Deciphering the Exact Sequence of Endogenous Soluble B Cell Maturation Antigen and Unbiased Quantitation in Multiple Myeloma Patient Samples by LC-MS.

BACKGROUND: B-cell maturation antigen is a pivotal therapeutic target for multiple myeloma (MM). Membrane-bound BCMA can be cleaved by γ-secretase and shed as soluble BCMA (sBCMA). sBCMA can act as a neutralizing sink to compete with drug, as well as serve as a diagnostic/prognostic biomarker for MM. Antibody-capture based methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoaffinity-liquid chromatography-multiple reaction monitoring (IA-LC-MRM), have been reported and well adopted to measure sBCMA in clinical samples. However, both methods are biased by capturing antibodies. METHODS: We have used various LC-MS workflows to characterize and quantify endogenous sBCMA in MM patient samples, including bottom-up peptide mapping, intact analysis, IA-based, and reagent-free (RF)-LC-MRM quantitation. RESULTS: We have confirmed that sBCMA contains a variable N-terminus and a C-terminus that extends to the transmembrane domain, ending at amino acid 61. Leveraging an in-house synthesized G-1-61 sBCMA recombinant standard, we developed a RF-LC-MRM method for unbiased sBCMA quantitation in MM patient samples. By comparing the results from RF-LC-MRM with ELISA and IA-LC-MRM, we demonstrated that RF-LC-MRM measures a more complete pool of endogenous sBCMA compared to the antibody-based methods. CONCLUSIONS: This work fills the knowledge gap of the exact sequence of endogenous sBCMA for the first time, which differs from the current commercially available standard. Additionally, this work highlights the necessity of identifying the actual sequence of an endogenous soluble target such as sBCMA, both for bioanalytical purposes and to underpin pharmacodynamic measurements.

Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs.

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability."

Absorption, Distribution, Metabolism, and Excretion of [14C]-Sotorasib in Rats and Dogs: Interspecies Differences in Absorption, Protein Conjugation and Metabolism.

Sotorasib is a first-in-class, targeted covalent inhibitor of Kirsten rat sarcoma viral oncogene homolog (KRAS)G12C approved by the FDA to treat patients with locally advanced or metastatic non-small cell lung cancer with the KRASG12C mutation. The mass balance, excretion, and metabolism of [14C]-sotorasib was characterized in rats and dogs after a single dose of 60 or 500 mg/kg, respectively. Mean recovery was >90% for both species. Excretion of unchanged sotorasib was a minor pathway in rats, accounting for <4% of administered dose in urine and <7% of administered dose in feces. Approximately 66% of administered dose was recovered in the bile from bile duct cannulated rats as metabolites. Excretion of unchanged sotorasib was the major excretion pathway in dogs, likely caused by solubility-limited absorption. Major pathways of sotorasib biotransformation included glutathione conjugation and oxidative metabolism. In vitro experiments demonstrated that nonenzymatic conjugation (Michael addition) was the primary mechanism of the reaction with glutathione. Extended radioactivity profiles in blood and plasma were observed in rats, but not dogs, after dosing with [14C]-sotorasib. In vitro experiments demonstrated that sotorasib-protein adducts were observed with both rat hemoglobin and serum albumin, explaining the extended radioactivity profile. SIGNIFICANCE STATEMENT: This study characterized the mass balance, excretion, and metabolism of [14C]-sotorasib, a covalent Kirsten rat sarcoma viral oncogene homolog G12C inhibitor, in rats and dogs. Rapid absorption and extensive metabolism of sotorasib was observed in rats, while sotorasib was primarily excreted unchanged in dog feces, likely due to solubility-limited absorption. Protein adducts with rat hemoglobin and serum albumin were characterized, explaining observed extended blood and plasma radioactivity profiles. The primary biotransformation pathway, glutathione conjugation, was mediated through nonenzymatic conjugation.

Preclinical development and phase 1 trial of a novel siRNA targeting lipoprotein(a).

Compelling evidence supports a causal role for lipoprotein(a) (Lp(a)) in cardiovascular disease. No pharmacotherapies directly targeting Lp(a) are currently available for clinical use. Here we report the discovery and development of olpasiran, a first-in-class, synthetic, double-stranded, N-acetylgalactosamine-conjugated small interfering RNA (siRNA) designed to directly inhibit LPA messenger RNA translation in hepatocytes and potently reduce plasma Lp(a) concentration. Olpasiran reduced Lp(a) concentrations in transgenic mice and cynomolgus monkeys in a dose-responsive manner, achieving up to over 80% reduction from baseline for 5-8 weeks after administration of a single dose. In a phase 1 dose-escalation trial of olpasiran (ClinicalTrials.gov: NCT03626662 ), the primary outcome was safety and tolerability, and the secondary outcomes were the change in Lp(a) concentrations and olpasiran pharmacokinetic parameters. Participants tolerated single doses of olpasiran well and experienced a 71-97% reduction in Lp(a) concentration with effects persisting for several months after administration of doses of 9 mg or higher. Serum concentrations of olpasiran increased approximately dose proportionally. Collectively, these results validate the approach of using hepatocyte-targeted siRNA to potently lower Lp(a) in individuals with elevated plasma Lp(a) concentration.

