterianos/administración & dosificación , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Inyecciones Intravenosas , Masculino , Modelos Animales , Ratas , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunología , Distribución TisularRESUMEN
Immunoaffinity (IA) LC-MS/MS pharmacokinetic (PK) assays are widely used in the field for antibody drug conjugates (ADCs) containing peptide linkers that are enzymatically cleavable, such as MC-ValCit-PAB. Conjugate PK assay strategies for these ADCs involve cleavage with cathepsin B or papain to release and measure the antibody-conjugated drug (acDrug) concentration. However, robust acDrug PK methods for disulfide-linked self-immolating ADCs are lacking as they are a different conjugation modality. We developed acDrug PK assays for next-generation disulfide-linked ADCs involving immunoaffinity capture, chemical cleavage, and LC-MS/MS. Disulfide-linked ADCs captured from plasma were chemically reduced at basic pH to release the linker-drug, followed by self-immolation to liberate the active drug, and quantified by MRM LC-MS/MS. Herein, we detail the development and optimization of this chemical cleavage acDrug PK assay, resulting in robust accuracy and precision (±20%). The conjugation site of the linker-drug on the antibody was found to affect the kinetics of drug release. Multiple biophysical and chemical characteristics, such as tertiary structure, fractional solvent accessibility, pKa of the conjugation site, surrounding residue's pI, and electrostatic charge, may directly impact the drug release kinetics. Similar site-specific stability has been previously reported for ADCs in vivo. The assay development and qualification data for this original assay format are presented along with its application to multiple in vitro and in vivo studies across species.
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Anticuerpos Monoclonales/farmacocinética , Disulfuros/farmacocinética , Inmunoconjugados/farmacocinética , Anticuerpos Monoclonales/análisis , Cromatografía Liquida , Disulfuros/análisis , Humanos , Inmunoensayo , Inmunoconjugados/análisis , Estructura Molecular , Espectrometría de Masas en TándemRESUMEN
This work discloses the first examples of antibody-drug conjugates (ADCs) that are constructed from linker-drugs bearing dimeric seco-CBI payloads (duocarmycin analogs). Several homogeneous, CD22-targeting THIOMAB antibody-drug conjugates (TDCs) containing the dimeric seco-CBI entities are shown to be highly efficacious in the WSU-DLCL2 and BJAB mouse xenograft models. Surprisingly, the seco-CBI-containing conjugates are also observed to undergo significant biotransformation in vivo in mice, rats, and monkeys and thereby form 1:1 adducts with the Alpha-1-Microglobulin (A1M) plasma protein from these species. Variation of both the payload mAb attachment site and length of the linker-drug is shown to alter the rates of adduct formation. Subsequent experiments demonstrated that adduct formation attenuates the in vitro antiproliferation activity of the affected seco-CBI-dimer TDCs, but does not significantly impact the in vivo efficacy of the conjugates. In vitro assays employing phosphatase-treated whole blood suggest that A1M adduct formation is likely to occur if the seco-CBI-dimer TDCs are administered to humans. Importantly, protein adduct formation leads to the underestimation of total antibody (Tab) concentrations using an ELISA assay but does not affect Tab values determined via an orthogonal LC-MS/MS method. Several recommendations regarding bioanalysis of future in vivo studies involving related seco-CBI-containing ADCs are provided based on these collective findings.
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alfa-Globulinas/química , Antineoplásicos/farmacología , Inmunoconjugados/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Haplorrinos , Humanos , Inmunoconjugados/química , Ratones , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.
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Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/metabolismo , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Área Bajo la Curva , Benzodiazepinas/inmunología , Benzodiazepinas/uso terapéutico , Humanos , Inmunoconjugados/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Lectinas Tipo C/inmunología , Leucemia Mieloide/sangre , Macaca fascicularis , Tasa de Depuración Metabólica , Ratones , Pirroles/inmunología , Pirroles/uso terapéutico , Ratas , Receptores Mitogénicos/inmunología , Especificidad de la EspecieRESUMEN
Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology.