Multienzyme Kinetics and Sequential Metabolism.

Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYPs) enzymes are catalytically promiscuous and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appear to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein, is a discussion of two distinct type of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.

Case Study 12: Roadmap to Quantifying Ago2-Mediated siRNA Metabolic Activation Kinetics.

Therapeutic siRNA is a prodrug that requires Ago2-mediated site-specific hydrolysis of the sense strand before RNA interference can occur. Although this metabolic activation step was first described 15 years ago, the kinetics of this reaction, and its relationship to in vivo siRNA efficacy, remains unexplored in the literature. To provide a roadmap to address these gaps, we describe a liquid chromatography-mass spectrometry method to monitor formation of the cleaved sense-strand metabolites in a reconstituted system. In the absence of metabolite standards for quantitation, we apply an ionization efficiency correction across a panel of siRNA molecules and find that it improves in vitro-in vivo correlation in a transgenic mouse model. Finally, we lay out a case for why Michaelis-Menten kinetics will likely be inadequate for describing Ago2-mediated metabolic activation kinetics, and propose several alternative models that can be solved numerically and applied to quantitated kinetic data when it becomes available.

Universal Automated Immunoaffinity Purification-CE-MS Platform for Accelerating Next Generation Biologic Design.

As the pharmaceutical industry places greater emphasis on pairing biological pathways with appropriate therapeutic intervention, an increase in the use of biologic drugs has emerged. With increasing complexity of biotherapeutics, absorption, distribution, metabolism, and excretion (ADME) studies have also become increasingly complex. The characterization of ADME properties is critical to tuning the pharmacokinetic profiles of next generation biologics (NGBs). The knowledge of the fate of a drug is essential for the enhancement of the design processes, elongation of exposure at the desired site of action, and achieving efficacy with minimum toxicity. In vivo proteolytic cleavage of biotherapeutics may lead to undesirable in vivo properties, such as rapid clearance, low bioavailability, and loss of pharmacodynamic effect. All of these may affect drug efficacy and/or generate safety concerns through increases in immunogenicity or off-target toxicity. The work herein describes the development of a robust, fully automated immunoaffinity purification (IA)-capillary electrophoresis-mass spectrometry (CE-MS) workflow. The reagents were carefully optimized to maximize isolation yields while minimizing the number of experimental steps to analytical results. The result is the development of a comprehensive integrated platform for the characterization of a wide range of biotherapeutics, including peptibodies, monoclonal antibodies, and bispecific antibodies. Empowered by this automated IA-CE-MS approach, implementing biotransformation studies at an early drug discovery stage can speed up the drug development process.

Introducing an In Vitro Liver Stability Assay Capable of Predicting the In Vivo Pharmacodynamic Efficacy of siRNAs for IVIVC.

There has been a renewed interest in therapeutic small interfering RNAs (siRNAs) over the past few years. This is particularly the result of successful and efficient delivery of N-acetylgalactosamine (GalNAc)-conjugated siRNAs to the liver. In general, the lead selection process for siRNA drugs is faster and more straightforward than traditional small molecules. Nevertheless, many siRNAs of different sequences and chemical modification patterns must still be evaluated before arriving at a final candidate. One of the major difficulties in streamlining this workflow is the well-known phenomenon that the in vitro data obtained from oligonucleotides transfected into cells are not directly predictive of their in vivo activity. Consequently, all oligonucleotides with some degree of in vitro activity are typically screened in vivo before final lead selection. Here, we demonstrate that the stability of liver-targeting GalNAc-conjugated siRNAs in a mouse liver homogenate shows an acceptable correlation to their in vivo target knockdown efficacy. Therefore, we suggest the incorporation of an in vitro liver homogenate stability assay during the lead optimization process for siRNAs. The addition of this assay to a flow scheme may decrease the need for animal studies, and it could bring cost savings and increase efficiency in siRNA drug development.

POE Immunoassay: Plate-based oligonucleotide electro-chemiluminescent immunoassay for the quantification of nucleic acids in biological matrices.

Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems-neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications.

Strain-Dependent Variability of Early Discovery Small Molecule Pharmacokinetics in Mice: Does Strain Matter?

Drug discovery programs routinely perform pharmacokinetic (PK) studies in mice to prioritize lead compounds based on anticipated exposure-efficacy and exposure-toxicity relationships. Because of logistical and/or technical issues, the strain of mouse in early discovery PK studies may not always match the strain in toxicity or efficacy studies. This elicits the question do appreciable strain-dependent differences in PK parameters exist to an extent that would warrant conducting PK studies in a strain that matches efficacy and toxicity models? To understand the impact that strain may have on PK parameters, we selected eight marketed drugs with well characterized absorption, distribution, metabolism, and excretion properties and diverse structures to perform PK studies in three common mouse strains (Bagg Albino c, C57BL/6, and CD-1). Some statistical strain-dependent differences were observed; however, we found good general agreement of PK parameters between strains: 88%, 100%, 75%, 76%, 94%, and 88% of compounds were within twofold across strains for clearance, volume of distribution at steady state, t 1/2, C max, T max, and oral bioavailability, respectively. Overall, we recommend that an approach using a single strain of mouse is appropriate for discovery screening PK studies, provided that proper caution is exercised. SIGNIFICANCE STATEMENT: The mouse strain in discovery pharmacokinetic (PK) studies may not match the strain in efficacy and toxicology studies. Currently, there is a gap in the literature addressing whether differences in PK parameters across mouse strains exist such that multiple PK studies are warranted. The results from this study indicated that the PK properties of clinically used drugs between mouse strains are within an acceptable range such that single strain PK is appropriate.

Emerging siRNA Design Principles and Consequences for Biotransformation and Disposition in Drug Development.

After two decades teetering at the intersection of laboratory tool and therapeutic reality, with two siRNA drugs now clinically approved, this modality has finally come into fruition. Consistent with other emerging modalities, initial proof-of-concept efforts concentrated on coupling pharmacologic efficacy with desirable safety profiles. Consequently, thorough investigations of siRNA absorption, distribution, metabolism, and excretion (ADME) properties are lacking. Advancing ADME knowledge will aid establishment of in vitro-in vivo correlations and pharmacokinetic-pharmacodynamic relationships to optimize candidate selection through discovery and translation. Here, we outline the emerging siRNA design principles and discuss the consequences for siRNA disposition and biotransformation. We propose a conceptual framework for siRNA ADME evaluation, contextualizing the site of biotransformation product formation with PK-PD modulation, and end with a discussion around safety and regulatory considerations and future directions for this modality.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles.

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

Pharmacokinetic and Drug Metabolism Properties of Novel Therapeutic Modalities.

The discovery and development of novel pharmaceutical therapies is rapidly transitioning from a small molecule-dominated focus to a more balanced portfolio consisting of small molecules, monoclonal antibodies, engineered proteins (modified endogenous proteins, bispecific antibodies, and fusion proteins), oligonucleotides, and gene-based therapies. This commentary, and the special issue as a whole, aims to highlight these emerging modalities and the efforts underway to better understand their unique pharmacokinetic and absorption, disposition, metabolism, and excretion (ADME) properties. The articles highlighted herein can be broadly grouped into those focusing on the ADME properties of novel therapeutics, those exploring targeted-delivery strategies, and finally, those discussing oligonucleotide therapies. It is also evident that whereas the field in general continues to progress toward new and more complex molecules, a significant amount of effort is still being placed on antibody-drug conjugates. As therapeutic molecules become increasingly complex, a parallel demand for advancements in experimental and analytical tools will become increasingly evident, both to increase the speed and efficiency of identifying safe and efficacious molecules and simultaneously decreasing our dependence on in vivo studies in preclinical species. The research and commentary included in this special issue will provide researchers, clinicians, and the patients we serve more options in the ongoing fight against grievous illnesses and unmet medical needs. SIGNIFICANCE STATEMENT: Recent trends in drug discovery and development suggest a shift away from a small molecule-dominated approach to a more balanced portfolio that includes small molecules, monoclonal antibodies, engineered proteins, and gene therapies. The research presented in this special issue of Drug Metabolism and Disposition will serve to highlight advancements in the understanding of the mechanisms that govern the pharmacokinetic and drug metabolism properties of the novel therapeutic modalities.

Plasma and Liver Protein Binding of N-Acetylgalactosamine-Conjugated Small Interfering RNA.

Understanding small interfering RNA (siRNA) fraction unbound (f u) in relevant physiologic compartments is critical for establishing pharmacokinetic-pharmacodynamic relationships for this emerging modality. In our attempts to isolate the equilibrium free fraction of N-acetylgalactosamine-conjugated siRNA using classic small-molecule in vitro techniques, we found that the hydrodynamic radius was critical in determining the size exclusion limit requirements for f u isolation, largely validating the siRNA "rigid rod" hypothesis. With this knowledge, we developed an orthogonally validated 50 kDa molecular-mass cutoff ultrafiltration assay to quantify f u in biologic matrices including human, nonhuman primate, rat, and mouse plasma, and human liver homogenate. To enhance understanding of the siRNA-plasma interaction landscape, we examined the effects of various common oligonucleotide therapeutic modifications to the ribose and helix backbone on siRNA f u in plasma (f u,plasma) and found that chemical modifications can alter plasma protein binding by at least 20%. Finally, to gain insight into which specific plasma proteins bind to siRNA, we developed a qualitative screen to identify binding "hits" across a panel of select purified human plasma proteins.

Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics.

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3' exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

Quantification of siRNA-Antibody Conjugates in Biological Matrices by Triplex-Forming Oligonucleotide ELISA.

The potential repertoire of short interfering RNA (siRNA) therapeutics is expanding as targeting strategies evolve. One approach to enable organ-specific delivery has been to directly conjugate siRNA to a monoclonal antibody (siRNA-mAb), analogous to antibody-drug conjugates. Detection of intact siRNA-mAb conjugates presents a bioanalytical challenge given that certain synthetic nucleotide chemical modifications and low-temperature requirements render common oligonucleotide detection assays, such as reverse transcription-polymerase chain reaction, incompatible with the immunoassay component. To circumvent these issues, we developed a triplex-forming oligonucleotide ELISA using locked nucleic acid (LNA) containing oligonucleotide probes. We demonstrate that the incorporation of these LNAs allow for an enrichment and immobilization of siRNA directly conjugated to an antibody at nondenaturing temperatures. Without further requirement for extraction or amplification, we can sensitively and specifically detect intact siRNA-mAb conjugates in complex matrices such as serum and tissue homogenate.

Immunoaffinity capture coupled with capillary electrophoresis - mass spectrometry to study therapeutic protein stability in vivo.

Protein engineering is at an all-time high in biopharmaceutics. As a result, absorption, distribution, metabolism and excretion (ADME) of proteins has become more important to understand in the context of engineering strategies to optimize therapeutic properties of potential lead constructs. Immunoaffinity capture coupled with a newly developed capillary electrophoresis - mass spectrometry (CE-MS) system was used to characterize intact protein mass analysis of a wild type Fc-FGF21 construct and a sequence re-engineered Fc-FGF21 construct from an in vivo study. A number of truncated forms were observed and the time courses of the various proteolytic products were identified and compared between the two constructs. The abundances of the intact and truncated forms were used to provide the basis to semi-quantify ADME properties of the two protein forms. The use of this immunoaffinity capture followed by CE-MS based intact mass analysis workflow provided a qualitative and quantitative analysis of the pharmacokinetic profiles of the two proteins. The platform presented here holds great potential in characterization of the ADME properties of proteins.

SRM-based measurements of proprotein convertase subtilisin/kexin type 9 and lipoprotein(a) kinetics in nonhuman primate serum.

AIM: PCSK9 and Lp(a) have been identified as potential biomarkers for cardiovascular disease. The ability to measure protein turnover rates will provide insights into the dynamic properties of these proteins and lead to better understanding of their biological roles. We aimed to implement the stable isotope-labeled tracers ([2H3]-leucine) and develop a novel LC-selected reaction monitoring (SRM) mass spectrometry (MS) method to study the kinetics of PCSK9 and Lp(a). RESULTS: A sensitive method using immunoaffinity enrichment coupled with LC-SRM MS was developed to measure the production and degradation rates of PCSK9 and Lp(a) in naive nonhuman primate serum. Comparable results were obtained from two different routes of tracer administration. CONCLUSION: Immunoaffinity enrichment coupled with LC-SRM MS demonstrated success in in vivo kinetic measurements of proteins with relatively slow turnover rate (Lp[a]) or low abundance (PCSK9) in serum.

Altering Antibody-Drug Conjugate Binding to the Neonatal Fc Receptor Impacts Efficacy and Tolerability.

Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.

Intact mass analysis of monoclonal antibodies by capillary electrophoresis-Mass spectrometry.

Characterization of monoclonal antibody (mAb) therapeutics by intact mass analysis provides important information on sequence integrity and post-translational modifications. In order to obtain domain specific information, monoclonal antibodies are reduced to heavy and light chain components or enzymatically digested into smaller portions or peptides. Liquid chromatography (LC) is widely used for separation of the antibody fragments in line with mass spectrometry (MS) for characterization. Capillary electrophoresis (CE) is an analytical technique with high separation efficiency, high sensitivity, and minimal inter-run sample carryover. Combining the resolving power of CE with electrospray ionization (ESI) MS has great potential in regards to accurate mass characterization of protein therapeutics and has been a long sought-after approach. However, the intrinsic technical difficulty in coupling CE to MS has hindered the broad application of CE-MS across the biopharmaceutical industry. Recently, a CE-MS interface has been developed [1] and commercialized. Herein, we report implementation of this technology for coupling CE to an Agilent time-of-flight (TOF) mass spectrometer. CE-MS provides an attractive complement to LC-MS for separation and intact mass determination of mAbs and antibody-based therapeutics.