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Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Aptitud Genética , Proteínas Tirosina Fosfatasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Cloruro de Sodio/metabolismo , Estrés FisiológicoRESUMEN
We modified a dual-cell linear ion trap mass spectrometer to perform infrared multiphoton dissociation (IRMPD) in the low-pressure trap of a dual-cell quadrupole linear ion trap (dual-cell QLT) and perform large-scale IRMPD analyses of complex peptide mixtures. Upon optimization of activation parameters (precursor q-value, irradiation time, and photon flux), IRMPD subtly, but significantly, outperforms resonant-excitation collisional-activated dissociation (CAD) for peptides identified at a 1% false-discovery rate (FDR) from a yeast tryptic digest (95% confidence, p = 0.019). We further demonstrate that IRMPD is compatible with the analysis of isobaric-tagged peptides. Using fixed QLT rf amplitude allows for the consistent retention of reporter ions, but necessitates the use of variable IRMPD irradiation times, dependent upon precursor mass to charge (m/z). We show that IRMPD activation parameters can be tuned to allow for effective peptide identification and quantitation simultaneously. We thus conclude that IRMPD performed in a dual-cell ion trap is an effective option for the large-scale analysis of both unmodified and isobaric-tagged peptides.
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Rayos Infrarrojos , Proteómica , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Bases de Datos Factuales , Espectrometría de Masas , Péptidos/análisis , FotonesRESUMEN
We describe a mass spectrometry method, QuantMode, which improves accuracy of isobaric tag-based quantification by alleviating the pervasive problem of precursor interference, simultaneous isolation and fragmentation of impurities, through gas-phase purification. QuantMode analysis of a yeast sample 'contaminated' with interfering human peptides showed substantially improved quantitative accuracy compared to a standard scan, with a small loss of spectral identifications. This technique enables large-scale, multiplexed quantitative proteomics using isobaric tagging.
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Gases , Proteínas/química , Proteoma , Humanos , Espectrometría de Masas en Tándem/métodosRESUMEN
The transcriptome and proteome change dynamically as cells respond to environmental stress; however, prior proteomic studies reported poor correlation between mRNA and protein, rendering their relationships unclear. To address this, we combined high mass accuracy mass spectrometry with isobaric tagging to quantify dynamic changes in ~2500 Saccharomyces cerevisiae proteins, in biological triplicate and with paired mRNA samples, as cells acclimated to high osmolarity. Surprisingly, while transcript induction correlated extremely well with protein increase, transcript reduction produced little to no change in the corresponding proteins. We constructed a mathematical model of dynamic protein changes and propose that the lack of protein reduction is explained by cell-division arrest, while transcript reduction supports redistribution of translational machinery. Furthermore, the transient 'burst' of mRNA induction after stress serves to accelerate change in the corresponding protein levels. We identified several classes of post-transcriptional regulation, but show that most of the variance in protein changes is explained by mRNA. Our results present a picture of the coordinated physiological responses at the levels of mRNA, protein, protein-synthetic capacity, and cellular growth.
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Perfilación de la Expresión Génica/métodos , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatografía Liquida , Regulación Fúngica de la Expresión Génica , Modelos Teóricos , Análisis de Secuencia por Matrices de Oligonucleótidos , Concentración Osmolar , Proteoma/genética , Proteómica/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Masas en TándemRESUMEN
Using a large set of high mass accuracy and resolution ETD tandem mass spectra, we characterized ETD-induced neutral losses. From these data we deduced the chemical formula for 20 of these losses. Many of them have been previously observed in electron-capture dissociation (ECD) spectra, such as losses of the side chains of arginine, aspartic acid, glutamic acid, glutamine, asparagine, leucine, histidine, and carbamidomethylated cysteine residues. With this information, we examined the diagnostic value of these amino acid-specific losses. Among 1285 peptide-spectrum matches, 92.5% have agreement between neutral loss-derived peptide amino acid composition and the peptide sequences. Moreover, we show that peptides can be uniquely identified by using only the accurate precursor mass and amino acid composition based on neutral losses; the median number of sequence candidates from an accurate mass query is reduced from 21 to 8 by adding side chain loss information. Besides increasing confidence in peptide identification, our findings suggest the potential use of these diagnostic losses in ETD spectra to improve false discovery rate estimation and to enhance the performance of scoring functions in database search algorithms.
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Aminoácidos/química , Fragmentos de Péptidos/química , Espectrometría de Masas en Tándem/métodos , Aminoácidos/metabolismo , Cationes/química , Extractos Celulares , Bases de Datos de Proteínas , Electrones , Células Madre Embrionarias , Humanos , Metaloendopeptidasas/metabolismo , Fragmentos de Péptidos/metabolismoRESUMEN
Here we present the Coon OMSSA Proteomic Analysis Software Suite (COMPASS): a free and open-source software pipeline for high-throughput analysis of proteomics data, designed around the Open Mass Spectrometry Search Algorithm. We detail a synergistic set of tools for protein database generation, spectral reduction, peptide false discovery rate analysis, peptide quantitation via isobaric labeling, protein parsimony and protein false discovery rate analysis, and protein quantitation. We strive for maximum ease of use, utilizing graphical user interfaces and working with data files in the original instrument vendor format. Results are stored in plain text comma-separated value files, which are easy to view and manipulate with a text editor or spreadsheet program. We illustrate the operation and efficacy of COMPASS through the use of two LC-MS/MS data sets. The first is a data set of a highly annotated mixture of standard proteins and manually validated contaminants that exhibits the identification workflow. The second is a data set of yeast peptides, labeled with isobaric stable isotope tags and mixed in known ratios, to demonstrate the quantitative workflow. For these two data sets, COMPASS performs equivalently or better than the current de facto standard, the Trans-Proteomic Pipeline.
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Algoritmos , Proteómica/estadística & datos numéricos , Programas Informáticos , Cromatografía Liquida , Biología Computacional , Interpretación Estadística de Datos , Bases de Datos de Proteínas/estadística & datos numéricos , Proteínas/aislamiento & purificación , Proteínas/normas , Proteómica/métodos , Proteómica/normas , Estándares de Referencia , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Espectrometría de Masas en Tándem/estadística & datos numéricosRESUMEN
Using a newly developed dual-cell quadrupole linear ion trap-orbitrap hybrid mass spectrometer (dcQLT-orbitrap), we demonstrate the utility of collecting high-resolution tandem mass spectral data for large-scale shotgun proteomics. Multiple nanoLC-MS/MS experiments on both an older generation quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) and the dcQLT-orbitrap, using both resonant-excitation CAD and beam-type CAD (HCD), were performed. Resulting from various technological advances (e.g., a stacked ring ion guide AP inlet, a dual cell QLT), the dcQLT-orbitrap exhibited increased duty cycle (approximately 1.5-2 times) and sensitivity for both CAD (ion trap detection) and HCD (orbitrap detection) methods. As compared to the older system, the dcQLT-orbitrap produced significantly more unique peptide identifications for both methods (approximately 30% improvement for CAD and approximately 115% improvement for HCD). The sizable improvement of the HCD method on the dcQLT-orbitrap system outperforms the current standard method of CAD with ion trap detection for large-scale analysis. Finally, we demonstrate that the increased HCD performance translates to a direct and substantial improvement in protein quantitation precision using isobaric tags.
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Análisis de Fourier , Proteínas/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
The lantibiotic synthetases LctM and HalM2 are bifunctional enzymes that catalyze both the dehydration of serine and threonine residues and the Michael-type additions of cysteine residues to the resulting dehydroamino acids in their substrate peptides. Using Fourier transform mass spectrometry to analyze these activities in vitro, the dehydration is shown to take place by a distributive mechanism, with build-up of intermediates observed in electrospray mass spectra. The cyclization activity of HalM2 was monitored through alkylation of free cysteines in intermediates, providing access to the regioselectivity of lanthionine ring formation using high-resolution tandem mass spectrometry. HalM2 is shown to catalyze the cyclization process in a largely N- to C-terminal directional fashion, forming a total of four lanthionine rings in its HalA2 substrate. These studies advance a model for lantibiotic production where substrate binding via an N-terminal leader results in dehydration and cyclization on similar time scales and with a high, though not strict, propensity for N-to-C directionality.